WorldWideScience

Sample records for vitro protein ligation

  1. A set of ligation-independent in vitro translation vectors for eukaryotic protein production

    Directory of Open Access Journals (Sweden)

    Endo Yaeta

    2008-03-01

    Full Text Available Abstract Background The last decade has brought the renaissance of protein studies and accelerated the development of high-throughput methods in all aspects of proteomics. Presently, most protein synthesis systems exploit the capacity of living cells to translate proteins, but their application is limited by several factors. A more flexible alternative protein production method is the cell-free in vitro protein translation. Currently available in vitro translation systems are suitable for high-throughput robotic protein production, fulfilling the requirements of proteomics studies. Wheat germ extract based in vitro translation system is likely the most promising method, since numerous eukaryotic proteins can be cost-efficiently synthesized in their native folded form. Although currently available vectors for wheat embryo in vitro translation systems ensure high productivity, they do not meet the requirements of state-of-the-art proteomics. Target genes have to be inserted using restriction endonucleases and the plasmids do not encode cleavable affinity purification tags. Results We designed four ligation independent cloning (LIC vectors for wheat germ extract based in vitro protein translation. In these constructs, the RNA transcription is driven by T7 or SP6 phage polymerase and two TEV protease cleavable affinity tags can be added to aid protein purification. To evaluate our improved vectors, a plant mitogen activated protein kinase was cloned in all four constructs. Purification of this eukaryotic protein kinase demonstrated that all constructs functioned as intended: insertion of PCR fragment by LIC worked efficiently, affinity purification of translated proteins by GST-Sepharose or MagneHis particles resulted in high purity kinase, and the affinity tags could efficiently be removed under different reaction conditions. Furthermore, high in vitro kinase activity testified of proper folding of the purified protein. Conclusion Four newly

  2. In vivo and in vitro protein ligation by naturally occurring and engineered split DnaE inteins.

    Directory of Open Access Journals (Sweden)

    A Sesilja Aranko

    Full Text Available BACKGROUND: Protein trans-splicing by naturally occurring split DnaE inteins is used for protein ligation of foreign peptide fragments. In order to widen biotechnological applications of protein trans-splicing, it is highly desirable to have split inteins with shorter C-terminal fragments, which can be chemically synthesized. PRINCIPAL FINDINGS: We report the identification of new functional split sites in DnaE inteins from Synechocystis sp. PCC6803 and from Nostoc punctiforme. One of the newly engineered split intein bearing C-terminal 15 residues showed more robust protein trans-splicing activity than naturally occurring split DnaE inteins in a foreign context. During the course of our experiments, we found that protein ligation by protein trans-splicing depended not only on the splicing junction sequences, but also on the foreign extein sequences. Furthermore, we could classify the protein trans-splicing reactions in foreign contexts with a simple kinetic model into three groups according to their kinetic parameters in the presence of various reducing agents. CONCLUSION: The shorter C-intein of the newly engineered split intein could be a useful tool for biotechnological applications including protein modification, incorporation of chemical probes, and segmental isotopic labelling. Based on kinetic analysis of the protein splicing reactions, we propose a general strategy to improve ligation yields by protein trans-splicing, which could significantly enhance the applications of protein ligation by protein trans-splicing.

  3. Expressed protein ligation for a large dimeric protein

    NARCIS (Netherlands)

    Karagöz, G.E.; Sinnige, T; Hsieh, O.; Rüdiger, S.G.D.

    2011-01-01

    Expressed protein ligation (EPL) is a protein engineering tool for post-translational ligation of protein or peptide fragments. This technique allows modification of specific parts of proteins, opening possibilities for incorporating probes for biophysical applications such as nuclear magnetic

  4. Protein biomarker validation via proximity ligation assays.

    Science.gov (United States)

    Blokzijl, A; Nong, R; Darmanis, S; Hertz, E; Landegren, U; Kamali-Moghaddam, M

    2014-05-01

    The ability to detect minute amounts of specific proteins or protein modifications in blood as biomarkers for a plethora of human pathological conditions holds great promise for future medicine. Despite a large number of plausible candidate protein biomarkers published annually, the translation to clinical use is impeded by factors such as the required size of the initial studies, and limitations of the technologies used. The proximity ligation assay (PLA) is a versatile molecular tool that has the potential to address some obstacles, both in validation of biomarkers previously discovered using other techniques, and for future routine clinical diagnostic needs. The enhanced specificity of PLA extends the opportunities for large-scale, high-performance analyses of proteins. Besides advantages in the form of minimal sample consumption and an extended dynamic range, the PLA technique allows flexible assay reconfiguration. The technology can be adapted for detecting protein complexes, proximity between proteins in extracellular vesicles or in circulating tumor cells, and to address multiple post-translational modifications in the same protein molecule. We discuss herein requirements for biomarker validation, and how PLA may play an increasing role in this regard. We describe some recent developments of the technology, including proximity extension assays, the use of recombinant affinity reagents suitable for use in proximity assays, and the potential for single cell proteomics. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. © 2013.

  5. Generating site-specifically modified proteins via a versatile and stable nucleophilic carbon ligation.

    Science.gov (United States)

    Kudirka, Romas; Barfield, Robyn M; McFarland, Jesse; Albers, Aaron E; de Hart, Gregory W; Drake, Penelope M; Holder, Patrick G; Banas, Stefanie; Jones, Lesley C; Garofalo, Albert W; Rabuka, David

    2015-02-19

    There is a need for facile chemistries that allow for chemo- and regioselectivity in bioconjugation reactions. To address this need, we are pioneering site-specific bioconjugation methods that use formylglycine as a bioorthogonal handle on a protein surface. Here we introduce aldehyde-specific bioconjugation chemistry, the trapped-Knoevenagel ligation. The speed and stability of the trapped-Knoevenagel ligation further advances the repertoire of aldehyde-based bioconjugations and expands the toolbox for site-specific protein modifications. The trapped-Knoevenagel ligation reaction can be run at near neutral pH in the absence of catalysts to produce conjugates that are stable under physiological conditions. Using this new ligation, we generated an antibody-drug conjugate that demonstrates excellent efficacy in vitro and in vivo. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Semisynthesis of Ribonuclease A using Intein-Mediated Protein Ligation

    OpenAIRE

    Ulrich Arnold; Hinderaker, Matthew P.; Raines, Ronald T.

    2002-01-01

    The introduction of non-natural amino acid residues or modules into proteins provides a new means to explore the basis for conformational stability, folding/unfolding behavior, or biological function. We exploited intein-mediated protein ligation to produce a semisynthetic ribonuclease A. Of the 124 residues of RNase A, residues 1–94 were linked to an intein. After expression of the fusion protein and thiol-induced cleavage, the RNase A(1–94) fragment possessed a C-terminal thioester. A pepti...

  7. Semisynthesis of Ribonuclease A using Intein-Mediated Protein Ligation

    Directory of Open Access Journals (Sweden)

    Ulrich Arnold

    2002-01-01

    Full Text Available The introduction of non-natural amino acid residues or modules into proteins provides a new means to explore the basis for conformational stability, folding/unfolding behavior, or biological function. We exploited intein-mediated protein ligation to produce a semisynthetic ribonuclease A. Of the 124 residues of RNase A, residues 1–94 were linked to an intein. After expression of the fusion protein and thiol-induced cleavage, the RNase A(1–94 fragment possessed a C-terminal thioester. A peptide identical to the C-terminal residues 95–124 of RNase A (with residue 95 being cysteine was successfully ligated to that thioester thereby reconstituting full-length wild-type RNase A. In mass spectrometry, this semisynthetic RNase A proved to be undistinguishable from the control protein, namely recombinant wild-type RNase A. Recombinant wild-type RNase A was obtained by expression of RNase A(1–124–intein fusion protein followed by thiol-induced cleavage and hydrolysis of the thioester. Both proteins showed thermal stabilities (Tm and catalytic activities comparable to the wild-type enzyme, indicating that both proteins folded properly. These results might serve as basis for the semisynthesis of RNase A variants containing non-natural modules in the aforementioned peptide.

  8. Sensitive detection of aggregated prion protein via proximity ligation.

    Science.gov (United States)

    Hammond, Maria; Wik, Lotta; Deslys, Jean-Philippe; Comoy, Emmanuel; Linné, Tommy; Landegren, Ulf; Kamali-Moghaddam, Masood

    2014-01-01

    The DNA assisted solid-phase proximity ligation assay (SP-PLA) provides a unique opportunity to specifically detect prion protein (PrP) aggregates by investigating the collocation of 3 or more copies of the specific protein. We have developed an SP-PLA that can detect PrP aggregates in brain homogenates from infected hamsters even after a 10(7)-fold dilution. In contrast, brain homogenate from uninfected animals did not generate a detectable signal at 100-fold higher concentration. Using either of the 2 monoclonal anti-PrP antibodies, 3F4 and 6H4, we successfully detected low concentrations of aggregated PrP. The presented results provide a proof of concept that this method might be an interesting tool in the development of diagnostic approaches of prion diseases.

  9. Convergent synthesis of proteins by kinetically controlled ligation

    Science.gov (United States)

    Kent, Stephen; Pentelute, Brad; Bang, Duhee; Johnson, Erik; Durek, Thomas

    2010-03-09

    The present invention concerns methods and compositions for synthesizing a polypeptide using kinetically controlled reactions involving fragments of the polypeptide for a fully convergent process. In more specific embodiments, a ligation involves reacting a first peptide having a protected cysteyl group at its N-terminal and a phenylthioester at its C-terminal with a second peptide having a cysteine residue at its N-termini and a thioester at its C-termini to form a ligation product. Subsequent reactions may involve deprotecting the cysteyl group of the resulting ligation product and/or converting the thioester into a thiophenylester.

  10. Recovery of the Zika virus through an in vitro ligation approach.

    Science.gov (United States)

    Deng, Cheng-Lin; Zhang, Qiu-Yan; Chen, Dong-Dong; Liu, Si-Qing; Qin, Cheng-Feng; Zhang, Bo; Ye, Han-Qing

    2017-07-01

    In this study, an in vitro ligation method was developed to assemble a full-length infectious cDNA clone of the Zika virus (ZIKV). Four contiguous cDNA subclones covering the complete ZIKV genome were constructed with unique BglI restriction sites at the ends of each fragment. The BglI restriction sites only allow in vitro ligation to happen between interconnecting restriction sites from adjacent cDNA fragments, resulting in an intact full-length cDNA of ZIKV. RNA transcripts derived from the full-length cDNA were infectious. The recombinant virus replicated as efficiently as the wild-type virus with similar growth kinetics and plaque morphologies in Vero and C6/36 cells. Both viruses were inhibited by NITD008 treatment. This in vitro ligation method will facilitate manipulation of the viral genome through genetic modifications of four separated subclones of ZIKV for the rapid and rational development of candidate vaccines and viral replication study.

  11. [Effect of bile duct ligation and recanalization on rat hepatocyte epithelial-mesenchymal phenotype and NOX4 protein expression].

    Science.gov (United States)

    Lou, An-Ni; Pan, Chun-Qiu; Li, Yang; Yang, Ren-Qiang; Li, Xu

    2015-10-01

    To observe epithelial-mesenchymal phenotypes and oxidative stress related protein expressions of the liver cells in a rat model of liver fibrosis induced by bile duct ligation and recanalization. Twenty-four male Wistar rats were randomized into 4 groups, including a sham-operated group, two bile duct ligation groups with ligation for 2 and 4 weeks, and a bile duct ligation group with a 2-week ligation followed by a 2-week recanalization. HE staining and Masson staining were used to assess liver fibrosis in the rats, and immunohistochemistry and Western blotting were employed to detect expressions of the epithelial and mesenchymal marker proteins and oxidative stress-related proteins. Compared with the sham-operated group, the rats with bile duct ligation showed obvious liver fibrosis, which worsened as the ligation time extended, accompanied by significantly increased expression of α-SMA, collagen I, NOX(4) and vimetin and reduced E-cadherin expression. Compared with the rats with bile duct ligation for 4 weeks, the rats in bile duct ligation-recanalization group showed obviously lessened liver fibrosis, significantly lowered expressions of NOX(4) and mesenchymal cell maker proteins, and enhanced expressions of epithelial cell marker proteins. Bile duct ligation up-regulates mesenchymal phenotype-related proteins and NOX(4) protein expression and down-regulates the expression of epithelial phenotype-related proteins, and these changes can be reversed by subsequent bile duct recanalization.

  12. Covalent attachment of proteins to solid supports and surfaces via Sortase-mediated ligation.

    Directory of Open Access Journals (Sweden)

    Lilyan Chan

    Full Text Available BACKGROUND: There is growing interest in the attachment of proteins to solid supports for the development of supported catalysts, affinity matrices, and micro devices as well as for the development of planar and bead based protein arrays for multiplexed assays of protein concentration, interactions, and activity. A critical requirement for these applications is the generation of a stable linkage between the solid support and the immobilized, but still functional, protein. METHODOLOGY: Solid supports including crosslinked polymer beads, beaded agarose, and planar glass surfaces, were modified to present an oligoglycine motif to solution. A range of proteins were ligated to the various surfaces using the Sortase A enzyme of S. aureus. Reactions were carried out in aqueous buffer conditions at room temperature for times between one and twelve hours. CONCLUSIONS: The Sortase A transpeptidase of S. aureus provides a general, robust, and gentle approach to the selective covalent immobilization of proteins on three very different solid supports. The proteins remain functional and accessible to solution. Sortase mediated ligation is therefore a straightforward methodology for the preparation of solid supported enzymes and bead based assays, as well as the modification of planar surfaces for microanalytical devices and protein arrays.

  13. Multiplexed homogeneous proximity ligation assays for high throughput protein biomarker research in serological material

    DEFF Research Database (Denmark)

    Lundberg, Martin; Thorsen, Stine Buch; Assarsson, Erika

    2011-01-01

    specificity, even in multiplex, by its dual recognition feature, its proximity requirement, and most importantly by using unique sequence specific reporter fragments on both antibody-based probes. To illustrate the potential of this protein detection technology, a pilot biomarker research project......A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays (PLA) in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub pM sensitivity each consuming...

  14. Proteomics-based analysis of lung injury-induced proteins in a mouse model of common bile duct ligation.

    Science.gov (United States)

    Sakaue, Tomohisa; Shikata, Fumiaki; Utsunomiya, Kaho; Fukae, Shunya; Kurata, Mie; Nakaoka, Hirotomo; Okazaki, Mikio; Kawanishi, Yujiro; Kojima, Ai; Higashiyama, Shigeki; Izutani, Hironori

    2017-06-01

    Lung injury is a life-threatening complication in patients with liver dysfunction. We recently provided an experimental lung injury model in mouse with common bile duct ligation. In this study, we aimed to characterize the pathologic and biochemical features of lung tissues in common bile duct ligation mice using a proteomic approach. Common bile ducts of BALB/c mice, 8 weeks of age, were ligated operatively. CD31-expressing pulmonary cells were sorted with immunomagnetic microbeads, and protein profiles were examined by 2-dimensional gel electrophoresis. Based on the results of protein identification, immunohistochemistry and quantitative reverse transcription polymerase chain reaction were carried out in pulmonary and hepatic tissues. Two-dimensional gel electrophoresis revealed 3 major inflammation-associated proteins exhibiting considerable increases in the number of CD31-positive pulmonary cells after common bile duct ligation. Mass spectrometry analysis identified these proteins as SerpinB1a (48 kDa), ANXA1 (46 kDa), and S100A9 (16 kDa). Furthermore, the 3 proteins were more highly expressed in dilated pulmonary blood vessels of common bile duct ligation mice, in which neutrophils and monocytes were prominent, as shown by immunohistochemistry. More importantly, SerpinB1a mRNA and protein were significantly upregulated in the liver, whereas S100A9 and ANXA1 mRNA and protein were upregulated in the lungs, as shown by quantitative reverse transcription polymerase chain reaction and Western blotting. We identified 3 proteins that were highly expressed in the lung after common bile duct ligation using a proteomics-based approach. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Efficient Synthesis of Peptide and Protein Functionalized Pyrrole-Imidazole Polyamides Using Native Chemical Ligation

    Directory of Open Access Journals (Sweden)

    Brian M. G. Janssen

    2015-06-01

    Full Text Available The advancement of DNA-based bionanotechnology requires efficient strategies to functionalize DNA nanostructures in a specific manner with other biomolecules, most importantly peptides and proteins. Common DNA-functionalization methods rely on laborious and covalent conjugation between DNA and proteins or peptides. Pyrrole-imidazole (Py–Im polyamides, based on natural minor groove DNA-binding small molecules, can bind to DNA in a sequence specific fashion. In this study, we explore the use of Py–Im polyamides for addressing proteins and peptides to DNA in a sequence specific and non-covalent manner. A generic synthetic approach based on native chemical ligation was established that allows efficient conjugation of both peptides and recombinant proteins to Py–Im polyamides. The effect of Py–Im polyamide conjugation on DNA binding was investigated by Surface Plasmon Resonance (SPR. Although the synthesis of different protein-Py–Im-polyamide conjugates was successful, attenuation of DNA affinity was observed, in particular for the protein-Py–Im-polyamide conjugates. The practical use of protein-Py–Im-polyamide conjugates for addressing DNA structures in an orthogonal but non-covalent manner, therefore, remains to be established.

  16. Application of meta- and para- phenylenediamine as enhanced oxime ligation catalysts for protein labeling, PEGylation, immobilization and release

    Science.gov (United States)

    Mahmoodi, Mohammad M.; Rashidian, Mohammad; Zhang, Yi; Distefano, Mark D.

    2015-01-01

    Meta- and para- phenylenediamines have recently been shown to catalyze oxime and hydrazone ligation reactions at rates much faster than aniline, a commonly used catalyst. Here, it is demonstrated how these new catalysts can be used in a generally applicable procedure for fluorescent labeling, PEGylation, immobilization and release of aldehyde and ketone functionalized proteins. The chemical orthogonality of phenylenediamine-catalyzed oxime ligation versus copper catalyzed click reaction has also been harnessed for simultaneous dual labeling of bifunctional proteins containing both aldehyde and alkyne groups in high yield. PMID:25640893

  17. The comparison of frictional resistance in titanium, self-ligating stainless steel, and stainless steel brackets using stainless steel and TMA archwires: An in vitro study

    OpenAIRE

    Khalid, Syed Altaf; Kumar, Vadivel; Jayaram, Prithviraj

    2012-01-01

    Aim: The aim of the study was to compare the frictional resistance of titanium, self-ligating stainless steel, and conventional stainless steel brackets, using stainless steel and titanium molybdenum alloy (TMA) archwires. Materials and Methods: We compared the frictional resistance in 0.018 slot and 0.022 slot of the three brackets - titanium, self-ligating stainless steel, and conventional stainless steel - using stainless steel archwires and TMA archwires. An in vitro study of simulated ca...

  18. In situ proximity ligation assays indicate that hemochromatosis proteins Hfe and transferrin receptor 2 (Tfr2) do not interact.

    Science.gov (United States)

    Rishi, Gautam; Crampton, Emily M; Wallace, Daniel F; Subramaniam, V Nathan

    2013-01-01

    The hemochromatosis associated proteins HFE and Transferrin Receptor 2 (TFR2) have been shown to be important for the proper regulation of hepcidin. A number of in vitro studies using transient overexpression systems have suggested that an interaction between HFE and TFR2 is required for the regulation of hepcidin. This model of iron sensing which centers upon the requirement for an interaction between HFE and TFR2 has recently been questioned with in vivo studies in mice from our laboratory and others which suggest that Hfe and Tfr2 can regulate hepcidin independently of each other. To re-examine the postulated interaction between Hfe and Tfr2 we developed a novel expression system in which both proteins are stably co-expressed and used the proximity ligation assay to examine the interactions between Hfe, Tfr1 and Tfr2 at a cellular level. We were able to detect the previously described interaction between Hfe and Tfr1, and heterodimers between Tfr1 and Tfr2; however no interaction between Hfe and Tfr2 was observed in our system. The results from this study indicate that Hfe and Tfr2 do not interact with each other when they are stably expressed at similar levels. Furthermore, these results support in vivo studies which suggest that Hfe and Tfr2 can independently regulate hepcidin.

  19. In situ proximity ligation assays indicate that hemochromatosis proteins Hfe and transferrin receptor 2 (Tfr2 do not interact.

    Directory of Open Access Journals (Sweden)

    Gautam Rishi

    Full Text Available The hemochromatosis associated proteins HFE and Transferrin Receptor 2 (TFR2 have been shown to be important for the proper regulation of hepcidin. A number of in vitro studies using transient overexpression systems have suggested that an interaction between HFE and TFR2 is required for the regulation of hepcidin. This model of iron sensing which centers upon the requirement for an interaction between HFE and TFR2 has recently been questioned with in vivo studies in mice from our laboratory and others which suggest that Hfe and Tfr2 can regulate hepcidin independently of each other. To re-examine the postulated interaction between Hfe and Tfr2 we developed a novel expression system in which both proteins are stably co-expressed and used the proximity ligation assay to examine the interactions between Hfe, Tfr1 and Tfr2 at a cellular level. We were able to detect the previously described interaction between Hfe and Tfr1, and heterodimers between Tfr1 and Tfr2; however no interaction between Hfe and Tfr2 was observed in our system. The results from this study indicate that Hfe and Tfr2 do not interact with each other when they are stably expressed at similar levels. Furthermore, these results support in vivo studies which suggest that Hfe and Tfr2 can independently regulate hepcidin.

  20. Minocycline restores cognitive-relative altered proteins in young bile duct-ligated rat prefrontal cortex.

    Science.gov (United States)

    Li, Shih-Wen; Chen, Yu-Chieh; Sheen, Jiunn-Ming; Hsu, Mei-Hsin; Tain, You-Lin; Chang, Kow-Aung; Huang, Li-Tung

    2017-07-01

    Bile duct ligation (BDL) model is used to study hepatic encephalopathy accompanied by cognitive impairment. We employed the proteomic analysis approach to evaluate cognition-related proteins in the prefrontal cortex of young BDL rats and analyzed the effect of minocycline on these proteins and spatial memory. BDL was induced in young rats at postnatal day 17. Minocycline as a slow-release pellet was implanted into the peritoneum. Morris water maze test and two-dimensional liquid chromatography-tandem mass spectrometry were used to evaluate spatial memory and prefrontal cortex protein expression, respectively. We used 2D/LC-MS/MS to analyze for affected proteins in the prefrontal cortex of young BDL rats. Results were verified with Western blotting, immunohistochemistry, and quantitative real-time PCR. The effect of minocycline in BDL rats was assessed. BDL induced spatial deficits, while minocycline rescued it. Collapsin response mediator protein 2 (CRMP2) and manganese-dependent superoxide dismutase (MnSOD) were upregulated and nucleoside diphosphate kinase B (NME2) was downregulated in young BDL rats. BDL rats exhibited decreased levels of brain-derived neurotrophic factor (BDNF) mRNA as compared with those by the control. However, minocycline treatment restored CRMP2 and NME2 protein expression, BDNF mRNA level, and MnSOD activity to control levels. We demonstrated that BDL altered the expression of CRMP2, NME2, MnSOD, and BDNF in the prefrontal cortex of young BDL rats. However, minocycline treatment restored the expression of the affected mediators that are implicated in cognition. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Chemoselective ligation

    Energy Technology Data Exchange (ETDEWEB)

    Saxon, Eliana [Albany, CA; Bertozzi, Carolyn R [Berkeley, CA

    2011-05-10

    The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

  2. Chemoselective ligation

    Science.gov (United States)

    Saxon, Eliana; Bertozzi, Carolyn

    2006-10-17

    The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

  3. Chemoselective ligation

    Energy Technology Data Exchange (ETDEWEB)

    Saxon, Eliana [Albany, CA; Bertozzi, Carolyn Ruth [Berkeley, CA

    2011-12-13

    The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

  4. Chemoselective ligation

    Energy Technology Data Exchange (ETDEWEB)

    Saxon, Eliana (Albany, CA); Bertozzi, Carolyn R. (Berkeley, CA)

    2010-02-23

    The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g. on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

  5. Chemoselective ligation

    Energy Technology Data Exchange (ETDEWEB)

    Saxon, Eliana [Albany, CA; Bertozzi, Carolyn [Berkeley, CA

    2003-05-27

    The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

  6. Chemoselective ligation

    Energy Technology Data Exchange (ETDEWEB)

    Saxon, Eliana [Albany, CA; Bertozzi, Carolyn R [Berkeley, CA

    2011-04-12

    The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

  7. Chemoselective ligation

    Energy Technology Data Exchange (ETDEWEB)

    Saxon, Eliana (Albany, CA); Bertozzi, Carolyn Ruth (Berkeley, CA)

    2010-11-23

    The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

  8. Efficient Bioconjugation of Protein Capture Agents to Biosensor Surfaces Using Aniline-Catalyzed Hydrazone Ligation

    Science.gov (United States)

    Byeon, Ji-Yeon; Limpoco, F. T.; Bailey, Ryan C.

    2010-01-01

    Aniline-catalyzed hydrazone ligation between surface immobilized hydrazines and aldehyde-modified antibodies is shown to be an efficient method for attaching protein capture agents to model oxide-coated biosensor substrates. Silicon photonic microring resonators are used to directly evaluate the efficiency of this surface bioconjugate reaction at various pHs and in the presence or absence of aniline as a nucleophilic catalyst. It is found that aniline significantly increases the net antibody loading for surfaces functionalized over a pH range from 4.5 to 7.4, allowing derivatization of substrates with reduced incubation time and sample consumption. This increase in antibody loading directly results in more sensitive antigen detection when functionalized microrings are employed in a label-free immunoassay. Furthermore, these experiments also reveal an interesting pH dependent non-covalent binding trend that plays an important role in dictating the amount of antibody attached onto the substrate, highlighting the competing contributions of the bioconjugate reaction rate and the dynamic interactions that control opportunities for a solution-phase biomolecule to react with a substrate-bound reagent. PMID:20809595

  9. Differential gene expression and adherence of Escherichia coli O157:H7 in vitro and in ligated pig intestines.

    Directory of Open Access Journals (Sweden)

    Xianhua Yin

    Full Text Available BACKGROUND: Escherichia coli O157:H7 strain 86-24 grown in MacConkey broth (MB shows almost no adherence to cultured epithelial cells but adheres well in pig ligated intestines. This study investigated the mechanisms associated with the difference between in-vitro and in-vivo adherence of the MB culture. METHODOLOGY/PRINCIPAL FINDINGS: It was found that decreased adherence in vitro by bacteria grown in MB was mainly due to lactose, possibly implicating the involvement of carbon catabolite repression (CCR. Expression of selected virulence-related genes associated with adherence and CCR was then examined by quantitative PCR. When bacteria were grown in MB and Brain Heart Infusion with NaHCO(3 (BHIN plus lactose, pH was reduced to 5.5-5.9 and there was a significant decrease in expression of the locus of enterocyte effacement (LEE genes eae, tir, espD, grlA/R and ler, and an increase in cya (cAMP, and two negative regulators of the LEE, gadE and hfq. Putative virulence genes stcE, hlyA, ent and nleA were also decreased in vitro. Reversal of these changes was noted for bacteria recovered from the intestine, where transcripts for qseF and fis and putative virulence factors AidA(15, TerC and Ent/EspL2 were significantly increased, and transcripts for AIDA(48, Iha, UreC, Efa1A, Efa1B, ToxB, EhxA, StcE, NleA and NleB were expressed at high levels. CONCLUSIONS/SIGNIFICANCE: Presence of lactose resulted in decreased expression of LEE genes and the failure of EHEC O157:H7 to adhere to epithelial cells in vitro but this repression was overcome in vivo. CCR and/or acidic pH may have played a role in repression of the LEE genes. Bacterial pathogens need to integrate their nutritional metabolism with expression of virulence genes but little is known of how this is done in E. coli O157:H7. This study indicates one aspect of the subject that should be investigated further.

  10. Tubal Ligation

    Science.gov (United States)

    ... details of the procedure Discuss the causes and probability of sterilization failure Share information about tubal ligation ... a not-for-profit organization and proceeds from Web advertising help support our mission. Mayo Clinic does ...

  11. Selective labeling of polypeptides using protein farnesyltransferase via rapid oxime ligation.

    Science.gov (United States)

    Rashidian, Mohammad; Dozier, Jonathan K; Lenevich, Stepan; Distefano, Mark D

    2010-12-21

    An aldehyde-containing alternative substrate for protein farnesyltransferase was prepared and shown to be enzymatically incorporated into a peptide and a protein. The protein was subsequently immobilized onto aminooxy-functionalized agarose beads or labeled with a fluorophore. This method for protein modification provides an alternative to the commonly employed Cu(I)-catalyzed click reaction.

  12. Analysis of close associations of uropod-associated proteins in human T-cells using the proximity ligation assay

    Directory of Open Access Journals (Sweden)

    Tommy Baumann

    2013-10-01

    Full Text Available We have shown previously that the raft-associated proteins flotillin-1 and -2 are rapidly recruited to the uropods of chemoattractant-stimulated human neutrophils and T-cells and are involved in cell polarization. Other proteins such as the adhesion receptor PSGL-1, the actin-membrane linker proteins ezrin/radixin/moesin (ERM and the signaling enzyme phosphatidylinositol-4-phosphate 5-kinase type Iγ90 (PIPKIγ90 also accumulate in the T-cell uropod. Using the in situ proximity ligation assay (PLA we now have investigated putative close associations of these proteins in human freshly isolated T-cells before and after chemokine addition. The PLA allows in situ subcellular localization of close proximity of endogenous proteins at single-molecule resolution in fixed cells. It allows detection also of weaker and transient complexes that would not be revealed with co-immunoprecipitation approaches. We previously provided evidence for heterodimer formation of tagged flotillin-1 and -2 in T-cells before and after chemokine addition using fluorescence resonance energy transfer (FRET. We now confirm these findings using PLA for the endogenous flotillins in fixed human T-cells. Moreover, in agreement with the literature, our PLA findings confirm a close association of endogenous PSGL-1 and ERM proteins both in resting and chemokine-activated human T-cells. In addition, we provide novel evidence using the PLA for close associations of endogenous activated ERM proteins with PIPKIγ90 and of endogenous flotillins with PSGL-1 in human T-cells, before and after chemokine addition. Our findings suggest that preformed clusters of these proteins coalesce in the uropod upon cell stimulation.

  13. Saponin fraction from Astragalus membranaceus roots protects mice against polymicrobial sepsis induced by cecal ligation and puncture by inhibiting inflammation and upregulating protein C pathway.

    Science.gov (United States)

    Gao, Xing-hua; Xu, Xian-xiang; Pan, Rong; Li, Ying; Luo, Yu-bin; Xia, Yu-feng; Murata, Kazuya; Matsuda, Hideaki; Dai, Yue

    2009-10-01

    Sepsis remains the leading cause of death in intensive care units. Uncontrolled systemic inflammation and an impaired protein C pathway are two important contributors to sepsis pathophysiology. Based on the beneficial effects of the saponin fraction from Astragalus membranaceus roots (SAM) against inflammation, liver dysfunction, and endothelium injury, we investigated the potential protective roles and underlying mechanisms of SAM on polymicrobial sepsis induced by cecal ligation and puncture (CLP) in mice. SAM, orally administered 1 h before and after CLP, significantly elevated the survival rate of mice. At 96 h after CLP operation, all mice in the model group died, whereas 33.3% of mice in the SAM (400 mg/kg)-treated group survived. SAM attenuated both inflammatory factors and their abilities to induce tissue dysfunction, which was mainly evidenced by decreased infiltration of polymorphonuclear leukocytes, tissue edema, and lung wet-to-dry weight ratio, lowered levels of myeloperoxidase (MPO), nitric oxide (NO), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in serum, as well as downregulated expressions of iNOS and IL-1beta mRNA in livers. Furthermore, we addressed the effects of SAM on the protein C (PC) pathway, closely linked with sepsis. In CLP-induced septic mice, SAM elevated the impaired expression of PC mRNA in livers. In vitro, SAM reversed the decreased expressions of thrombomodulin (TM) and endothelial PC receptor (EPCR) mRNA induced by lipopolysaccharide (LPS) in endothelial cells. These findings suggest that SAM is able to restore the impaired protein C pathway. Taken together, the current study demonstrates that SAM has protective effects on polymicrobial sepsis in mice. The mechanisms of action involve anti-inflammation and upregulation of the PC pathway.

  14. WaaL of Pseudomonas aeruginosa utilizes ATP in in vitro ligation of O antigen onto lipid A-core.

    Science.gov (United States)

    Abeyrathne, Priyanka D; Lam, Joseph S

    2007-09-01

    waaL has been implicated as the gene that encodes the O-antigen ligase. To date, in vitro biochemical evidence to prove that WaaL possesses ligase activity has been lacking due to the difficulty of purifying WaaL and unavailability of substrates. Here we describe the purification of WaaL, a membrane protein with 11 potential transmembrane segments from Pseudomonas aeruginosa, and the development of an in vitro O-antigen ligase assay. WaaL was expressed in a P. aeruginosa wbpL knockout strain, which is defective in its initial glycosyltransferase for O-antigen biosynthesis. This approach allowed the purification of WaaL without contaminating O-antigen-undecaprenol-phosphate (Und-P) molecules. Purified WaaL resolved to a monomer (35 kDa) and a dimer (70 kDa) band in SDS-PAGE. The substrates for the O-antigen ligase assay, O-antigen-Und-P and lipid A-core were prepared from a waaL mutant. ATP at 2-4 mM is optimum for the O-ligase activity, and ATP hydrolysis by WaaL follows Michaelis-Menten kinetics. Site-directed mutagenesis analysis indicated that the periplasmic loop region of WaaL is important for ligase activity. A waaL mutant of P. aeruginosa could not be cross-complemented by waaL of Escherichia coli, which suggested that each of these proteins has specificity for its cognate core oligosaccharide.

  15. Influence of ligation method on friction resistance of lingual brackets with different second-order angulations: an in vitro study

    Directory of Open Access Journals (Sweden)

    Graziane Olímpio Pereira

    Full Text Available ABSTRACT Objective: To evaluate stainless steel archwire static friction in active and passive self-ligating lingual and conventional brackets with second-order angulations. Methods: Two conventional lingual brackets for canines (STb light/Ormco; PSWb/Tecnident, and two self-ligating brackets, one active (In-Ovation L/GAC and the other passive (3D/ Forestadent, were evaluated. A stainless steel archwire was used at 0°, 3° and 5° angulations. Metal ligatures, conventional elastic ligatures, and low friction elastic ligatures were also tested. A universal testing machine applied friction between brackets and wires, simulating sliding mechanics, to produce 2-mm sliding at 3 mm/minute speed. Results: Two-way analysis of variance demonstrated a significant effect of the interaction between brackets and angulations (p < 0.001. Tukey test indicated that the highest frictional resistance values were observed at 5° angulation for In-Ovation L, PSWb bracket with non conventional ligature, and STb bracket with metal ligature. As for 3D, PSWb with conventional or metal ligatures, and STb brackets with non conventional ligature, showed significantly lower static frictional resistance with 0° angulation. At 0° angulation, STb brackets with metal ties, In-Ovation L brackets and 3D brackets had the lowest frictional resistance. Conclusions: As the angulation increased from 0° to 3°, static friction resistance increased. When angulation increased from 3° to 5°, static friction resistance increased or remained the same. Self-ligating 3D and In-Ovation L brackets, as well as conventional STb brackets, seem to be the best option when sliding mechanics is used to perform lingual orthodontic treatment.

  16. Influence of ligation method on friction resistance of lingual brackets with different second-order angulations: an in vitro study

    Science.gov (United States)

    Pereira, Graziane Olímpio; Gimenez, Carla Maria Melleiro; Prieto, Lucas; Prieto, Marcos Gabriel do Lago; Basting, Roberta Tarkany

    2016-01-01

    ABSTRACT Objective: To evaluate stainless steel archwire static friction in active and passive self-ligating lingual and conventional brackets with second-order angulations. Methods: Two conventional lingual brackets for canines (STb light/Ormco; PSWb/Tecnident), and two self-ligating brackets, one active (In-Ovation L/GAC) and the other passive (3D/ Forestadent), were evaluated. A stainless steel archwire was used at 0°, 3° and 5° angulations. Metal ligatures, conventional elastic ligatures, and low friction elastic ligatures were also tested. A universal testing machine applied friction between brackets and wires, simulating sliding mechanics, to produce 2-mm sliding at 3 mm/minute speed. Results: Two-way analysis of variance demonstrated a significant effect of the interaction between brackets and angulations (p bracket with non conventional ligature, and STb bracket with metal ligature. As for 3D, PSWb with conventional or metal ligatures, and STb brackets with non conventional ligature, showed significantly lower static frictional resistance with 0° angulation. At 0° angulation, STb brackets with metal ties, In-Ovation L brackets and 3D brackets had the lowest frictional resistance. Conclusions: As the angulation increased from 0° to 3°, static friction resistance increased. When angulation increased from 3° to 5°, static friction resistance increased or remained the same. Self-ligating 3D and In-Ovation L brackets, as well as conventional STb brackets, seem to be the best option when sliding mechanics is used to perform lingual orthodontic treatment. PMID:27653262

  17. Chemical synthesis of a polypeptide backbone derived from the primary sequence of the cancer protein NY-ESO-1 enabled by kinetically controlled ligation and pseudoprolines.

    Science.gov (United States)

    Harris, Paul W R; Brimble, Margaret A

    2015-03-01

    The cancer protein NY-ESO-1 has been shown to be one of the most promising vaccine candidates although little is known about its cellular function. Using a chemical protein strategy, the 180 amino acid polypeptide, tagged with an arginine solubilizing tail, was assembled in a convergent manner from four unprotected peptide α-thioester peptide building blocks and one cysteinyl polypeptide, which were in turn prepared by Boc and Fmoc solid phase peptide synthesis (SPPS) respectively. To facilitate the assembly by ligation chemistries, non-native cysteines were introduced as chemical handles into the polypeptide fragments; pseudoproline dipeptides and microwave assisted Fmoc SPPS were crucial techniques to prepare the challenging hydrophobic C-terminal fragment. Three sequential kinetically controlled ligations, which exploited the reactivity between peptide arylthioesters and peptide alkylthioesters, were then used in order to assemble the more tractable N-terminal region of NY-ESO-1. The ensuing 147 residue polypeptide thioester then underwent successful final native chemical ligation with the very hydrophobic C-terminal polypeptide bearing an N-terminal cysteine affording the 186 residue polypeptide as an advanced intermediate en route to the native NY-ESO-1 protein. © 2015 Wiley Periodicals, Inc.

  18. Protein release from hippocampus in vitro.

    Science.gov (United States)

    Hesse, G W; Hofstein, R; Shashoua, V E

    1984-07-02

    Physiologically viable slices of rat hippocampus in vitro continuously release protein into the superfusion medium at a rate of about 2 micrograms/mg tissue/h. Assays of a cytoplasmic marker enzyme (lactate dehydrogenase) indicate that this material is not the result of cell lysis. Pulse-chase experiments using [3H]valine indicate that a substantial fraction of the newly synthesized proteins eventually appear in the incubation medium (18.7% +/- 3% of the total TCA precipitable radioactivity during a 6-h superfusion) and that the releasable protein pool has an apparent half-life of about 4 h. Simultaneous labeling of newly synthetized proteins with [3H]fucose and [14C]valine showed a 3-fold higher ratio of [3H]fucose to [14C]valine in the released protein fraction compared to the soluble cytoplasmic protein and to the crude membrane protein fraction, suggesting that the soluble released proteins are more highly glycosylated than the proteins retained in the tissue. Electrophoretic migration patterns on SDS-polyacrylamide gels with both labeled and unlabeled proteins show differences between the released proteins and the soluble cytoplasmic proteins of the tissue. Several molecular weights between 14 kdalton and 86 kdalton appear to be characteristic of the released protein fraction. These results suggest that a distinct group of proteins and glycoproteins exists in hippocampal tissue which is destined to be selectively released into the extracellular space.

  19. Semi-synthesis of murine prion protein by native chemical ligation and chemical activation for preparation of polypeptide-α-thioester.

    Science.gov (United States)

    Shi, Lei; Chen, Huai; Zhang, Si-Yu; Chu, Ting-Ting; Zhao, Yu-Fen; Chen, Yong-Xiang; Li, Yan-Mei

    2017-06-01

    Prions are suspected as pathogen of the fatal transmissible spongiform encephalopathies. Strategies to access homogenous prion protein (PrP) are required to fully comprehend the molecular mechanism of prion diseases. However, the polypeptide fragments from PrP show a high tendency to form aggregates, which is a gigantic obstacle of protein synthesis and purification. In this study, murine prion sequence 90 to 230 that is the core three-dimensional structure domain was constructed from three segments murine PrP (mPrP)(90-177), mPrP(178-212), and mPrP(213-230) by combining protein expression, chemical synthesis and chemical ligation. The protein sequence 90 to 177 was obtained from expression and finally converted into the polypeptide hydrazide by chemical activation of a cysteine in the tail. The other two polypeptide fragments of the C-terminal were obtained by chemical synthesis, which utilized the strategies of isopeptide and pseudoproline building blocks to complete the synthesis of such difficult sequences. The three segments were finally assembled by sequentially using native chemical ligation. This strategy will allow more straightforward access to homogeneously modified PrP variants. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  20. Correlative Förster Resonance Electron Transfer-Proximity Ligation Assay (FRET-PLA) Technique for Studying Interactions Involving Membrane Proteins.

    Science.gov (United States)

    Ivanusic, Daniel; Denner, Joachim; Bannert, Norbert

    2016-08-01

    This unit provides a guide and detailed protocol for studying membrane protein-protein interactions (PPI) using the acceptor-sensitized Förster resonance electron transfer (FRET) method in combination with the proximity ligation assay (PLA). The protocol in this unit is focused on the preparation of FRET-PLA samples and the detection of correlative FRET/PLA signals as well as on the analysis of FRET-PLA data and interpretation of correlative results when using cyan fluorescent protein (CFP) as a FRET donor and yellow fluorescent protein (YFP) as a FRET acceptor. The correlative application of FRET and PLA combines two powerful tools for monitoring PPI, yielding results that are more reliable than with either technique alone. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  1. Identification of nitric oxide synthase as a thiolate-ligated heme protein using magnetic circular dichroism spectroscopy. Comparison with cytochrome P-450-CAM and chloroperoxidase.

    Science.gov (United States)

    Sono, M; Stuehr, D J; Ikeda-Saito, M; Dawson, J H

    1995-08-25

    Nitric oxide (NO) has recently been recognized as an important biomolecule playing diverse physiological roles. It is synthesized in several different tissues from L-Arg and O2, using NADPH as an electron donor, by a family of heme-containing catalytically self-sufficient monooxygenases known as nitric oxide synthases (NOS). Recently, the CO complex of reduced NOS has been shown to exhibit an absorption maximum near 450 nm, a characteristic spectral feature of cytochrome P-450 (P-450). Yet, the amino acid sequences of NOS and P-450 have no homology. To further probe the active site heme coordination structure and the heme environment of NOS, we have employed magnetic circular dichroism (MCD) and CD spectroscopy in the present study. MCD spectra of several derivatives of rat brain neuronal NOS strikingly resemble those of analogous derivatives of bacterial P-450-CAM and fungal chloroperoxidase, two known thiolate-ligated heme proteins. Given the proven fingerprinting capability of MCD spectroscopy, this provides convincing evidence for endogenous thiolate (cysteinate) ligation to the heme iron of NOS. Furthermore, the heme-related Soret CD bands of NOS (positive) and P-450s (negative), as represented by P-450-CAM, are almost mirror images, whereas chloroperoxidase exhibits totally different CD band shapes. This suggests that the active sites of NOS and P-450 may share some common structural features, but significant distinctions exist between their heme environments in certain aspects such as hydrophobicity or size.

  2. Sensitivity and specificity of in situ proximity ligation for protein interaction analysis in a model of steatohepatitis with Mallory-Denk bodies.

    Directory of Open Access Journals (Sweden)

    Bernhard Zatloukal

    Full Text Available The in situ proximity ligation assay (isPLA is an increasingly used technology for in situ detection of protein interactions, post-translational modifications, and spatial relationships of antigens in cells and tissues, in general. In order to test its performance we compared isPLA with immunofluorescence microscopy by analyzing protein interactions in cytoplasmic protein aggregates, so-called Mallory Denk bodies (MDBs. These structures represent protein inclusions in hepatocytes typically found in human steatohepatitis and they can be generated in mice by feeding of 3,5-diethoxy-carbonyl-1,4-dihydrocollidine (DDC. We investigated the colocalization of all three key MDB components, namely keratin 8 (K8, keratin 18 (K18, and p62 (sequestosome 1 by isPLA and immunofluorescence microscopy. Sensitivity and specificity of isPLA was assessed by using Krt8-/- and Krt18-/- mice as biological controls, along with a series of technical controls. isPLA signal visualization is a robust technology with excellent sensitivity and specificity. The biological relevance of signals generated critically depends on the performance of antibodies used, which requires careful testing of antibodies like in immunofluorescence microscopy. There is a clear advantage of isPLA in visualizing protein co-localization, particularly when antigens are present at markedly different concentrations. Furthermore, isPLA is superior to confocal microscopy with respect to spatial resolution of colocalizing antigens. Disadvantages compared to immunofluorescence are increased costs and longer duration of the laboratory protocol.

  3. In vitro hemodynamic model of the arm arteriovenous circulation to study hemodynamics of native arteriovenous fistula and the distal revascularization and interval ligation procedure.

    Science.gov (United States)

    Varble, Nicole; Day, Steven; Phillips, Daniel; Mix, Doran; Schwarz, Karl; Illig, Karl A; Chandra, Ankur

    2014-05-01

    Experimental modeling of arteriovenous hemodialysis fistula (AVF) hemodynamics is challenging. Mathematical modeling struggles to accurately represent the capillary bed and venous circulation. In vivo animal models are expensive and labor intensive. We hypothesized that an in vitro, physiologic model of the extremity arteriovenous circulation with provisions for AVF and distal revascularization and interval ligation (DRIL) configurations could be created as a platform for hemodynamic modeling and testing. An anatomic, upper extremity arteriovenous model was constructed of tubing focusing on the circulation from the subclavian artery to subclavian vein. Tubing material, length, diameter, and wall thickness were selected to match vessel compliance and morphology. All branch points were constructed at physiologic angles. The venous system and capillary bed were modeled using tubing and one-way valves and compliance chambers. A glycerin/water solution was created to match blood viscosity. The system was connected to a heart simulator. Pressure waveforms and flows were recorded at multiple sites along the model for the native circulation, brachiocephalic AVF configuration, and the AVF with DR without and with IL (DR no IL and DRIL). A preset mean cardiac output of 4.2 L/min from the heart simulator yielded a subclavian artery pressure of 125/55 mm Hg and a brachial artery pressure of 121/54 mm Hg with physiologic arterial waveforms. Mean capillary bed perfusion pressure was 41 mm Hg, and mean venous pressure in the distal brachial vein was 17 mm Hg with physiologic waveforms. AVF configuration resulted in a 15% decrease in distal pressure and a 65% decrease in distal flow to the hand. DR no IL had no change in distal pressure with a 27% increase in distal flow. DRIL resulted in a 3% increase in distal pressure and a 15% increase in distal flow to the hand above that of DR no IL. Flow through the DR bypass decreased from 329 mL/min to 55 mL/min with the addition of IL

  4. In situ demonstration of CD40- and CD154-positive cells in psoriatic lesions and keratinocyte production of chemokines by CD40 ligation in vitro

    NARCIS (Netherlands)

    Pasch, Marcel C.; Timár, Krisztina K.; van Meurs, Marjan; Heydendael, Vera M. R.; Bos, Jan D.; Laman, Jon D.; Asghar, Syed S.

    2004-01-01

    In psoriatic lesions, T cells and keratinocytes are in an activated state. Ligation of CD40 expressed on activated keratinocytes with CD154 expressed on activated T cells is thought to be involved in the pathogenesis of psoriasis. However, the presence of CD40(+) and CD154(+) cells in psoriatic skin

  5. In situ demonstration of CD40- and CD154-positive cells in psoriatic lesions and keratinocyte production of chemokines by CD40 ligation in vitro.

    NARCIS (Netherlands)

    Pasch, M.C.; Timar, K.K.; Meurs, M. van; Heydendael, V.M.; Bos, J.D.; Laman, J.D.; Asghar, S.S.

    2004-01-01

    In psoriatic lesions, T cells and keratinocytes are in an activated state. Ligation of CD40 expressed on activated keratinocytes with CD154 expressed on activated T cells is thought to be involved in the pathogenesis of psoriasis. However, the presence of CD40(+) and CD154(+) cells in psoriatic skin

  6. Click nucleic acid ligation: applications in biology and nanotechnology.

    Science.gov (United States)

    El-Sagheer, Afaf H; Brown, Tom

    2012-08-21

    Biochemical strategies that use a combination of synthetic oligonucleotides, thermostable DNA polymerases, and DNA ligases can produce large DNA constructs up to 1 megabase in length. Although these ambitious targets are feasible biochemically, comparable technologies for the chemical synthesis of long DNA strands lag far behind. The best available chemical approach is the solid-phase phosphoramidite method, which can be used to assemble DNA strands up to 150 bases in length. Beyond this point, deficiencies in the chemistry make it impossible to produce pure DNA. A possible alternative approach to the chemical synthesis of large DNA strands is to join together carefully purified synthetic oligonucleotides by chemical methods. Click ligation by the copper-catalyzed azide-alkyne (CuAAC) reaction could facilitate this process. In this Account, we describe the synthesis, characterization, and applications of oligonucleotides prepared by click ligation. The alkyne and azide oligonucleotide strands can be prepared by standard protocols, and the ligation reaction is compatible with a wide range of chemical modifications to DNA and RNA. We have employed click ligation to synthesize DNA constructs up to 300 bases in length and much longer sequences are feasible. When the resulting triazole linkage is placed in a PCR template, various DNA polymerases correctly copy the entire base sequence. We have also successfully demonstrated both in vitro transcription and rolling circle amplification through the modified linkage. This linkage has shown in vivo biocompatibility: an antibiotic resistance gene containing triazole linkages functions in E. coli . Using click ligation, we have synthesized hairpin ribozymes up to 100 nucleotides in length and a hammerhead ribozyme with the triazole linkage located at the substrate cleavage site. At the opposite end of the length scale, click-ligated, cyclic mini-DNA duplexes have been used as models to study base pairing. Cyclic duplexes have

  7. A proximity ligation assay using transiently transfected, epitope-tagged proteins: application for in situ detection of dimerized receptor tyrosine kinases.

    Science.gov (United States)

    Gajadhar, Aaron; Guha, Abhijit

    2010-02-01

    The development of small molecule and antibody inhibitors targeting the interaction of receptor tyrosine kinases (RTKs), such as epidermal growth factor receptor (EGFR), is of high pharmacological and biological interest. Unfortunately, conventional biochemical techniques using cell or tissue lysates and co-immunoprecipitation experiments to investigate EGFR dimerization are not always conclusive. Here we describe a series of technical and biological validation experiments demonstrating the utility of a proximity ligation assay (PLA)-based methodology for in situ visualization and quantification of ligand-dependent EGFR receptor dimerization in intact cells. Using the PLA approach combined with a universally applicable epitope tagging strategy, we detected EGFR dimers in cells transiently co-expressing FLAG-tagged and MYC-tagged human EGFRs. Our data strongly suggest that PLA can be used to detect ligand-dependent EGFR dimerization and this signal is generated in a protein interaction-based manner, rather than solely due to proximity of target proteins. This application represents a generalized RTK expression strategy for protein-interaction analysis in a transient expression system where antibody epitopes are not known or not unique enough to discriminate between interaction partners. This assay also holds promise as a general RTK dimerization screening tool in tissue specimens to identify potential dimerization inhibitors with clinical relevance.

  8. Nanoparticle-protein corona in invertebrate in vitro testing

    DEFF Research Database (Denmark)

    Hayashi, Yuya; Miclaus, Teodora; Scavenius, Carsten

    2013-01-01

    , and the primary cells were thus exposed to silver nanoparticles with pre-formed corona of serum albumin (a major serum protein). Here we have profiled proteins forming the hard corona around silver nanoparticles (OECD reference materials, 15 nm and 75 nm) using gel electrophoresis techniques to identify proteins...... for evaluation of the protein corona in invertebrate in vitro setting....

  9. Tubal Ligation Reversal

    Science.gov (United States)

    ... and other factors. Success rates may be as high as 80 percent or as low as near 40 percent depending on your circumstances. Tubal ligation reversal is abdominal surgery, which carries a risk of infection, bleeding and ...

  10. Utilization of Nitrophenylphosphates and Oxime-Based Ligation for the Development of Nanomolar Affinity Inhibitors of the Yersinia pestis Outer Protein H (YopH) Phosphatase

    Energy Technology Data Exchange (ETDEWEB)

    Bahta, Medhanit; Lountos, George T.; Dyas, Beverly; Kim, Sung-Eun; Ulrich, Robert G.; Waugh, David S.; Burke, Jr., Terrence R. (NIH); (USARL)

    2012-08-10

    Our current study reports the first K{sub M} optimization of a library of nitrophenylphosphate-containing substrates for generating an inhibitor lead against the Yersinia pestis outer protein phosphatase (YopH). A high activity substrate identified by this method (K{sub M} = 80 {micro}M) was converted from a substrate into an inhibitor by replacement of its phosphate group with difluoromethylphosphonic acid and by attachment of an aminooxy handle for further structural optimization by oxime ligation. A cocrystal structure of this aminooxy-containing platform in complex with YopH allowed the identification of a conserved water molecule proximal to the aminooxy group that was subsequently employed for the design of furanyl-based oxime derivatives. By this process, a potent (IC{sub 50} = 190 nM) and nonpromiscuous inhibitor was developed with good YopH selectivity relative to a panel of phosphatases. The inhibitor showed significant inhibition of intracellular Y. pestis replication at a noncytotoxic concentration. The current work presents general approaches to PTP inhibitor development that may be useful beyond YopH.

  11. Signal regulatory protein alpha ligation induces macrophage nitric oxide production through JAK/STAT- and phosphatidylinositol 3-kinase/Rac1/NAPDH oxidase/H2O2-dependent pathways.

    Science.gov (United States)

    Alblas, Jacqueline; Honing, Henk; de Lavalette, Chantal Renardel; Brown, Marion H; Dijkstra, Christine D; van den Berg, Timo K

    2005-08-01

    Signal regulatory protein alpha (SIRPalpha) is a glycoprotein receptor that recruits and signals via the tyrosine phosphatases SHP-1 and SHP-2. In macrophages SIRPalpha can negatively regulate the phagocytosis of host cells and the production of tumor necrosis factor alpha. Here we provide evidence that SIRPalpha can also stimulate macrophage activities, in particular the production of nitric oxide (NO) and reactive oxygen species. Ligation of SIRPalpha by antibodies or soluble CD47 triggers inducible nitric oxide synthase expression and production of NO. This was not caused by blocking negative-regulatory SIRPalpha-CD47 interactions. SIRPalpha-induced NO production was prevented by inhibition of the tyrosine kinase JAK2. JAK2 was found to associate with SIRPalpha in macrophages, particularly after SIRPalpha ligation, and SIRPalpha stimulation resulted in JAK2 and STAT1 tyrosine phosphorylation. Furthermore, SIRPalpha-induced NO production required the generation of hydrogen peroxide (H(2)O(2)) by a NADPH oxidase (NOX) and the phosphatidylinositol 3-kinase (PI3-K)-dependent activation of Rac1, an intrinsic NOX component. Finally, SIRPalpha ligation promoted SHP-1 and SHP-2 recruitment, which was both JAK2 and PI3-K dependent. These findings demonstrate that SIRPalpha ligation induces macrophage NO production through the cooperative action of JAK/STAT and PI3-K/Rac1/NOX/H(2)O(2) signaling pathways. Therefore, we propose that SIRPalpha is able to function as an activating receptor.

  12. Decreased C-reactive protein induces abnormal vascular structure in a rat model of liver dysfunction induced by bile duct ligation

    Directory of Open Access Journals (Sweden)

    Ji Hye Jun

    2016-09-01

    Full Text Available Background/Aims Chronic liver disease leads to liver fibrosis, and although the liver does have a certain regenerative capacity, this disease is associated with dysfunction of the liver vessels. C-reactive protein (CRP is produced in the liver and circulated from there for metabolism. CRP was recently shown to inhibit angiogenesis by inducing endothelial cell dysfunction. The objective of this study was to determine the effect of CRP levels on angiogenesis in a rat model of liver dysfunction induced by bile duct ligation (BDL. Methods The diameter of the hepatic vein was analyzed in rat liver tissues using hematoxylin and eosin (H&E staining. The expression levels of angiogenic factors, albumin, and CRP were analyzed by real-time PCR and Western blotting. A tube formation assay was performed to confirm the effect of CRP on angiogenesis in human umbilical vein endothelial cells (HUVECs treated with lithocholic acid (LCA and siRNA-CRP. Results The diameter of the hepatic portal vein increased significantly with the progression of cirrhosis. The expression levels of angiogenic factors were increased in the cirrhotic liver. In contrast, the expression levels of albumin and CRP were significantly lower in the liver tissue obtained from the BDL rat model than in the normal liver. The CRP level was correlated with the expression of albumin in hepatocytes treated with LCA and siRNA-CRP. Tube formation was significantly decreased in HUVECs when they were treated with LCA or a combination of LCA and siRNA-CRP. Conclusion CRP seems to be involved in the abnormal formation of vessels in hepatic disease, and so it could be a useful diagnostic marker for hepatic disease.

  13. Tubal ligation - slideshow

    Science.gov (United States)

    ... GO GO About MedlinePlus Site Map FAQs Customer Support Health Topics Drugs & Supplements Videos & Tools Español You Are Here: Home → Medical Encyclopedia → Tubal ligation - Series—Normal anatomy URL of this page: //medlineplus.gov/ency/presentations/ ...

  14. Variation in In Vitro Digestibility of Barley Protein

    DEFF Research Database (Denmark)

    Buchmann, N. B.

    1979-01-01

    In vitro digestibility of protein was measured with pepsin/pancreatin in 321 spring barley lines grown in the field. The variation in digestibility was far less than the variation in the protein content. A small environmental influence on the digestibility was found. Two entries had slightly...... impaired digestibilities; these findings were partially verified in a repeated field trial, but were not confirmed in vivo. In vitro digestibilities of barleys grown in pots at various N-levels were positively correlated with protein or hordein content. In vitro digestibility was negatively correlated...... with the fibre content in decortification fractions of Bomi and the high-lysine mutant Risø 1508. The digestibility was positively correlated with the hordein concentration for the Bomi fractions, but not for the 1508 fractions....

  15. DNA Nanoparticles for Improved Protein Synthesis In Vitro.

    Science.gov (United States)

    Galinis, Robertas; Stonyte, Greta; Kiseliovas, Vaidotas; Zilionis, Rapolas; Studer, Sabine; Hilvert, Donald; Janulaitis, Arvydas; Mazutis, Linas

    2016-02-24

    The amplification and digital quantification of single DNA molecules are important in biomedicine and diagnostics. Beyond quantifying DNA molecules in a sample, the ability to express proteins from the amplified DNA would open even broader applications in synthetic biology, directed evolution, and proteomics. Herein, a microfluidic approach is reported for the production of condensed DNA nanoparticles that can serve as efficient templates for in vitro protein synthesis. Using phi29 DNA polymerase and a multiple displacement amplification reaction, single DNA molecules were converted into DNA nanoparticles containing up to about 10(4)  clonal gene copies of the starting template. DNA nanoparticle formation was triggered by accumulation of inorganic pyrophosphate (produced during DNA synthesis) and magnesium ions from the buffer. Transcription-translation reactions performed in vitro showed that individual DNA nanoparticles can serve as efficient templates for protein synthesis in vitro. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Relationship between Molecular Structure Characteristics of Feed Proteins and Protein In vitro Digestibility and Solubility.

    Science.gov (United States)

    Bai, Mingmei; Qin, Guixin; Sun, Zewei; Long, Guohui

    2016-08-01

    The nutritional value of feed proteins and their utilization by livestock are related not only to the chemical composition but also to the structure of feed proteins, but few studies thus far have investigated the relationship between the structure of feed proteins and their solubility as well as digestibility in monogastric animals. To address this question we analyzed soybean meal, fish meal, corn distiller's dried grains with solubles, corn gluten meal, and feather meal by Fourier transform infrared (FTIR) spectroscopy to determine the protein molecular spectral band characteristics for amides I and II as well as α-helices and β-sheets and their ratios. Protein solubility and in vitro digestibility were measured with the Kjeldahl method using 0.2% KOH solution and the pepsin-pancreatin two-step enzymatic method, respectively. We found that all measured spectral band intensities (height and area) of feed proteins were correlated with their the in vitro digestibility and solubility (p≤0.003); moreover, the relatively quantitative amounts of α-helices, random coils, and α-helix to β-sheet ratio in protein secondary structures were positively correlated with protein in vitro digestibility and solubility (p≤0.004). On the other hand, the percentage of β-sheet structures was negatively correlated with protein in vitro digestibility (pproteins are closely related to their in vitro digestibility at 28 h and solubility. Furthermore, the α-helix-to-β-sheet ratio can be used to predict the nutritional value of feed proteins.

  17. Effect of self-ligating bracket type and vibration on frictional force and stick-slip phenomenon in diverse tooth displacement conditions: an in vitro mechanical analysis.

    Science.gov (United States)

    Seo, Yu-Jin; Lim, Bum-Soon; Park, Young Guk; Yang, Il-Hyung; Ahn, Seok-Joon; Kim, Tae-Woo; Baek, Seung-Hak

    2015-10-01

    To evaluate the effects of self-ligating bracket (SLB) type and vibration on frictional force and stick-slip phenomenon (SSP) in diverse tooth displacement conditions when a levelling/alignment wire was drawn. A total of 16 groups were tested (n = 10/group): Two types of SLBs [active SLB (ASLB, In-Ovation R) and passive SLB (PSLB, Damon Q)]; vibration (30 Hz and 0.25 N) and non-vibration conditions; and 4 types of displacement [2mm lingual displacement of the maxillary right lateral incisor (LD), 2mm gingival displacement of the maxillary right canine (GD), combination of LD and GD (LGD), and control]. After applying artificial saliva to the typodont system, 0.018 copper nickel-titanium archwire was drawn by Instron with a speed of 0.5mm/min for 5 minutes at 36.5°C. After static/kinetic frictional forces (SFF/KFF), and frequency/amplitude of SSP were measured, statistical analysis was performed. ASLB exhibited higher SFF, KFF, and SSP amplitude (all P Vibration decreased SFF, KFF, and SSP amplitude and increased SSP frequency in control and all displacement groups (all P vibration condition (P vibration conditions, ASLB demonstrated higher SSP amplitude than PSLB in all displacement groups (all P vibration; all P vibration). Even in tooth displacement conditions, vibration significantly reduced SFF, KFF, SSP amplitude, and increased SPP frequency in both PSLB and ASLB. However, in vivo studies would be needed to confirm the clinical significance. © The Author 2014. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  18. Protein turnover during in vitro tissue engineering.

    Science.gov (United States)

    Li, Qiyao; Chang, Zhen; Oliveira, Gisele; Xiong, Maiyer; Smith, Lloyd M; Frey, Brian L; Welham, Nathan V

    2016-03-01

    Repopulating acellular biological scaffolds with phenotypically appropriate cells is a promising approach for regenerating functional tissues and organs. Under this tissue engineering paradigm, reseeded cells are expected to remodel the scaffold by active protein synthesis and degradation; however, the rate and extent of this remodeling remain largely unknown. Here, we present a technique to measure dynamic proteome changes during in vitro remodeling of decellularized tissue by reseeded cells, using vocal fold mucosa as the model system. Decellularization and recellularization were optimized, and a stable isotope labeling strategy was developed to differentiate remnant proteins constituting the original scaffold from proteins newly synthesized by reseeded cells. Turnover of matrix and cellular proteins and the effects of cell-scaffold interaction were elucidated. This technique sheds new light on in vitro tissue remodeling and the process of tissue regeneration, and is readily applicable to other tissue and organ systems. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. In Vitro Digestibility of Rapeseed and Bovine Whey Protein Mixtures.

    Science.gov (United States)

    Joehnke, Marcel Skejovic; Rehder, Alina; Sørensen, Susanne; Bjergegaard, Charlotte; Sørensen, Jens Christian; Markedal, Keld Ejdrup

    2018-01-24

    Partial replacement of animal protein sources with plant proteins is highly relevant for the food industry, but potential effects on protein digestibility need to be established. In this study, the in vitro protein digestibility (IVPD) of four protein sources and their mixtures (50:50 w/w ratio) was investigated using a transient pepsin hydrolysis (1 h) followed by pancreatin (1 h). The protein sources consisted of napin-rich rapeseed (Brassica napus L.) protein concentrates (RPCs; RP1, RP2) prepared in pilot scale and major bovine whey proteins (WPs; α-LA, alpha-lactalbumin; β-LG, beta-lactoglobulin). IVPD of individual protein sources was higher for WPs compared to RPCs. The RP2/β-LG mixture resulted in an unexpected high IVPD equivalent to β-LG protein alone. Protein mixtures containing RP1 showed a new IVPD response type due to the negative influence of a high trypsin inhibitor activity (TIA) level. Improved IVPD of RP1 alone and in protein mixtures was obtained by lowering the TIA level using dithiothreitol (DTT). These results showed that napin-rich protein products prepared by appropriate processing can be combined with specific WPs in mixtures to improve the IVPD.

  20. In vitro relative protein digestibility and lipoxygenase activity used as ...

    African Journals Online (AJOL)

    Proximate composition, In Vitro Relative Protein Digestibility of 5 varieties of soybean (glycine max), was done. Meals from whole seeds and dehulled seeds were both investigated. Stability to photoxidation was also monitored on the oils over a period after sunlight exposure and finally the extent of oil oxidation was ...

  1. Therapeutically important proteins from in vitro plant tissue culture systems.

    Science.gov (United States)

    Doran, Pauline M

    2013-01-01

    Plant cells cultured in liquid medium in bioreactors are now being used commercially to produce biopharmaceutical proteins. The emergence of in vitro plant cell culture as a production vehicle reflects the importance of key biosafety and biocontainment concerns affecting the competitiveness of alternative systems such as mammalian cell culture and agriculture. Food plant species are particularly attractive as hosts for in vitro protein production: the risk of transgene escape and food chain contamination is eliminated using containment facilities, while regulatory approval for oral delivery of drugs may be easier than if non-edible species were used. As in whole plants, proteolysis in cultured plant cells can lead to significant degradation of foreign proteins after synthesis; however, substantial progress has been made to counter the destructive effects of proteases in plant systems. Although protein secretion into the culture medium is advantageous for product recovery and purification, measures are often required to minimise extracellular protease activity and product losses due to irreversible surface adsorption. Disposable plastic bioreactors, which are being used increasingly in mammalian cell bioprocessing, are also being adopted for plant cell culture to allow rapid scale-up and generation of saleable product. This review examines a range of technical and regulatory issues affecting the choice of industrial production platform for foreign proteins, and assesses progress in the development of in vitro plant systems for biopharmaceutical production.

  2. Zinc and/or cadmium accumulation in Gynura pseudochina (L.) DC. studied in vitro and the effect on crude protein

    Science.gov (United States)

    Panitlertumpai, Natthawoot; Nakbanpote, Woranan; Sangdee, Aphidech; Thumanu, Kanjana; Nakai, Izumi; Hokura, Akiko

    2013-03-01

    Gynura pseudochina (L.) DC. is a zinc (Zn)/cadmium (Cd) hyperaccumulative plant. The aim of this study was to examine the tolerance of G. pseudochina (L.) DC. for Zn and/or Cd accumulation and protein expression. An in vitro tissue culture system was used to control the environment and effects of the microorganisms. Treatments with higher Zn and Cd concentrations increased chlorosis and the accumulation of metals in the root and shoot. Cd treatment at low levels induced the growth of the plant, and the translocation factor was high. A dual treatment with Cd and Zn decreased the metals' toxicity and demonstrated the plant's proclivity to accumulate Cd. The SDS-PAGE and FT-IR analyses showed the effect of the metals' toxicity on protein expression and secondary structure. Moreover, using XAFS techniques, it was demonstrated that treatment with a high Zn concentration (100 mg l-1) resulted in tetrahedral coordination with mixed S/O ligation in the protein extract as compared with Znsbnd O coordination in the protein extract from the control plant cultured in the presence of trace levels of Zn (0.04 mg l-1). This research suggested that G. pseudochina (L.) DC. had properties to tolerate a high Zn and Cd concentration, related to the sulphur proteins.

  3. TCR/CD3 ligation of a TCR-transgenic T lymphoma blocks its proliferation in vitro but does not affect its growth in vivo

    DEFF Research Database (Denmark)

    Reimann, J; Rudolphi, A; Tcherepnev, G

    1994-01-01

    A backcrossed mouse line was established homozygous for the autosomal recessive mutation scid (severe combined immunodeficiency) and carrying T cells which express transgenic (tg) T cell receptor (TCR) alpha and beta chains that mediate H-2 class I (Db)-restricted recognition of a male (H...... of TL1 cells resembled that of small-to-medium lymphoblasts. The cells had the following phenotype: CD3 + TCR alpha T+TCR beta T+CD4-CD8- CD44-CD45RB+LECAM-1 + IL-2R- and low H-2 expression. Exposure of TL1 cells to TCR-binding monoclonal antibodies or lectins blocked in their in vitro proliferation...

  4. In vitro Determination of Extracellular Proteins from Xylella fastidiosa

    Science.gov (United States)

    Mendes, Juliano S.; Santiago, André S.; Toledo, Marcelo A. S.; Horta, Maria A. C.; de Souza, Alessandra A.; Tasic, Ljubica; de Souza, Anete P.

    2016-01-01

    The phytopathogen Xylella fastidiosa causes economic losses in important agricultural crops. Xylem vessel occlusion caused by biofilm formation is the major mechanism underlying the pathogenicity of distinct strains of X. fastidiosa. Here, we provide a detailed in vitro characterization of the extracellular proteins of X. fastidiosa. Based on the results, we performed a comparison with a strain J1a12, which cannot induce citrus variegated chlorosis symptoms when inoculated into citrus plants. We then extend this approach to analyze the extracellular proteins of X. fastidiosa in media supplemented with calcium. We verified increases in extracellular proteins concomitant with the days of growth and, consequently, biofilm development (3–30 days). Outer membrane vesicles carrying toxins were identified beginning at 10 days of growth in the 9a5c strain. In addition, a decrease in extracellular proteins in media supplemented with calcium was observed in both strains. Using mass spectrometry, 71 different proteins were identified during 30 days of X. fastidiosa biofilm development, including proteases, quorum-sensing proteins, biofilm formation proteins, hypothetical proteins, phage-related proteins, chaperones, toxins, antitoxins, and extracellular vesicle membrane components. PMID:28082960

  5. Protein profile of mouse ovarian follicles grown in vitro.

    Science.gov (United States)

    Anastácio, Amandine; Rodriguez-Wallberg, Kenny A; Chardonnet, Solenne; Pionneau, Cédric; Fédérici, Christian; Almeida Santos, Teresa; Poirot, Catherine

    2017-12-01

    emphasize proteins with different expression profiles between the three follicular stages. Supplementary western blot analysis (using new biological replicates) was performed to confirm the expression variations of three proteins during follicle development in vitro. It was found that 609 out of 1401 identified proteins were common to the three follicle developmental stages investigated. Some proteins were identified uniquely at one stage: 71 of the 775 identified proteins in SF, 181 of 1092 in SMR and 192 of 1100 in AF. Additional qualitative and quantitative analysis highlighted 44 biological processes over-represented in our samples compared to the Mus musculus gene database. In particular, it was possible to identify proteins implicated in the cell cycle, calcium ion binding and glycolysis, with specific expressions and abundance, throughout in vitro follicle development. Data are available via ProteomeXchange with identifier PXD006227. The proteome analyses described in this study were performed after in vitro development. Despite fractionation of the samples before LC-MS/MS, proteomic approaches are not exhaustive, thus proteins that are not identified in a group are not necessarily absent from that group, although they are likely to be less abundant. This study allowed a general view of proteins implicated in follicle development in vitro and it represents the most complete catalog of the whole follicle proteome available so far. Not only were well known proteins of the oocyte identified but also proteins that are probably expressed only in granulosa cells. This study was supported by the Portuguese Foundation for Science and Technology, FCT (PhD fellowship SFRH/BD/65299/2009 to A.A.), the Swedish Childhood Cancer Foundation (PR 2014-0144 to K.A.R-.W.) and Stockholm County Council to K.A.R-.W. The authors of the study have no conflict of interest to report.

  6. Assembly of a biocompatible triazole-linked gene by one-pot click-DNA ligation

    Science.gov (United States)

    Kukwikila, Mikiembo; Gale, Nittaya; El-Sagheer, Afaf H.; Brown, Tom; Tavassoli, Ali

    2017-11-01

    The chemical synthesis of oligonucleotides and their enzyme-mediated assembly into genes and genomes has significantly advanced multiple scientific disciplines. However, these approaches are not without their shortcomings; enzymatic amplification and ligation of oligonucleotides into genes and genomes makes automation challenging, and site-specific incorporation of epigenetic information and/or modified bases into large constructs is not feasible. Here we present a fully chemical one-pot method for the assembly of oligonucleotides into a gene by click-DNA ligation. We synthesize the 335 base-pair gene that encodes the green fluorescent protein iLOV from ten functionalized oligonucleotides that contain 5ʹ-azide and 3ʹ-alkyne units. The resulting click-linked iLOV gene contains eight triazoles at the sites of chemical ligation, and yet is fully biocompatible; it is replicated by DNA polymerases in vitro and encodes a functional iLOV protein in Escherichia coli. We demonstrate the power and potential of our one-pot gene-assembly method by preparing an epigenetically modified variant of the iLOV gene.

  7. Chemical aminoacylation of tRNAs with fluorinated amino acids for in vitro protein mutagenesis

    Directory of Open Access Journals (Sweden)

    Shijie Ye

    2010-04-01

    Full Text Available This article describes the chemical aminoacylation of the yeast phenylalanine suppressor tRNA with a series of amino acids bearing fluorinated side chains via the hybrid dinucleotide pdCpA and ligation to the corresponding truncated tRNA species. Aminoacyl-tRNAs can be used to synthesize biologically relevant proteins which contain fluorinated amino acids at specific sites by means of a cell-free translation system. Such engineered proteins are expected to contribute to our understanding of discrete fluorines’ interaction with canonical amino acids in a native protein environment and to enable the design of fluorinated proteins with arbitrary desired properties.

  8. RX-207, a Small Molecule Inhibitor of Protein Interaction with Glycosaminoglycans (SMIGs), Reduces Experimentally Induced Inflammation and Increases Survival Rate in Cecal Ligation and Puncture (CLP)-Induced Sepsis.

    Science.gov (United States)

    Juhas, Stefan; Harris, Nicholas; Il'kova, Gabriela; Rehák, Pavol; Zsila, Ferenc; Yurgenzon Kogan, Faina; Lahmy, Orly; Zhuk, Regina; Gregor, Paul; Koppel, Juraj

    2018-02-01

    The fused quinazolinone derivative, RX-207, is chemically and functionally related to small molecule inhibitors of protein binding to glycosaminoglycans (SMIGs). Composed of a planar aromatic amine scaffold, it inhibits protein binding to glycosaminoglycans (GAGs). RX-207 reduced neutrophil migration in thioglycollate-induced peritonitis (37%), inhibited carrageenan-induced paw edema (32%) and cerulein-induced pancreatitis (28%), and increased animal survival in the mouse model of cecal ligation and puncture (CLP)-induced sepsis (60%). The mechanism of RX-207 action, analyzed by UV spectroscopy, confirmed that which was elucidated for chemically related anti-inflammatory SMIGs. RX-207 binding to cell surface GAGs can account for the inhibition of neutrophil recruitment via the micro-vasculature and as a consequence, the reduction of neutrophil mediated tissue damage in the animal models of inflammation and improved survival of mice in CLP-induced sepsis.

  9. Synthesizing and modifying peptides for chemoselective ligation and assembly into quantum dot-peptide bioconjugates.

    Science.gov (United States)

    Algar, W Russ; Blanco-Canosa, Juan B; Manthe, Rachel L; Susumu, Kimihiro; Stewart, Michael H; Dawson, Philip E; Medintz, Igor L

    2013-01-01

    Quantum dots (QDs) are well-established as photoluminescent nanoparticle probes for in vitro or in vivo imaging, sensing, and even drug delivery. A critical component of this research is the need to reliably conjugate peptides, proteins, oligonucleotides, and other biomolecules to QDs in a controlled manner. In this chapter, we describe the conjugation of peptides to CdSe/ZnS QDs using a combination of polyhistidine self-assembly and hydrazone ligation. The former is a high-affinity interaction with the inorganic surface of the QD; the latter is a highly efficient and chemoselective reaction that occurs between 4-formylbenzoyl (4FB) and 2-hydrazinonicotinoyl (HYNIC) moieties. Two methods are presented for modifying peptides with these functional groups: (1) solid phase peptide synthesis; and (2) solution phase modification of pre-synthesized, commercial peptides. We further describe the aniline-catalyzed ligation of 4FB- and HYNIC-modified peptides, in the presence of a fluorescent label on the latter peptide, as well as subsequent assembly of the ligated peptide to water-soluble QDs. Many technical elements of these protocols can be extended to labeling peptides with other small molecule reagents. Overall, the bioconjugate chemistry is robust, selective, and modular, thereby potentiating the controlled conjugation of QDs with a diverse array of biomolecules for various applications.

  10. IN VITRO RUMINAL PROTEIN DEGRADATION AND MICROBIAL PROTEIN FORMATION OF SEED LEGUMES

    Science.gov (United States)

    Seed legumes such as peas, lupins, and faba beans are important feeds for dairy cows in Europe and other regions. Ruminal protein degradability was quantified using the inhibitor in vitro (IIV) system for samples of 5 seed legumes: 2 peas (cv. Alembo and Helena), 1 white lupin (Lupinus albus, cv. Mu...

  11. In vitro nuclear interactome of the HIV-1 Tat protein.

    LENUS (Irish Health Repository)

    Gautier, Virginie W

    2009-01-01

    BACKGROUND: One facet of the complexity underlying the biology of HIV-1 resides not only in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. HIV-1 encodes the regulatory protein Tat (86-101aa), which is essential for HIV-1 replication and primarily orchestrates HIV-1 provirus transcriptional regulation. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. However they can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. To obtain a comprehensive nuclear interaction map of Tat in T-cells, we have designed a proteomic strategy based on affinity chromatography coupled with mass spectrometry. RESULTS: Our approach resulted in the identification of a total of 183 candidates as Tat nuclear partners, 90% of which have not been previously characterised. Subsequently we applied in silico analysis, to validate and characterise our dataset which revealed that the Tat nuclear interactome exhibits unique signature(s). First, motif composition analysis highlighted that our dataset is enriched for domains mediating protein, RNA and DNA interactions, and helicase and ATPase activities. Secondly, functional classification and network reconstruction clearly depicted Tat as a polyvalent protein adaptor and positioned Tat at the nexus of a densely interconnected interaction network involved in a range of biological processes which included gene expression regulation, RNA biogenesis, chromatin structure, chromosome organisation, DNA replication and nuclear architecture. CONCLUSION: We have completed the in vitro Tat nuclear interactome and have highlighted its modular network properties and particularly those involved in the coordination of gene expression by Tat. Ultimately, the highly specialised set of molecular interactions identified will

  12. Temporal differential proteomes of Clostridium difficile in the pig ileal-ligated loop model.

    Directory of Open Access Journals (Sweden)

    Tavan Janvilisri

    Full Text Available The impact of Clostridium difficile infection (CDI on healthcare is becoming increasingly recognized as it represents a major cause of nosocomial diarrhea. A rising number of CDI cases and outbreaks have been reported worldwide. Here, we developed the pig ileal-ligated loop model for semi-quantitative analysis comparing temporal differential proteomes in C. difficile following in vivo incubation with in vitro growth using isobaric tags for relative and absolute quantification (iTRAQ. Proteins retrieved from the in vitro cultures and the loop contents after 4, 8, and 12 h in vivo incubation were subjected to in-solution digestion, iTRAQ labeling, two-dimensional liquid chromatography/tandem mass spectrometry and statistical analyses. From a total of 1152 distinct proteins identified in this study, 705 proteins were available for quantitative measures at all time points in both biological and technical replicates; 109 proteins were found to be differentially expressed. With analysis of clusters of orthologous group and protein-protein network interactions, we identified the proteins that might play roles in adaptive responses to the host environment, hence enhancing pathogenicity during CDI. This report represents the quantitative proteomic analysis of C. difficile that demonstrates time-dependent protein expression changes under conditions that mimic in vivo infection and identifies potential candidates for diagnostic or therapeutic measures.

  13. Proximity ligation in situ assay for monitoring the global DNA methylation in cells

    National Research Council Canada - National Science Library

    Hervouet, Eric; Hulin, Philippe; Vallette, François M; Cartron, Pierre-François

    2011-01-01

    .... We here present the development of the detection of protein-protein interactions via the adaptation of the proximity ligation in situ technology to evaluate the DNA methylation status in cells since...

  14. Chemoenzymatic Site-Specific Reversible Immobilization and Labeling of Proteins from Crude Cellular Extract Without Prior Purification Using Oxime and Hydrazine Ligation

    Science.gov (United States)

    Mahmoodi, Mohammad M.; Rashidian, Mohammad; Dozier, Jonathan K.; Distefano, Mark D.

    2014-01-01

    In a facile and potentially general method for protein modification at the C-terminus, aldehyde-modified proteins, obtained from enzymatic protein prenylation, react rapidly with hydrazide and aminooxy surfaces and fluorophores at neutral pH and in micromolar concentration ranges of reagents. This strategy was used for fluorescent labeling of eGFP-CVIA, as a model protein, with aminooxy and hydrazide fluorophores or PEGs, and immobilization onto and subsequent release of the protein from hydrazide-functionalized agarose beads using hydrazone-oxime exchange. This method is described in detail here and provides site-specifically PEGylated or fluorescently labeled proteins starting from crude cellular extract in three steps: prenylation, capture, and release. PMID:23839992

  15. Modulation of collateral artery growth in a porcine hindlimb ligation model using MCP-1

    NARCIS (Netherlands)

    Voskuil, Michiel; van Royen, Niels; Hoefer, Imo E.; Seidler, Randolph; Guth, Brian D.; Bode, Christoph; Schaper, Wolfgang; Piek, Jan J.; Buschmann, Ivo R.

    2003-01-01

    For an appropriate extrapolation to patients with peripheral arterial obstructive disease, we tested the efficacy of monocyte chemoattractant protein 1 (MCP-1) treatment in a porcine hindlimb ligation model. In 40 minipigs, a femoral artery ligation was performed. Control animals were examined

  16. ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro.

    Science.gov (United States)

    Cherwa, James E; Tyson, Joshua; Bedwell, Gregory J; Brooke, Dewey; Edwards, Ashton G; Dokland, Terje; Prevelige, Peter E; Fane, Bentley A

    2017-01-01

    During ϕX174 morphogenesis, 240 copies of the external scaffolding protein D organize 12 pentameric assembly intermediates into procapsids, a reaction reconstituted in vitro In previous studies, ϕX174 strains resistant to exogenously expressed dominant lethal D genes were experimentally evolved. Resistance was achieved by the stepwise acquisition of coat protein mutations. Once resistance was established, a stimulatory D protein mutation that greatly increased strain fitness arose. In this study, in vitro biophysical and biochemical methods were utilized to elucidate the mechanistic details and evolutionary trade-offs created by the resistance mutations. The kinetics of procapsid formation was analyzed in vitro using wild-type, inhibitory, and experimentally evolved coat and scaffolding proteins. Our data suggest that viral fitness is correlated with in vitro assembly kinetics and demonstrate that in vivo experimental evolution can be analyzed within an in vitro biophysical context. Experimental evolution is an extremely valuable tool. Comparisons between ancestral and evolved genotypes suggest hypotheses regarding adaptive mechanisms. However, it is not always possible to rigorously test these hypotheses in vivo We applied in vitro biophysical and biochemical methods to elucidate the mechanistic details that allowed an experimentally evolved virus to become resistant to an antiviral protein and then evolve a productive use for that protein. Moreover, our results indicate that the respective roles of scaffolding and coat proteins may have been redistributed during the evolution of a two-scaffolding-protein system. In one-scaffolding-protein virus assembly systems, coat proteins promiscuously interact to form heterogeneous aberrant structures in the absence of scaffolding proteins. Thus, the scaffolding protein controls fidelity. During ϕX174 assembly, the external scaffolding protein acts like a coat protein, self-associating into large aberrant spherical

  17. Synthesis of PAF, an antifungal protein from P. chrysogenum, by native chemical ligation: native disulfide pattern and fold obtained upon oxidative refolding.

    Science.gov (United States)

    Váradi, Györgyi; Tóth, Gábor K; Kele, Zoltán; Galgóczy, László; Fizil, Ádám; Batta, Gyula

    2013-09-16

    The folding of disulfide proteins is of considerable interest because knowledge of this may influence our present understanding of protein folding. However, sometimes even the disulfide pattern cannot be unequivocally determined by the available experimental techniques. For example, the structures of a few small antifungal proteins (PAF, AFP) have been disclosed recently using NMR spectroscopy but with some ambiguity in the actual disulfide pattern. For this reason, we carried out the chemical synthesis of PAF. Probing different approaches, the oxidative folding of the synthetic linear PAF yielded a folded protein that has identical structure and antifungal activity as the native PAF. In contrast, unfolded linear PAF was inactive, a result that may have implications concerning its redox state in the mode of action.

  18. Sortase-Mediated Ligation of Purely Artificial Building Blocks

    OpenAIRE

    Xiaolin Dai; Diana M. Mate; Ulrich Glebe; Tayebeh Mirzaei Garakani; Andrea Körner; Ulrich Schwaneberg; Alexander Böker

    2018-01-01

    Sortase A (SrtA) from Staphylococcus aureus has been often used for ligating a protein with other natural or synthetic compounds in recent years. Here we show that SrtA-mediated ligation (SML) is universally applicable for the linkage of two purely artificial building blocks. Silica nanoparticles (NPs), poly(ethylene glycol) and poly(N-isopropyl acrylamide) are chosen as synthetic building blocks. As a proof of concept, NP–polymer, NP–NP, and polymer–polymer structures are formed by SrtA cata...

  19. Synthesis of a designed transmembrane protein by thioether ligation of solubilised segments : Nα-haloacetylated peptides survived resin cleavage using TFA with EDT as scavenger

    NARCIS (Netherlands)

    Englebretsen, D.R; Choma, C.T.; Robillard, G.T.

    1998-01-01

    Nα-haloacetylated peptides made by Fmoc solid phase synthesis survived cleavage when EDT was used as a cleavage component. Two segments of a desgned transmembrane protein, one bromoacetylated, the other containing a cysteine, and each bearing a "solubilising tail" peptide, were synthesised by Fmoc

  20. Co-Localization of Insulin-Like Growth Factor Binding Protein-1, Casein Kinase-2β, and Mechanistic Target of Rapamycin in Human Hepatocellular Carcinoma Cells as Demonstrated by Dual Immunofluorescence and in Situ Proximity Ligation Assay.

    Science.gov (United States)

    Singal, Sahil S; Nygard, Karen; Dhruv, Manthan R; Biggar, Kyle; Abu Shehab, Majida; Shun-Cheng Li, Shawn; Jansson, Thomas; Gupta, Madhulika B

    2017-10-14

    Insulin-like growth factor binding protein (IGFBP)-1 influences fetal growth by modifying insulin-like growth factor-I (IGF-I) bioavailability. IGFBP-1 phosphorylation, which markedly increases its affinity for IGF-I, is regulated by mechanistic target of rapamycin (mTOR) and casein kinase (CSNK)-2. However, the underlying molecular mechanisms remain unknown. We examined the cellular localization and potential interactions of IGFBP-1, CSNK-2β, and mTOR as a prerequisite for protein-protein interaction. Analysis of dual immunofluorescence images indicated a potential perinuclear co-localization between IGFBP-1 and CSNK-2β and a nuclear co-localization between CSNK-2β and mTOR. Proximity ligation assay (PLA) indicated proximity between IGFBP-1 and CSNK-2β as well as mTOR and CSNK-2β but not between mTOR and IGFBP-1. Three-dimensional rendering of the PLA images validated that IGFBP-1 and CSNK-2β interactions were in the perinuclear region and mTOR and CSNK-2β interactions were predominantly perinuclear rather than nuclear as indicated by mTOR and CSNK-2β co-localization. Compared with control, hypoxia and rapamycin treatment showed markedly amplified PLA signals for IGFBP-1 and CSNK-2β (approximately 18-fold, P = 0.0002). Stable isotope labeling with multiple reaction monitoring-mass spectrometry demonstrated that hypoxia and rapamycin treatment increased IGFBP-1 phosphorylation at Ser98/Ser101/Ser119/Ser174 but most considerably (106-fold) at Ser169. We report interactions between CSNK-2β and IGFBP-1 as well as mTOR and CSNK-2β, providing strong evidence of a mechanistic link between mTOR and IGF-I signaling, two critical regulators of cell growth via CSNK-2. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  1. An in vitro Evaluation of Friction Characteristics of Conventional Stainless Steel and Self-ligating Stainless Steel Brackets with different Dimensions of Archwires in Various Bracket-archwire Combination.

    Science.gov (United States)

    Sridharan, K; Sandbhor, Shailesh; Rajasekaran, U B; Sam, George; Ramees, M Mohamed; Abraham, Esther A

    2017-08-01

    The purpose of this research is to compare the frictional attributes of stainless steel conventional brackets and self-ligating stainless steel brackets with different dimensions of archwires. The test was carried with two sets of maxillary brackets: (1) Conventional stainless steel (Victory Series), (2) stainless steel self-ligating (SmartClip) without first premolar brackets. Stainless steel, nickel-titanium (NiTi), and beta-Ti which are the types of orthodontic wire alloys were tested in this study. To monitor the frictional force, a universal testing machine (Instron 33R 4467) that comprises 10 kg tension load cell was assigned on a range of 1 kg and determined from 0 to 2 kg, which allows moving of an archwire along the brackets. One-way analysis of variance was used to test the difference between groups. To analyze the statistical difference between the two groups, Student's t-test was used. For Victory Series in static friction, p-value was 0.946 and for kinetic friction it was 0.944; at the same time for SmartClip, the p value for static and kinetic frictional resistance was 0.497 and 0.518 respectively. Hence, there was no statistically significant difference between the NiTi and stainless steel archwires. It is concluded that when compared with conventional brackets with stainless steel ligatures, self-ligating brackets can produce significantly less friction during sliding. Beta-Ti archwires expressed high amount of frictional resistance and the stainless steel archwires comprise low frictional resistance among all the archwire materials. In orthodontics, frictional resistance has always had a major role. Its ability to impair tooth movement leads to the need for higher forces to move the teeth and it extends the treatment time which results in loss of posterior anchorage. Friction in orthodontics is related with sliding mechanics when a wire is moving through one or a series of bracket slots.

  2. Degradation of two soluble proteins--casein and egg protein by a macro in vitro method.

    Science.gov (United States)

    Udén, P

    2013-08-01

    Degradation of casein and egg protein was studied with whole rumen contents (RC) in a macro in vitro system to elucidate previous findings of initial rapid disappearance of soluble proteins in vitro. Five to 7.5 kg of RC from a dry and/or a lactating cow were incubated with buffer and casein or egg protein for 180 min with frequent sampling. Degradation was measured as loss of trichloroacetic acid precipitable N (TCA-N) from the inocula. Normal (39°C) and low (2°C) temperature incubations were examined in Exp. 1, using 1 g of TCA-N from casein. Four levels of casein (0-12 g TCA-N) in Exp. 2 and four levels of egg albumin (0-24 g TCA-N) in Exp. 3 were fermented at 39°C. Initial recovery of casein TCA-N was 106% at 2°C and 56% at 39°C (Exp. 1). Casein (TCA-N) recovered initially increased in Exp. 2 from 21% at 3 g to 86% at 12 g TCA-N, while absolute loss remained relatively constant at 358 mg TCA-N/kg RC (SD=47). Fractional degradation rate was highest (0.03/min) at the intermediate dosage level. In the absence of rumen fluid (Exp. 4), no casein was lost. Initial egg protein recovery was on average 103% (Exp. 3). Recovery seemed unaffected by dosage level, and absolute degradation rate was relatively constant over time and increased with dosage level (p<0.001) from 1.48 to 2.95 mg TCA-N/(kg RC × min). Maximum degradation rate [mg TCA-N/(kg RC × min)] and affinity constant (mg TCA-N/kg RC) were estimated at 261 and 1650, respectively. It is concluded that a surprisingly constant amount of casein disappears immediately from warm rumen fluid and that this does not occur either with chilled RC, in the absence of rumen fluid, or when replaced with egg protein. The mechanisms for this disappearance are yet to be discovered. © 2012 Blackwell Verlag GmbH.

  3. Identification of maturation and protein synthesis related proteins from porcine oocytes during in vitro maturation

    Directory of Open Access Journals (Sweden)

    Seo Kang

    2011-06-01

    Full Text Available Abstract Background In vitro maturation (IVM of mammalian oocytes is divided into the GV (germinal vesicle stage, MI (metaphase I stage and MII (metaphase II stage stages, and only fully mature oocytes have acquired the ability to be fertilized and initiate zygotic development. These observations have been mostly based on morphological evaluations, but the molecular events governing these processes are not fully understood. The aim of the present study was to better understand the processes involved in the molecular regulation of IVM using 2-DE analysis followed by mass spectrometry to identify proteins that are differentially expressed during oocyte IVM. Result A total of 16 up-regulated and 12 down-regulated proteins were identified. To investigate the IVM process, we specifically focused on the proteins that were up-regulated during the MII stage when compared with the GV stage, which included PRDX 2, GST, SPSY, myomegalin, PED4D, PRKAB 1, and DTNA. These up-regulated proteins were functionally involved in redox regulation and the cAMP-dependent pathway, which are essential for the intracellular signaling involved in oocyte maturation. Interestingly, the PDE4D and its partner, myomegalin, during the MII stage was consistently confirmed up-regulation by western blot analyses. Conclusion These results could be used to better understand some aspects of the molecular mechanisms underlying porcine oocyte maturation. This study identified some regulatory proteins that may have important roles in the molecular events involved in porcine oocyte maturation, particularly with respect to the regulation of oocyte meiotic resumption, MII arrest and oocyte activation. In addition, this study may have beneficial applications not only to basic science with respect to the improvement of oocyte culture conditions but also to mammalian reproductive biotechnology with potential implications.

  4. Characterization of the Endothelial Cell Cytoskeleton following HLA Class I Ligation

    Science.gov (United States)

    Ziegler, Mary E.; Souda, Puneet; Jin, Yi-Ping; Whitelegge, Julian P.; Reed, Elaine F.

    2012-01-01

    Background Vascular endothelial cells (ECs) are a target of antibody-mediated allograft rejection. In vitro, when the HLA class I molecules on the surface of ECs are ligated by anti-HLA class I antibodies, cell proliferation and survival pathways are activated and this is thought to contribute to the development of antibody-mediated rejection. Crosslinking of HLA class I molecules by anti-HLA antibodies also triggers reorganization of the cytoskeleton, which induces the formation of F-actin stress fibers. HLA class I induced stress fiber formation is not well understood. Methodology and Principal Findings The present study examines the protein composition of the cytoskeleton fraction of ECs treated with HLA class I antibodies and compares it to other agonists known to induce alterations of the cytoskeleton in endothelial cells. Analysis by tandem mass spectrometry revealed unique cytoskeleton proteomes for each treatment group. Using annotation tools a candidate list was created that revealed 12 proteins, which were unique to the HLA class I stimulated group. Eleven of the candidate proteins were phosphoproteins and exploration of their predicted kinases provided clues as to how these proteins may contribute to the understanding of HLA class I induced antibody-mediated rejection. Three of the candidates, eukaryotic initiation factor 4A1 (eIF4A1), Tropomyosin alpha 4-chain (TPM4) and DDX3X, were further characterized by Western blot and found to be associated with the cytoskeleton. Confocal microscopy analysis showed that class I ligation stimulated increased eIF4A1 co-localization with F-actin and paxillin. Conclusions/Significance Colocalization of eIF4A1 with F-actin and paxillin following HLA class I ligation suggests that this candidate protein could be a target for understanding the mechanism(s) of class I mediated antibody-mediated rejection. This proteomic approach for analyzing the cytoskeleton of ECs can be applied to other agonists and various cells types

  5. In vitro pH-Stat protein hydrolysis of feed ingredients for Atlantic cod, Gadus morhua. 2. In vitro protein digestibility of common and alternative feed ingredients

    NARCIS (Netherlands)

    Tibbetts, S.; Verreth, J.A.J.; Lall, S.P.

    2011-01-01

    Using enzyme fractions isolated from the pyloric caeca of farmed Atlantic cod, the in vitro degree of protein hydrolysis (DH) of numerous conventional and novel feed ingredients were measured by a closed-system pH-Stat assay. Regression equations describing the relationship between in vivo apparent

  6. Intracellular analysis of in vitro modified HIV Tat protein

    NARCIS (Netherlands)

    Koken, S. E.; Greijer, A. E.; Verhoef, K.; van Wamel, J.; Bukrinskaya, A. G.; Berkhout, B.

    1994-01-01

    Human immunodeficiency viruses HIV-1 and HIV-2 encode a Tat protein that specifically activates transcription from the viral long terminal repeat. To characterize the properties of the Tat proteins, we have expressed them in Escherichia coli. The purified Tat protein was biochemically analyzed and

  7. Recent Progress using the Staudinger Ligation for Radiolabeling Applications.

    Science.gov (United States)

    Mamat, Constantin; Gott, Matthew; Steinbach, Jörg

    2017-09-11

    The increasing application of positron emission tomography (PET) and single photon emission computer tomography (SPECT) in radiopharmacy and nuclear medicine has stimulated the development of a multitude of novel and versatile bioorthogonal conjugation techniques. Currently, there is particular interest in radiolabeling biologically active, high molecular weight compounds like peptides, proteins or antibodies, but also for the labeling of small organic compounds. An enormous challenge in radiolabeling these biologically active molecules is that the introduction of radiohalogens like fluorine-18 as well as various radiometals proceeds under harsh conditions, which could destroy the biomolecule. The Staudinger Ligation is one of the most powerful bioorthogonal conjugation techniques. The reaction proceeds over wide temperature and pH ranges; an amide (peptide) bond is formed as the ligation unit, which minimizes distortion of the structure; no isomers are obtained; and the reaction proceeds without any metal catalyst. Due to this adaptability, this robust ligation type is a perfect candidate with a high potential for various applications in the field of radiopharmacy for the labeling of biomolecules under mild conditions. This review summarizes recent research concerning the implementation of the Staudinger Ligation for radiolabeling applications. This article is protected by copyright. All rights reserved.

  8. Purification of RNA-Protein Splicing Complexes Using a Tagged Protein from In Vitro Splicing Reaction Mixture.

    Science.gov (United States)

    Kataoka, Naoyuki

    2016-01-01

    In eukaryotes, pre-mRNA splicing is an essential step for gene expression. Splicing reactions have been well investigated by using in vitro splicing reactions with extracts prepared from cultured cells. Here, we describe protocols for the preparation of splicing-competent extracts from cells expressing a tagged spliceosomal protein. The whole-cell extracts are able to splice exogenously added pre-mRNA and the RNA-protein complex formed in the in vitro splicing reaction can be purified by immunoprecipitation using antibodies against the peptide tag on the splicing protein. The method described here to prepare splicing-active extracts from whole cells is particularly useful when studying pre-mRNA splicing in various cell types, and the expression of a tagged spliceosomal protein allows one to purify and analyze the RNA-protein complexes by simple immunoprecipitation.

  9. In vitro determination of dietary protein and amino acid digestibility for humans.

    Science.gov (United States)

    Butts, Christine A; Monro, John A; Moughan, Paul J

    2012-08-01

    The development, refinement and validation of in vitro digestibility assays for dietary protein and amino acids for single stomached mammals are reviewed. The general principles of in vitro digestibility assays and their limitations are discussed. In vitro protein digestibility assays must be accurate, rapid, cheap, simple, robust, adaptable and relevant to the processes of digestion, absorption, and metabolism. Simple in vitro methods have the potential to give useful measures of in vivo amino acid and protein digestibility for humans. In vitro methods, including the complex multi-component models of digestion simulating the various physical and chemical processes, require independent validation with in vivo data from the target species or an acceptable animal model using the most appropriate in vivo measure of digestibility. For protein sources devoid of anti-nutritional factors or plant fibre, true ileal digestibility is the recommended in vivo baseline, while for plant proteins the recommended in vivo assay is real ileal digestibility. More published comparative studies are required to adequately validate in vitro digestibility assays.

  10. Protein Engineering by Combined Computational and In Vitro Evolution Approaches.

    Science.gov (United States)

    Rosenfeld, Lior; Heyne, Michael; Shifman, Julia M; Papo, Niv

    2016-05-01

    Two alternative strategies are commonly used to study protein-protein interactions (PPIs) and to engineer protein-based inhibitors. In one approach, binders are selected experimentally from combinatorial libraries of protein mutants that are displayed on a cell surface. In the other approach, computational modeling is used to explore an astronomically large number of protein sequences to select a small number of sequences for experimental testing. While both approaches have some limitations, their combination produces superior results in various protein engineering applications. Such applications include the design of novel binders and inhibitors, the enhancement of affinity and specificity, and the mapping of binding epitopes. The combination of these approaches also aids in the understanding of the specificity profiles of various PPIs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Isolation of a 60 kDa protein with in vitro anticancer activity against ...

    African Journals Online (AJOL)

    A 60 kDa protein was purified from secreted purple fluid of A. dactylomela, a sea hare from Persian Gulf. The protein purification procedure consisted basically of ammonium sulfate precipitation, ion exchange chromatography using DEAE– Sepharose and ultra-filtration method. In vitro antiproliferative and cytotoxic activity ...

  12. Protein Corona: Impact of Lymph Versus Blood in a Complex In Vitro Environment.

    Science.gov (United States)

    Bonvin, Debora; Aschauer, Ulrich; Alexander, Duncan T L; Chiappe, Diego; Moniatte, Marc; Hofmann, Heinrich; Mionić Ebersold, Marijana

    2017-08-01

    In biological environments, the surface of nanoparticles (NPs) are modified by protein corona (PC) that determines their biological behavior. Unfortunately, in vitro tests still give different PC than in vivo tests causing in vitro-in vivo discrepancy; hence, in vitro studies are not indicative for the NPs' behavior in vivo. Here is demonstrated that PC in vitro is strongly influenced by the type of extracellular fluid (ECF), blood or lymph, by their high and low flow conditions and transitions between ECFs, and a combination of these parameters. As a result, this in vitro study approaches fluidic and dynamic variations to which NPs are exposed in vivo: different ECF that NPs encounter first in different injection routes, different transitions in-between ECFs during circulation, and simultaneous change in the exposed flow in these transitions. The most-abundant proteins in PCs are found to be not the most abundant in ECFs, but those having high affinity for binding to the surface of NPs. Moreover, some proteins are differently abundant in PCs at different flows, which indicate force-promoted binding, catch bonds. These results suggest that future in vitro studies should consider more complex incubation conditions to improve the in vitro-in vivo consistency necessary for translational research. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13.  In vitro renaturation of proteins from inclusion bodies

    Directory of Open Access Journals (Sweden)

    Dorota Porowińska

    2012-06-01

    Full Text Available Recombinant proteins and enzymes are commonly used in many areas of our life, such as diagnostics, industry and medicine, due to heterologous synthesis in prokaryotic expression systems. However, a high expression level of foreign protein in bacteria cells results in formation of inactive and insoluble aggregates – inclusion bodies.Reactivation of aggregated proteins is a complex and time-consuming process. Every protein requires experimental optimization of the process conditions. The choice of the refolding method depends on the type of recombinant protein and its physical, chemical and biological properties.Recovery of the activity of proteins accumulated in inclusion bodies can be divided into 4 steps: 1 inclusion bodies isolation, 2 solubilization of aggregates, 3 renaturation, 4 purification of catalytically active molecules.Efficiency of the refolding process depends on many physical factors and chemical and biological agents. The above parameters determine the time of the folding and prevent protein aggregation. They also assist the folding and have an influence on the solubility and stability of native molecules.To date, dilution, dialysis and chromatography are the most often used methods for protein refolding. 

  14. [In vitro renaturation of proteins from inclusion bodies].

    Science.gov (United States)

    Porowińska, Dorota; Marszałek, Ewelina; Wardęcka, Paulina; Komoszyński, Michał

    2012-06-11

    Recombinant proteins and enzymes are commonly used in many areas of our life, such as diagnostics, industry and medicine, due to heterologous synthesis in prokaryotic expression systems. However, a high expression level of foreign protein in bacteria cells results in formation of inactive and insoluble aggregates--inclusion bodies. Reactivation of aggregated proteins is a complex and time-consuming process. Every protein requires experimental optimization of the process conditions. The choice of the refolding method depends on the type of recombinant protein and its physical, chemical and biological properties. Recovery of the activity of proteins accumulated in inclusion bodies can be divided into 4 steps: 1) inclusion bodies isolation, 2) solubilization of aggregates, 3) renaturation, 4) purification of catalytically active molecules. Efficiency of the refolding process depends on many physical factors and chemical and biological agents. The above parameters determine the time of the folding and prevent protein aggregation. They also assist the folding and have an influence on the solubility and stability of native molecules. To date, dilution, dialysis and chromatography are the most often used methods for protein refolding.

  15. Formulation and in vitro characterization of protein-loaded liposomes

    Science.gov (United States)

    Kuzimski, Lauren

    Background/Objective: Protein-based drugs are increasingly used to treat a variety of conditions including cancer and cardio-vascular disease. Due to the immune system's innate ability to degrade the foreign particles quickly, protein-based treatments are generally short-lived. To address this limitation, the objective of the study was to: 1) develop protein-loaded liposomes; 2) characterize size, stability, encapsulation efficiency and rate of protein release; and 3) determine intracellular uptake and distribution; and 4) protein structural changes. Method: Liposomes were loaded with a fluorescent-albumin using freeze-thaw (F/T) methodology. Albumin encapsulation and release were quantified by fluorescence spectroscopic techniques. Flow cytometry was used to determine liposome uptake by macrophages. Epifluorescence microscopy was used to determine cellular distribution of liposomes. Stability was determined using dynamic light scattering by measuring liposome size over one month period. Protein structure was determined using circular dichroism (CD). Result: Encapsulation of albumin in liposome was ˜90% and was dependent on F/T rates, with fifteen cycles yielding the highest encapsulation efficacy (p < 0.05). Albumin-loaded liposomes demonstrated consistent size (<300nm). Release of encapsulated albumin in physiological buffer at 25°C was ˜60% in 72 h. Fluorescence imaging suggested an endosomal route of cellular entry for the FITC-albumin liposome with maximum uptake rates in immune cells (30% at 2hour incubation). CD suggested protein structure is minimally impacted by freeze-thaw methodology. Conclusion: Using F/T as a loading method, we were able to successfully achieve a protein-loaded liposome that was under 300nm, had encapsulation of ˜90%. Synthesized liposomes demonstrated a burst release of encapsulate protein (60%) at 72 hours. Cellular trafficking confirmed endosomal uptake, and minimal protein damage was noticed in CD.

  16. Hydrogels Formed by Oxo-ester Mediated Native Chemical Ligation.

    Science.gov (United States)

    Strehin, Iossif; Gourevitch, Dmitri; Zhang, Yong; Heber-Katz, Ellen; Messersmith, Phillip B

    2013-06-01

    Oxo-ester mediated native chemical ligation (OMNCL) is a variation of the more general native chemical ligation (NCL) reaction that is widely employed for chemoselective ligation of peptide fragments. While OMNCL has been used for a variety of peptide ligations and for biomolecular modification of surfaces, it is typically practiced under harsh conditions that are unsuitable for use in a biological context. In this report we describe the use of OMNCL for polymer hydrogel formation, in-vitro cell encapsulation, and in-vivo implantation. Multivalent polymer precursors containing N-hydroxysuccinimide (NHS) activated oxo-esters and N-cysteine (N-Cys) endgroups were chemically synthesized from branched poly(ethylene glycol) (PEG). Hydrogels formed rapidly at physiologic pH upon mixing of aqueous solutions of NHS and N-Cys functionalized PEGs. Quantitative 1H NMR experiments showed that the reaction proceeds through an OMNCL pathway involving thiol capture to form a thioester intermediate, followed by an S-to-N acyl rearrangement to yield an amide cross-link. pH and temperature were found to influence gelation rate, allowing tailoring of gelation times from a few seconds to a few minutes. OMNCL hydrogels initially swelled before contracting to reach an equilibrium increase in relative wet weight of 0%. This unique behavior impacted the gel stiffness and was attributed to latent formation of disulfide cross-links between network-bound Cys residues. OMNCL hydrogels were adhesive to hydrated tissue, generating a lap shear adhesion strength of 46 kPa. Cells encapsulated in OMNCL hydrogels maintained high viability, and in-situ formation of OMNCL hydrogel by subcutaneous injection in mice generated a minimal acute inflammatory response. OMNCL represents a promising strategy for chemical cross-linking of hydrogels in a biological context and is an attractive candidate for in-vivo applications such as wound healing, tissue repair, drug delivery, and tissue engineering.

  17. An Improved Method for Identifying Specific DNA-Protein-Binding Sites In Vitro.

    Science.gov (United States)

    Wang, Liangyan; Lu, Huizhi; Wang, Yunguang; Yang, Su; Xu, Hong; Cheng, Kaiying; Zhao, Ye; Tian, Bing; Hua, Yuejin

    2017-03-01

    Binding of proteins to specific DNA sequences is essential for a variety of cellular processes such as DNA replication, transcription and responses to external stimuli. Chromatin immunoprecipitation is widely used for determining intracellular DNA fragments bound by a specific protein. However, the subsequent specific or accurate DNA-protein-binding sequence is usually determined by DNA footprinting. Here, we report an alternative method for identifying specific sites of DNA-protein-binding (designated SSDP) in vitro. This technique is mainly dependent on antibody-antigen immunity, simple and convenient, while radioactive isotope labeling and optimization of partial degradation by deoxyribonuclease (DNase) are avoided. As an example, the specific binding sequence of a target promoter by DdrO (a DNA damage response protein from Deinococcus radiodurans) in vitro was determined by the developed method. The central sequence of the binding site could be easily located using this technique.

  18. Compact structure and proteins of pasta retard in vitro digestive evolution of branched starch molecular structure.

    Science.gov (United States)

    Zou, Wei; Sissons, Mike; Warren, Frederick J; Gidley, Michael J; Gilbert, Robert G

    2016-11-05

    The roles that the compact structure and proteins in pasta play in retarding evolution of starch molecular structure during in vitro digestion are explored, using four types of cooked samples: whole pasta, pasta powder, semolina (with proteins) and extracted starch without proteins. These were subjected to in vitro digestion with porcine α-amylase, collecting samples at different times and characterizing the weight distribution of branched starch molecules using size-exclusion chromatography. Measurement of α-amylase activity showed that a protein (or proteins) from semolina or pasta powder interacted with α-amylase, causing reduced enzymatic activity and retarding digestion of branched starch molecules with hydrodynamic radius (Rh)100nm. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Effect of radiation processing on antinutrients, in-vitro protein digestibility and protein efficiency ratio bioassay of legume seeds

    Science.gov (United States)

    El-Niely, Hania F. G.

    2007-06-01

    The effects of irradiation (dose levels of 5, 7.5 and 10 kGy) on nutritive characteristics of peas ( Pisum satinum L), cowpeas ( Vigna unguiculata L.Walp), lentils ( Lens culinaris Med), kidneybeans ( Phaseolus vulgaris L), and chickpeas ( Cicer arietinum L) were examined. Analyses included proximate composition, levels of anti-nutrients (phytic acid, tannins), available lysine (AL), in vitro protein digestibility (IVPD), and protein efficiency ratio (PER) in the growing rat. The results showed that moisture, crude protein, crude fat, crude fiber, and ash were unchanged by the irradiation. Radiation processing significantly ( pnutritional properties of these legumes.

  20. Colorimetric determination of in vitro feed protein degradation

    African Journals Online (AJOL)

    bound to soya-bean was bound to fish meal and corn gluten meal, respectively. Degradation values expressed as a percen- ... size of the diazonium chromophore. 'n Kolorimetriese metode vir die bepaling van ... technique involves treating feed proteins to form a highly coloured diazotized derivative, the colour of which is ...

  1. In vitro relative protein digestibility and lipoxygenase activity used as ...

    African Journals Online (AJOL)

    Nyala variety gave the highest relative protein digestibility both for the whole and dehulled seeds, ... (UV absorbance at 232 nm) of 11.26 after 12 hours exposure to sunlight and 62,0 .... dehulled B; seeds), heat treated (steamed) for 1 hour.

  2. Colorimetric determination of in vitro feed protein degradation

    African Journals Online (AJOL)

    A colorimetric method for determining protein degradation us' ing a diazonium chromophore was compared with the dacron bag technique. Diazotized f ish meal, maize gluten meal and soya-bean were incubated with strained rumen fluid and a suitable growth medium. Only 49o/o and 18% of the colour bound to soya-bean ...

  3. Influence of lipid extraction from different protein sources on in vitro digestibility

    OpenAIRE

    Sant'Ana,Rita de Cássia Oliveira; Mendes,Fabrícia Queiroz; Pires,Christiano Vieira; Oliveira,Maria Goreti de Almeida

    2011-01-01

    Proteins are the most abundant macromolecules in living cells and their primary role in the diet is to supply the body with essential amino acids in adequate quantities for the synthesis and maintenance of body tissues. The determination of protein digestibility of foods is an important factor to estimate their quality and the in vitro methodology is a fast and easy way to perform it. This study aimed to determine the influence of lipids on the in vitro digestibility of animal and vegetable p...

  4. Realizing Serine/Threonine Ligation: Scope and Limitations and Mechanistic Implication Thereof

    Directory of Open Access Journals (Sweden)

    Clarence T. T. Wong

    2014-05-01

    Full Text Available Serine/Threonine ligation (STL has emerged as an alternative tool for protein chemical synthesis, bioconjugations as well as macrocyclization of peptides of various sizes. Owning to the high abundance of Ser/Thr residues in natural peptides and proteins, STL is expected to find a wide range of applications in chemical biology research. Herein, we have fully investigated the compatibility of the serine/threonine ligation strategy for X-Ser/Thr ligation sites, where X is any of the 20 naturally occurring amino acids. Our studies have shown that 17 amino acids are suitable for ligation, while Asp, Glu, and Lys are not compatible. Among the working 17 C-terminal amino acids, the retarded reaction resulted from the bulky β-branched amino acid (Thr, Val and Ile is not seen under the current ligation condition. We have also investigated the chemoselectivity involving the amino group of the internal lysine which may compete with the N-terminal Ser/Thr for reaction with the C-terminal salicylaldehyde (SAL ester aldehyde group. The result suggested that the free internal amino group does not adversely slow down the ligation rate.

  5. Differential Synthesis in Vitro of Barley Aleurone and Starchy Endosperm Proteins

    DEFF Research Database (Denmark)

    Mundy, John; Hejgaard, Jørn; Hansen, Annette

    1986-01-01

    RNAs from isolated endosperm and aleurone tissues (developing and mature grain) and from cultured (germinating) aleurone layers treated with abscisic acid (ABA) and GA(3). B and C hordein polypeptides and the salt-soluble proteins beta-amylase, protein Z, protein C, the chymotrypsin inhibitors (CI-1 and 2......, expression of genes encoding ASI, PSI, protein C, and PAPI is tissue and stage-specific during seed development. Only ASI, CI-1, and PAPI were synthesized in significant amounts with mRNA from cultured aleurone layers. The levels of synthesis of PAPI and CI-1 were independent of hormone treatment......To widen the selection of proteins for gene expression studies in barley seeds, experiments were performed to identify proteins whose synthesis is differentially regulated in developing and germinating seed tissues. The in vitro synthesis of nine distinct barley proteins was compared using m...

  6. Effect of initial protein concentration and pH on in vitro gastric digestion of heated whey proteins.

    Science.gov (United States)

    Zhang, Sha; Vardhanabhuti, Bongkosh

    2014-02-15

    The in vitro digestion of heated whey protein aggregates having different structure and physicochemical properties was evaluated under simulated gastric conditions. Aggregates were formed by heating whey protein isolates (WPI) at 3-9% w/w initial protein concentration and pH 3.0-7.0. Results showed that high protein concentration led to formation of larger WPI aggregates with fewer remaining monomers. Aggregates formed at high protein concentrations showed slower degradation rate compared to those formed at low protein concentration. The effect of initial protein concentration on peptide release pattern was not apparent. Heating pH was a significant factor affecting digestion pattern. At pH above the isoelectric point, the majority of the proteins involved in the aggregation, and aggregates formed at pH 6.0 were more susceptible to pepsin digestion than at pH 7.0. At acidic conditions, only small amount of proteins was involved in the aggregation and heated aggregates were easily digested by pepsin, while the remaining unaggregated proteins were very resistant to gastric digestion. The potential physiological implication of these results on satiety was discussed. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Orchidopexy san ligation technique of orchidopexy

    Directory of Open Access Journals (Sweden)

    Jain Vishal

    2011-01-01

    Full Text Available Pediatric hernia surgery is the most common operation done by pediatric general surgeons and it is a core competency for general surgeons in the developing world. Herniotomy is performed for the surgical repair of hernia and along with orchiopexy for the closure of associated patent processus vaginalis. Traditionally, ligation of hernial sac during orchiopexy is considered mandatory to prevent postoperative development of hernia. The present report was designed to study the results of non-ligation of the hernial sac during orchiopexy. It was found that non-ligation has no untoward effect on early complications and recurrence rate on long-term follow-up. It is suggested that it is not necessary to ligate the hernial sac during orchiopexy in children.

  8. Amino acid composition, score and in vitro protein digestibility of foods commonly consumed in Norhwest Mexico

    OpenAIRE

    Graciela Caire-Juvera; Francisco A. Vázquez-Ortiz; Maria I. Grijalva-Haro

    2013-01-01

    A better knowledge of the amino acid composition of foods commonly consumed in different regions is essential to calculate their scores and, therefore, to predict their protein quality. This paper presents the amino acid composition, amino acid score and in vitro protein digestibility of fifteen foods that are commonly consumed in Northwest Mexico. The foods were prepared by the traditional methods and were analyzed by reverse-phase HPLC. The chemical score for each food was determined using ...

  9. Entropy driven chain effects on ligation chemistry

    OpenAIRE

    Pahnke, K.; Brandt, J.; Gryn'ova, G.; Lindner, P.; Schweins, R.; Schmidt, F.G.; Lederer, A; Coote, M.L.; Barner-Kowollik, C

    2015-01-01

    We report the investigation of fundamental entropic chain effects that enable the tuning of modular ligation chemistry – for example dynamic Diels–Alder (DA) reactions in materials applications – not only classically via the chemistry of the applied reaction sites, but also via the physical and steric properties of the molecules that are being joined. Having a substantial impact on the reaction equilibrium of the reversible ligation chemistry, these effects are important when tran...

  10. Protein biogenesis in Archaea: addressing translation initiation using an in vitro protein synthesis system for Haloferax volcanii.

    Science.gov (United States)

    Ring, Gabriela; Londei, Paola; Eichler, Jerry

    2007-05-01

    Translation initiation in Archaea combines aspects of the parallel process in Eukarya and Bacteria alongside traits unique to this domain. To better understand translation initiation in Archaea, an in vitro translation system from the haloarchaeon Haloferax volcanii has been developed. The ability to translate individual mRNAs both under the conditions used in previously developed poly(U)-dependent poly(Phe) synthesis systems as well as under physiological conditions was shown. Using the H. volcanii system, mRNAs proceeded by either 'strong' or 'weak' Shine-Dalgarno (SD) motifs, or completely lacking leader sequences were effectively translated. The in vitro haloarchaeal system also successfully translated mRNA from Bacteria, again either presenting a SD initiation motif or completely lacking a leader sequence. Thus, the ability to translate individual mRNAs in vitro offers a system to address translation initiation as well as other aspects of protein biogenesis in Archaea.

  11. In Vitro Digestibilities of Six Rumen Protected Fat-Protein Supplement Formulas

    Directory of Open Access Journals (Sweden)

    Lilis Hartati

    2012-01-01

    Full Text Available Abstract. The aim of the research was to evaluate the efficacy of protection method of rumen protected fat-protein supplements. In vitro digestibility test was carried out to examine nutrients digestibility of different supplement formula based on the sources of protein and oil. The research used two sources of fat namely crude palm oil (CPO and fish oil (FO and three sources of protein namely milk skim, soy flour, and soybean meal. Thus there were 6 combinations that subjected in the in vitro digestibility test. The observed variables were the digestibility of dry matter (DM, organic matter (OM, crude fat (CF, and crude protein (CP. Results indicated that the method for protecting protein and fat was effective. This was showed by low nutrients digestibility in the rumen and high nutrients digestibility in the post rumen. In conclusion the combination between skim milk and CPO gave the best results among the other supplement formula. Keywords: rumen protected nutrient, fat-protein supplement, rumen digestibility, in vitro Animal Production 14(1:1-5, January 2012

  12. Comparing protein VEGF inhibitors: In vitro biological studies

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Lanlan; Liang, Xiao Huan [Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080 (United States); Ferrara, Napoleone, E-mail: nf@gene.com [Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080 (United States)

    2011-05-06

    Highlights: {yields} VEGF is a mediator of angiogenesis. {yields} VEGF inhibitors have clinical applications in cancer and eye disorders. {yields} Five protein VEGF inhibitors were compared for their ability to inhibit. {yields} VEGF-induced activities in cultured endothelial cells. -- Abstract: VEGF inhibitors are widely used as a therapy for tumors and intravascular neovascular disorders, but limited and conflicting data regarding their relative biological potencies are available. The purpose of the study is to compare different protein VEGF inhibitors for their ability to inhibit VEGF-stimulated activities. We tested ranibizumab, the full-length variant of ranibizumab (Mab Y0317), bevacizumab, the VEGF-TrapR1R2 and Flt(1-3)-IgG in bioassays measuring VEGF-stimulated proliferation of bovine retinal microvascular endothelial cells or chemotaxis of human umbilical vein endothelial cells (HUVEC). The inhibitors were also compared for their ability to inhibit MAP kinase activation in HUVECs following VEGF addition. Ranibizumab, VEGF-TrapR1R2 and Flt(1-3)-IgG had very similar potencies in the bioassays tested. Bevacizumab was over 10-fold less potent than these molecules. Mab Y0317 was over 30-fold more potent than bevacizumab. The findings reported in this manuscript describe important intrinsic characteristics of several VEGF inhibitors that may be useful to design and interpret preclinical or clinical studies.

  13. Isolation of a 60 kDa protein with in vitro anticancer activity against ...

    African Journals Online (AJOL)

    SERVER

    2007-06-04

    Jun 4, 2007 ... Sea hares have greatly attracted the interest of all those investigating chemical defense substances. Most of these substances are low molecular weight compounds derived from algal diets. In vitro anticancer effect of a 60 kDa protein isolated from the purple fluid of Aplysia dactylomela on four.

  14. In vitro evaluation of utilizable crude protein using ruminal fluid in ...

    African Journals Online (AJOL)

    The prime objective of this study was to evaluate the effective utilizable crude protein (uCP) in leaves, whole and seeds-removed pods of Moringa stenopetala and Moringa oleifera using ruminal fluid in vitro. Samples were analyzed for proximate nutrients using official methods. The metabolizable energy (ME), organic ...

  15. Internal Activation of Peptidyl Prolyl Thioesters in Native Chemical Ligation.

    Science.gov (United States)

    Gui, Yue; Qiu, Lingqi; Li, Yaohao; Li, Hongxing; Dong, Suwei

    2016-04-13

    Prolyl thioesters have shown significantly lower reactivities in native chemical ligation (NCL) in comparison to that of the alanyl thioester. This report describes a mild and efficient internal activation protocol of peptidyl prolyl thioesters in NCL without using any thiol-based additives, where the introduction of a 4-mercaptan substituent on the C-terminal proline significantly improves the reactivity of prolyl thioesters via the formation of a bicyclic thiolactone intermediate. The kinetic data indicate that the reaction rate is comparable to that of the reported data of alanyl thioesters, and the mechanistic studies suggest that the ligation of two peptide segments proceeds through an NCL-like pathway instead of a direct aminolysis, which ensures the chemoselectivity and compatibility of various amino acid side chains. This 4-mercaptoprolyl thioester-based protocol also allows an efficient one-pot ligation-desulfurization procedure. The utility of this method has been further demonstrated in the synthesis of a proline-rich region of Wilms tumor protein 1.

  16. In vitro influence of dietary protein and fructooligosaccharides on metabolism of canine fecal microbiota.

    Science.gov (United States)

    Pinna, Carlo; Vecchiato, Carla Giuditta; Zaghini, Giuliano; Grandi, Monica; Nannoni, Eleonora; Stefanelli, Claudio; Biagi, Giacomo

    2016-03-12

    The present in vitro study investigated whether the utilization of fructooligosaccharides (FOS) may influence canine fecal microbial population in presence of diets differing in their protein content and digestibility. Fresh fecal samples were collected from five adult dogs, pooled, and incubated for 24 h with the undigested residue of three diets: 1, Low protein high digestibility diet (LP HD, crude protein (CP) 229 g/kg); 2, High protein high digestibility diet (HP HD, CP 304 g/kg); 3, High protein low digestibility diet (HP LD, CP 303 g/kg) that had been previously subjected to enzymatic digestion. In the in vitro fermentation study, there were six treatments: 1) LP HD; 2) HP HD 3) HP LD; 4) LP HD + FOS; 5) HP HD + FOS; 6) HP LD + FOS. Fructooligosaccharides were added at the final concentration of 1.5 g/L. Samples of fermentation fluid were collected at 6 and 24 h of incubation. Values of pH were reduced by FOS at 6 and 24 h (P protein digestibility and high dietary protein level resulted in higher pH at both sampling times (P protein digestibility resulted (P protein digestibility (+21 and 22%, respectively; P dietary protein level resulted in lower counts of lactobacilli and enterococci (-0.5 and -0.7 log cells/mL, respectively; P protein digestibility tended to increase counts of C. perfringens (+0.2 log cells/mL; P = 0.07). Results from the present study showed that diets rich in protein may exert negative influences on the canine intestinal ecosystem, slightly increasing the presence of ammonia and reducing counts of lactobacilli and enterococci. Moreover, the presence of poorly digestible protein resulted in lower concentrations of VFA. Conversely, administration of FOS may improve metabolism of canine intestinal microbiota, reducing ammonia concentrations and enhancing VFA production.

  17. Determination of Cell Wall Protein from Selected Feedstuffs and its Relationship with Ruminal Protein Digestibility in Vitro

    Directory of Open Access Journals (Sweden)

    Anuraga Jayanegara

    2016-08-01

    Full Text Available This study was aimed to analyze neutral detergent insoluble crude protein (NDICP and acid detergent insoluble crude protein (ADICP contents of various commonly used forage and concentrate feedstuffs in Indonesia. A number of forages and concentrates, i.e. gliricidia, trichantera, indigofera, calliandra, papaya leaves, cassava leaves, leucaena, rapeseed meal, corn gluten feed, soybean meal, copra meal, palm kernel meal, fish and bone meal and wheat bran were subjected to proximate analysis, cell wall nitrogen determination and in vitro rumen fermentation evaluation. Chemical composition analysis was done in duplicate. The in vitro incubation was conducted in 14 treatments and 3 replicates by following a randomized complete block design. Variables measured after the incubation were total volatile fatty acid (VFA, ammonia, dry matter digestibility (DMD, organic matter digestibility (OMD and crude protein digestibility (CPD. Results revealed that among the forages, cassava leaf had the highest proportion of NDICP while corn gluten feed was the highest among the concentrates. Different from that of NDICP, the highest proportion of ADICP was obtained in papaya leaf and copra meal for the forages and concentrates, respectively. Higher proportion of NDICP tended to decrease CPD (P<0.1. Although higher ADICP apparently tended to decrease CPD as well, the relationship was insignificant due to the higher variation of the dependent variable. It was concluded that cell wall protein proportion in feed may be used as an indicator to determine the quality of protein and its utilization in the rumen.

  18. An In Vitro Assay for Outer Membrane Protein Assembly by the BAM Complex.

    Science.gov (United States)

    Roman-Hernandez, Giselle; Bernstein, Harris D

    2015-01-01

    To elucidate the mechanism of a biochemical process it is often essential to reconstitute the reaction in vitro using the minimal set of factors required to drive the reaction to completion. Here, we describe a method to reconstitute the folding and membrane integration of bacterial outer membrane (OM) proteins that have a characteristic β-barrel structure. In this method the BAM complex, a heteroligomer that catalyzes the membrane integration of β-barrel proteins, is first purified and inserted into small lipid vesicles. Denatured OM proteins are then assembled and integrated into the vesicles in the presence of a molecular chaperone called SurA.

  19. Synthesis of Bacteriophage M13-Specific Proteins in a DNA-Dependent Cell-Free System II. In Vitro Synthesis of Biologically Active Gene 5 Protein

    Science.gov (United States)

    Konings, Ruud N. H.; Jansen, Josephine; Cuypers, Theo; Schoenmakers, John G. G.

    1973-01-01

    It is shown that gene 5 protein of bacteriophage M13 is one of the major proteins synthesized in vitro under the direction of M13 replicative-form DNA. By means of DNA-cellulose chromatography, this protein has been purified to homogeneity and its biological characteristics have been compared with those of its native counterpart. Like native gene 5 protein, the purified, in vitro-synthesized protein binds tightly and selectively to single-stranded, but not to double-stranded, DNAs. These results suggest that truly functional gene 5 protein is made in the cell-free system. Images PMID:4586780

  20. In vitro phosphorylation and acetylation of the murine pocket protein Rb2/p130.

    Directory of Open Access Journals (Sweden)

    Muhammad Saeed

    Full Text Available The retinoblastoma protein (pRb and the related proteins Rb2/p130 and 107 represent the "pocket protein" family of cell cycle regulators. A key function of these proteins is the cell cycle dependent modulation of E2F-regulated genes. The biological activity of these proteins is controlled by acetylation and phosphorylation in a cell cycle dependent manner. In this study we attempted to investigate the interdependence of acetylation and phosphorylation of Rb2/p130 in vitro. After having identified the acetyltransferase p300 among several acetyltransferases to be associated with Rb2/p130 during S-phase in NIH3T3 cells in vivo, we used this enzyme and the CDK4 protein kinase for in vitro modification of a variety of full length Rb2/p130 and truncated versions with mutations in the acetylatable lysine residues 1079, 128 and 130. Mutation of these residues results in the complete loss of Rb2/p130 acetylation. Replacement of lysines by arginines strongly inhibits phosphorylation of Rb2/p130 by CDK4; the inhibitory effect of replacement by glutamines is less pronounced. Preacetylation of Rb2/p130 strongly enhances CDK4-catalyzed phosphorylation, whereas deacetylation completely abolishes in vitro phosphorylation. In contrast, phosphorylation completely inhibits acetylation of Rb2/p130 by p300. These results suggest a mutual interdependence of modifications in a way that acetylation primes Rb2/p130 for phosphorylation and only dephosphorylated Rb2/p130 can be subject to acetylation. Human papillomavirus 16-E7 protein, which increases acetylation of Rb2/p130 by p300 strongly reduces phosphorylation of this protein by CDK4. This suggests that the balance between phosphorylation and acetylation of Rb2/p130 is essential for its biological function in cell cycle control.

  1. Estimation of Relationship Between In Situ and In Vitro Rumen Protein Degradability of Extruded Full Fat Soybean

    Directory of Open Access Journals (Sweden)

    Arzu Erol Tunç

    2017-10-01

    Full Text Available The objectives of this study were to estimate the protein degradability of extruded full fat soybean (ESB by in situ (nylon bag and in vitro enzymatic method and to develop an equation in order predict in situ degradability from in vitro values. In the study enzymatic technique; hydrolysis after 1 h (INV1 and after 24 h (INV24 by a purified protease extracted from Streptomyces griseus in a borate-phosphate buffer at pH 8 was used as in vitro method. Relationship between in situ effective protein degradability (INSE and in vitro degradability after 1 and 24 hours incubations (INV1 and INV24 were determined. In situ protein degradability was measured at 0, 2, 4, 8, 16, 24, and 48 and at 72 h incubations in the rumen of 3 Holstein cows. In the study INSE, INV1 and INV24 were determined as 58.05, 20.24 and 41.46% respectively. Despite there were differences between in situ and in vitro protein degradability values, correlation coefficients between in situ and in vitro protein degradability of ESB were high and regression equations for estimation of in situ from in vitro were found significant. As conclusion in vitro enzymatic protein degradability (INV1 and INV24 can be used for estimation of in situ effective protein degradability of extruded full fat soybean.

  2. Avaliação do atrito em braquetes autoligáveis submetidos à mecânica de deslizamento: um estudo in vitro Evaluation of friction in self-ligating brackets subjected to sliding mechanics: an in vitro study

    Directory of Open Access Journals (Sweden)

    Mariana Ribeiro Pacheco

    2011-02-01

    Full Text Available INTRODUÇÃO: o atrito gerado na interface braquete/fio durante a mecânica de deslizamento pode reduzir a eficiência da movimentação ortodôntica. O método de ligação do fio ao braquete exerce importante papel na determinação desse atrito. MÉTODOS: o presente estudo comparou a força de atrito gerada por quatro tipos de braquetes autoligáveis (Time®; Damon 2®; In-Ovation R® e Smart Clip® com um grupo de braquetes ortodônticos convencionais (Dynalock® associados a ligaduras elásticas tradicionais (Dispens-A-Stix®, que serviu como grupo controle. A força de atrito estático foi mensurada através da máquina universal de ensaios EMIC® DL 500 com dois fios de aço inoxidável com secção transversal 0,018" e 0,017" x 0,025". RESULTADOS: a análise de variância ANOVA e o teste de Tukey mostraram baixos níveis de atrito nos quatro braquetes autoligáveis associados ao fio 0,018" (PINTRODUCTION: Friction generated at the bracket/archwire interface during sliding mechanics can reduce the efficiency of orthodontic movement. The ligation method employed to tie the archwire to the bracket plays an important role in determining this friction. METHODS: This study compared the frictional force generated by four different types of self-ligating brackets (Time™, Damon 2™, In-Ovation R™ and Smart Clip™ with a group of conventional orthodontic brackets (Dynalock™ that require the use of traditional elastomeric ligatures (ExDispens-A-Stix™, which served as the control group. Static friction force was measured using an EMIC DL™ 500 universal testing machine using stainless steel round 0.018-in and rectangular 0.017x0.025-in archwires. RESULTS: ANOVA and Tukey's test showed low levels of friction in the four self-ligating brackets in tests with the 0.018-in wire (P <0.05. However, the results noted when the self-ligating brackets were tested using 0.017x 0.025-in archwires showed high resistance to sliding in the self-ligating

  3. Interspecies In Vitro Evaluation of Stereoselective Protein Binding for 3,4-Methylenedioxymethamphetamine

    Directory of Open Access Journals (Sweden)

    Wan Raihana Wan Aasim

    2017-01-01

    Full Text Available Abuse of 3,4-methylenedioxymethamphetamine (MDMA is becoming more common worldwide. To date, there is no information available on stereoselectivity of MDMA protein binding in humans, rats, and mice. Since stereoselectivity plays an important role in MDMA’s pharmacokinetics and pharmacodynamics, in this study we investigated its stereoselectivity in protein binding. The stereoselective protein binding of rac-MDMA was investigated using two different concentrations (20 and 200 ng/mL in human plasma and mouse and rat sera using an ultrafiltration technique. No significant stereoselectivity in protein binding was observed in both human plasma and rat serum; however, a significant stereoselective binding (p<0.05 was observed in mouse serum. Since the protein binding of MDMA in mouse serum is considerably lower than in humans and rats, caution should be exercised when using mice for in vitro studies involving MDMA.

  4. Synthesis of heteroglycoclusters by using orthogonal chemoselective ligations

    Directory of Open Access Journals (Sweden)

    Baptiste Thomas

    2012-03-01

    Full Text Available Synthetic heteroglycoclusters are being subjected to increasing interest due to their potential to serve as selective ligands for carbohydrate-binding proteins. In this paper, we describe an expedient strategy to prepare cyclopeptides displaying well-defined distributions and combinations of carbohydrates. By using both oxime ligation and copper(I-catalyzed alkyne–azide cycloaddition, two series of compounds bearing binary combinations of αMan, αFuc or βLac in an overall tetravalent presentation, and either 2:2 or 3:1 relative proportions, have been prepared.

  5. Relationship between Molecular Structure Characteristics of Feed Proteins and Protein In vitro Digestibility and Solubility

    OpenAIRE

    Bai, Mingmei; Qin, Guixin; Sun, Zewei; Long, Guohui

    2015-01-01

    The nutritional value of feed proteins and their utilization by livestock are related not only to the chemical composition but also to the structure of feed proteins, but few studies thus far have investigated the relationship between the structure of feed proteins and their solubility as well as digestibility in monogastric animals. To address this question we analyzed soybean meal, fish meal, corn distiller’s dried grains with solubles, corn gluten meal, and feather meal by Fourier transfor...

  6. Glycation of wood frog (Rana sylvatica) hemoglobin and blood proteins: in vivo and in vitro studies

    Science.gov (United States)

    MacDonald, Justin A.; Degenhardt, Thorsten; Baynes, John W.; Storey, Kenneth B.

    2010-01-01

    The effects of in vivo freezing and glucose cryoprotectant on protein glycation were investigated in the wood frog, Rana sylvatica. Our studies revealed no difference in the fructoselysine content of blood plasma sampled from control, 27 h frozen and 18 h thawed wood frogs. Glycated hemoglobin (GHb) decreased slightly with 48 h freezing exposure and was below control levels after 7 d recovery, while glycated serum albumin was unchanged by 48 h freezing but did increase after 7 d of recovery. In vitro exposure of blood lysates to glucose revealed that the GHb production in wood frogs was similar to that of the rat but was lower than in leopard frogs. We conclude that wood frog hemoglobin was glycated in vitro; however, GHb production was not apparent during freezing and recovery when in vivo glucose is highly elevated. It is possible that wood frog blood proteins have different in vivo susceptibilities to glycation. PMID:19540217

  7. The fate of a designed protein corona on nanoparticles in vitro and in vivo.

    Science.gov (United States)

    Bargheer, Denise; Nielsen, Julius; Gébel, Gabriella; Heine, Markus; Salmen, Sunhild C; Stauber, Roland; Weller, Horst; Heeren, Joerg; Nielsen, Peter

    2015-01-01

    A variety of monodisperse superparamagnetic iron oxide particles (SPIOs) was designed in which the surface was modified by PEGylation with mono- or bifunctional poly(ethylene oxide)amines (PEG). Using (125)I-labeled test proteins (transferrin, albumin), the binding and exchange of corona proteins was studied first in vitro. Incubation with (125)I-transferrin showed that with increasing grade of PEGylation the binding was substantially diminished without a difference between simply adsorbed and covalently bound protein. However, after incubation with excess albumin and subsequently whole plasma, transferrin from the preformed transferrin corona was more and more lost from SPIOs in the case of adsorbed proteins. If non-labeled transferrin was used as preformed corona and excess (125)I-labeled albumin was added to the reaction mixtures with different SPIOs, a substantial amount of label was bound to the particles with initially adsorbed transferrin but little or even zero with covalently bound transferrin. These in vitro experiments show a clear difference in the stability of a preformed hard corona with adsorbed or covalently bound protein. This difference seems, however, to be of minor importance in vivo when polymer-coated (59)Fe-SPIOs with adsorbed or covalently bound (125)I-labeled mouse transferrin were injected intravenously in mice. With both protein coronae the (59)Fe/(125)I-labelled particles were cleared from the blood stream within 30 min and appeared in the liver and spleen to a large extent (>90%). In addition, after 2 h already half of the (125)I-labeled transferrin from both nanodevices was recycled back into the plasma and into tissue. This study confirms that adsorbed transferrin from a preformed protein corona is efficiently taken up by cells. It is also highlighted that a radiolabelling technique described in this study may be of value to investigate the role of protein corona formation in vivo for the respective nanoparticle uptake.

  8. The fate of a designed protein corona on nanoparticles in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Denise Bargheer

    2015-01-01

    Full Text Available A variety of monodisperse superparamagnetic iron oxide particles (SPIOs was designed in which the surface was modified by PEGylation with mono- or bifunctional poly(ethylene oxideamines (PEG. Using 125I-labeled test proteins (transferrin, albumin, the binding and exchange of corona proteins was studied first in vitro. Incubation with 125I-transferrin showed that with increasing grade of PEGylation the binding was substantially diminished without a difference between simply adsorbed and covalently bound protein. However, after incubation with excess albumin and subsequently whole plasma, transferrin from the preformed transferrin corona was more and more lost from SPIOs in the case of adsorbed proteins. If non-labeled transferrin was used as preformed corona and excess 125I-labeled albumin was added to the reaction mixtures with different SPIOs, a substantial amount of label was bound to the particles with initially adsorbed transferrin but little or even zero with covalently bound transferrin. These in vitro experiments show a clear difference in the stability of a preformed hard corona with adsorbed or covalently bound protein. This difference seems, however, to be of minor importance in vivo when polymer-coated 59Fe-SPIOs with adsorbed or covalently bound 125I-labeled mouse transferrin were injected intravenously in mice. With both protein coronae the 59Fe/125I-labelled particles were cleared from the blood stream within 30 min and appeared in the liver and spleen to a large extent (>90%. In addition, after 2 h already half of the 125I-labeled transferrin from both nanodevices was recycled back into the plasma and into tissue. This study confirms that adsorbed transferrin from a preformed protein corona is efficiently taken up by cells. It is also highlighted that a radiolabelling technique described in this study may be of value to investigate the role of protein corona formation in vivo for the respective nanoparticle uptake.

  9. In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag

    DEFF Research Database (Denmark)

    Visone, Valeria; Han, Wenyuan; Perugino, Giuseppe

    2017-01-01

    , and measure protein activity and kinetics in living cells. Whereas several tags are suitable for protein imaging in mesophilic organisms, the application of this approach to microorganisms living at high temperature has lagged behind. Archaea provide an excellent and unique model for understanding basic cell......, and allowed visualization of the enzyme in living cells. To the best of our knowledge, this is the first report of in vivo imaging of any protein of a thermophilic archaeon, filling an important gap in available tools for cell biology studies in these organisms....... to production of a functional H5 protein, which was successfully labeled with appropriate fluorescent molecules and visualized in cell extracts as well as in Δogt live cells. H5 was fused to reverse gyrase, a peculiar thermophile-specific DNA topoisomerase endowed with positive supercoiling activity...

  10. Phosphorylation in vitro of eukaryotic initiation factors IF-E2 and IF-E3 by protein kinases

    DEFF Research Database (Denmark)

    Issinger, O G; Benne, R; Hershey, J W

    1976-01-01

    Purified protein synthesis initiation factors IF-E2 and IF-E3 from rabbit reticulocytes were phosphorylated in vitro with protein kinases isolated from the same source. The highest levels of phosphorylation resulted from incubation of the factors with a cyclic nucleotide-independent protein kinase...

  11. Protein enrichment, cellulase production and in vitro digestion improvement of pangolagrass with solid state fermentation.

    Science.gov (United States)

    Hu, Chan-Chin; Liu, Li-Yun; Yang, Shang-Shyng

    2012-02-01

    Pangolagrass, Digitaria decumbens Stent, is a major grass for cow feeding, and may be a good substrate for protein enrichment. To improve the quality of pangolagrass for animal feeding, cellulolytic microbes were isolated from various sources and cultivated with solid state fermentation to enhance the protein content, cellulase production and in vitro digestion. The microbes, culture conditions and culture media were studied. Cellulolytic microbes were isolated from pangolagrass and its extracts, and composts. Pangolagrass supplemented with nitrogen and minerals was used to cultivate the cellulolytic microbes with solid state fermentation. The optimal conditions for protein enrichment and cellulase activity were pangolagrass substrate at initial moisture 65-70%, initial pH 6.0-8.0, supplementation with 2.5% (NH(4))(2)SO(4), 2.5% KH(2)PO(4) and K(2)HPO(4) mixture (2:1, w/w) and 0.3% MgSO(4).7H(2)O and cultivated at 30(o)C for 6 days. The protein content of fermented pangolagrass increased from 5.97-6.28% to 7.09-16.96% and the in vitro digestion improved from 4.11-4.38% to 6.08-19.89% with the inoculation of cellulolytic microbes by solid state fermentation. Each 1 g of dried substrate yielded Avicelase 0.93-3.76 U, carboxymethylcellulase 1.39-4.98 U and β-glucosidase 1.20-6.01 U. The isolate Myceliophthora lutea CL3 was the strain found to be the best at improving the quality of pangolagrass for animal feeding with solid state fermentation. Solid state fermentation of pangolagrass inoculated with appropriate microbes is a feasible process to enrich protein content, increase in vitro digestibility and improve the quality for animal feeding. Copyright © 2011. Published by Elsevier B.V.

  12. A highly efficient catalyst for oxime ligation and hydrazone-oxime exchange suitable for bioconjugation.

    Science.gov (United States)

    Rashidian, Mohammad; Mahmoodi, Mohammad M; Shah, Rachit; Dozier, Jonathan K; Wagner, Carston R; Distefano, Mark D

    2013-03-20

    Imine-based reactions are useful for a wide range of bioconjugation applications. Although aniline is known to catalyze the oxime ligation reaction under physiological conditions, it suffers from slow reaction kinetics, specifically when a ketone is being used or when hydrazone-oxime exchange is performed. Here, we report on the discovery of a new catalyst that is up to 15 times more efficient than aniline. That catalyst, m-phenylenediamine (mPDA), was initially used to analyze the kinetics of oxime ligation on aldehyde- and ketone-containing small molecules. While mPDA is only modestly more effective than aniline when used in equal concentrations (~2-fold), its much greater aqueous solubility relative to aniline allows it to be used at higher concentrations, resulting in significantly more efficient catalysis. In the context of protein labeling, it was first used to site-specifically label an aldehyde-functionalized protein through oxime ligation, and its kinetics were compared to reaction with aniline. Next, a protein was labeled with an aldehyde-containing substrate in crude cell lysate, captured with hydrazide-functionalized beads and then the kinetics of immobilized protein release via hydrazone-oxime exchange were analyzed. Our results show that mPDA can release and label 15 times more protein than aniline can in 3 h. Then, using the new catalyst, ciliary neurotrophic factor, a protein with therapeutic potential, was successfully labeled with a fluorophore in only 5 min. Finally, a protein containing the unnatural amino acid, p-acetyl phenylalanine, a ketone-containing residue, was prepared and PEGylated efficiently via oxime ligation using mPDA. This new catalyst should have a significant impact on the field of bioconjugation, where oxime ligation and hydrazone-oxime exchange are commonly employed.

  13. A Highly Efficient Catalyst for Oxime Ligation and Hydrazone-Oxime Exchange Suitable for Bioconjugation

    Science.gov (United States)

    Rashidian, Mohammad; Mahmoodi, Mohammad M.; Shah, Rachit; Dozier, Jonathan K.; Wagner, Carston R.; Distefano, Mark D.

    2014-01-01

    Imine-based reactions are useful for a wide range of bioconjugation applications. Although aniline is known to catalyze the oxime ligation reaction under physiological conditions, it suffers from slow reaction kinetics, specifically when a ketone is being used or when hydrazone-oxime exchange is performed. Here, we report on the discovery of a new catalyst that is up to 15 times more efficient than aniline. That catalyst, m-phenylenediamine (mPDA), was initially used to analyze the kinetics of oxime ligation on aldehyde- and ketone-containing small molecules. While mPDA is only modestly more effective than aniline when used in equal concentrations (~ 2-fold), its much greater aqueous solubility relative to aniline allows it to be used at higher concentrations, resulting in significantly more efficient catalysis. In the context of protein labeling, it was first used to site-specifically label an aldehyde-functionalized protein through oxime ligation, and its kinetics were compared to reaction with aniline. Next, a protein was labeled with an aldehyde-containing substrate in crude cell lysate, captured with hydrazide-functionalized beads and then the kinetics of immobilized protein release via hydrazone-oxime exchange were analyzed. Our results show that mPDA can release and label 15 times more protein than aniline can in 3 h. Then, using the new catalyst, ciliary neurotrophic factor, a protein with therapeutic potential, was successfully labeled with a fluorophore in only 5 min. Finally, a protein containing the unnatural amino acid, p-acetyl phenylalanine, a ketone-containing residue, was prepared and PEGylated efficiently via oxime ligation using mPDA. This new catalyst should have a significant impact on the field of bioconjugation, where oxime ligation and hydrazone-oxime exchange are commonly employed. PMID:23425124

  14. Phase behaviour and in vitro hydrolysis of wheat starch in mixture with whey protein.

    Science.gov (United States)

    Yang, Natasha; Liu, Yingting; Ashton, John; Gorczyca, Elisabeth; Kasapis, Stefan

    2013-04-15

    Network formation of whey protein isolate (WPI) with increasing concentrations of native wheat starch (WS) has been examined. Small deformation dynamic oscillation in shear and modulated temperature differential scanning calorimetry enabled analysis of binary mixtures at the macro- and micromolecular level. Following heat induced gelation, textural hardness was measured by undertaking compression tests. Environmental scanning electron microscopy provided tangible information on network morphology of polymeric constituents. Experiments involving in vitro starch digestion also allowed for indirect assessment of phase topology in the binary mixture. The biochemical component of this work constitutes an attempt to utilise whey protein as a retardant to the enzymatic hydrolysis of starch in a model system with α-amylase enzyme. During heating, rheological profiles of binary mixtures exhibited dramatic increases in G' at temperatures more closely related to those observed for single whey protein rather than pure starch. Results from this multidisciplinary approach of analysis, utilising rheology, calorimetry and microscopy, argue for the occurrence of phase separation phenomena in the gelled systems. There is also evidence of whey protein forming the continuous phase with wheat starch being the discontinuous filler, an outcome that is explored in the in vitro study of the enzymatic hydrolysis of starch. Copyright © 2012. Published by Elsevier Ltd.

  15. Crude protein, fibre and phytic acid in vitro digestibility of selected legume and buckwheat samples

    Directory of Open Access Journals (Sweden)

    Petra Vojtíšková

    2013-01-01

    Full Text Available The aim of this study was to determine crude protein, fibre and phytic acid in vitro digestibility of selected legumes and buckwheat products. All analyses except the phytic acid contents were performed in the line with the Commission Regulation (EC No. 152/2009. A modified version of Holt’s Method was used for phytic acid (phytate determination. None of all samples contained more than 11% of moisture. Soybeans are rich in crude protein; they contain nearly 40% of this compound. The content of crude protein in buckwheat flours was about 14%. The highest amount of phytate was found in common beans and soybeans-about 2 g/100 g of dry matter. On the other hand, the lowest phytate content was observed in buckwheat pasta (F. esculentum groats was 1.9 g per 100 g of dry matter. In vitro digestibility was determined using an incubator Daisy and pepsin enzymes and the combination of pepsin and pancreatin. The highest coefficient of crude protein digestibility was discovered to be in peels and wholemeal flour. The greatest fibre digestibility coefficients were obtained for peels, which contain about 65% of fibre in their dry matter. When pepsin was used, a higher phytic acid digestibility coefficient for G. max, Ph. vulgaris, peels, flour, groats and broken groats was observed; while when the combination of pepsin and pancreatin was used, higher phytic acid digestibility coefficients for peas, lentil and wholemeal flour were observed.

  16. In vitro affinity screening of protein and peptide binders by megavalent bead surface display.

    Science.gov (United States)

    Diamante, Letizia; Gatti-Lafranconi, Pietro; Schaerli, Yolanda; Hollfelder, Florian

    2013-10-01

    The advent of protein display systems has provided access to tailor-made protein binders by directed evolution. We introduce a new in vitro display system, bead surface display (BeSD), in which a gene is mounted on a bead via strong non-covalent (streptavidin/biotin) interactions and the corresponding protein is displayed via a covalent thioether bond on the DNA. In contrast to previous monovalent or low-copy bead display systems, multiple copies of the DNA and the protein or peptide of interest are displayed in defined quantities (up to 10(6) of each), so that flow cytometry can be used to obtain a measure of binding affinity. The utility of the BeSD in directed evolution is validated by library selections of randomized peptide sequences for binding to the anti-hemagglutinin (HA) antibody that proceed with enrichments in excess of 10(3) and lead to the isolation of high-affinity HA-tags within one round of flow cytometric screening. On-bead K(d) measurements suggest that the selected tags have affinities in the low nanomolar range. In contrast to other display systems (such as ribosome, mRNA and phage display) that are limited to affinity panning selections, BeSD possesses the ability to screen and rank binders by their affinity in vitro, a feature that hitherto has been exclusive to in vivo multivalent cell display systems (such as yeast display).

  17. In vitro bioaccessibility of proteins and lipids of pH-shift processed Nannochloropsis oculata microalga.

    Science.gov (United States)

    Cavonius, L R; Albers, E; Undeland, I

    2016-04-01

    The pH-shift process fractionates biomass into soluble proteins and insoluble fractions, followed by precipitation and recovery of the solubilized proteins. Nannochloropsis oculata in seawater was subjected to the pH-shift process, followed by digestion of various intermediates and product fractions of the process, using the Infogest in vitro digestion model (Minekus et al., 2014) with added gastric lipase. As measures for protein and lipid accessibility, degrees of protein hydrolysis and fatty acid liberation were assessed post-digestion and compared to the amounts of peptide bonds and total fatty acids present in the raw materials. Results showed that neither proteins nor lipids of intact Nannochloropsis cells were accessible to the mammalian digestive enzymes used in the digestion model. Cell disruption, and to a lesser extent, further pH-shift processing with protein solubilisation at pH 7 or pH 10, increased the accessibility of lipids. For proteins, differences amongst the pH-shift processed materials were non-significant, though pre-freezing the product prior to digestion increased the accessibility from 32% to 47%. For fatty acids, pH-shift process-products gave rise to 43% to 52% lipolysis, with higher lipolysis for products solubilised at pH 10 as opposed to pH 7. Our results indicate the importance of processing to produce an algal product that has beneficial nutritional properties when applied as food or feed.

  18. Penentuan Daya Cerna Protein In Vitro Ikan Bawal (Colossoma Macropomum pada Umur Panen Berbeda

    Directory of Open Access Journals (Sweden)

    Dede Saputra

    2014-12-01

    Full Text Available Digestibility is part of the sample consumed and not released into the feces. This study discusses about the protein digestibility of tambaqui fish (Colossoma macropomum at different ages and sizes in vitro. Protein digestibility is the ability of the organisms to break down proteins into simple molecular compounds. At the initial stage, all samples used were dried, then distilled water at pH 8 added, and later the mixture was homogenized for three minutes. After the mixture was homogeneous, enzymes and distilled water were added. Samples were then centrifuged and the absorbance of the sample was measured using absorption spectrophotometry. The results obtained during the observations showed that the digestibility of proteins in the casein showed a value of 100%, small pomfret fish for 28.37%, pomfret fish at 58.42%, and large pomfret fish amount to 88.39%. This shows the larger size of the fish, then the digestibility of protein will be higher. In addition, the lower the pH, the protein digestibility will be higher for hydrolysis of proteins requires an acidic environment of pH conditions.

  19. In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A

    Science.gov (United States)

    Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

    2015-01-01

    A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5’-end including the 5’-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer. PMID:26221730

  20. Artery ligation in the treatment of hemorrhoidal disease

    NARCIS (Netherlands)

    Schuurman, J.P.

    2012-01-01

    The aim of this thesis was to study the working principle in relation to the outcome of the artery ligation procedure; a treatment for hemorrhoidal disease. Hemorrhoidal artery ligation, known as HAL (hemorrhoidal artery ligation) or THD (transanal hemorrhoidal dearterialization) procedure, is a

  1. Non-histone chromosomal proteins. Their isolation and role in determining specificity of transcription in vitro.

    Science.gov (United States)

    Blüthmann, H; Mrozek, S; Gierer, A

    1975-10-15

    We describe a method for fractionation of chromatin components by selective dissociation with salt in buffers containing 5 M urea in combination with cromatography on hydroxyapatite at 4 degrees C. This results in two histone and four non-histone fractions which are recovered in high yield and with minimal proteolytic contamination. Template capacity measurements of the isolated chromatins and pre-saturation competition hybridization experiments support the idea that a group of non-histone proteins activate the transcription of specific DNA sequences which were not transcribed from purified DNA to the same extent. In reconstitution experiments a non-histone protein fraction, NH4, prepared from lymphocyte chromatin by hydroxyapatite chromatography is shown to cause transcription in vitro of lymphocyte-specific RNA sequences. A subfraction with a molecular weight of 30 000 comprising 40% of the NH4 fraction protein is characteristic for this tissue and not found in liver chromatin.

  2. Freezing of in vitro produced bovine embryos in animal protein-free medium containing vegetal peptones.

    Science.gov (United States)

    George, F; Vrancken, M; Verhaeghe, B; Verhoeye, F; Schneider, Y-J; Massip, A; Donnay, I

    2006-09-15

    Successful cryopreservation is essential for a large-scale dispersal of bovine in vitro produced (IVP) embryos that have been shown to be more sensitive to cryopreservation than their in vivo counterparts. On the other hand, the use of animal proteins in freezing media increases sanitary risks. We first replaced animal proteins, such as bovine serum albumin (BSA) in the freezing medium by plant-derived peptides (vegetal peptones). A batch of wheat peptones was selected after a preliminary experiment showing the absence of toxicity of concentrationsanimal protein-free freezing medium containing 1.8 mg/mL peptones. No beneficial effect of adding 1 mg/mL sodium hyaluronate or 100 microM beta-mercaptoethanol was observed on embryo survival or quality. In conclusion, we have demonstrated that vegetal peptones can replace BSA in freezing media without affecting blastocyst survival and quality.

  3. Prospective Randomized Clinical Trial on Suction Elastic Band Ligator Versus Forceps Ligator in the Treatment of Haemorrhoids

    Directory of Open Access Journals (Sweden)

    A.R. Mohd Ramzisham

    2005-10-01

    Conclusion: Suction band ligation is superior to forceps ligation for the treatment of second- and third-degree haemorrhoids in terms of pain tolerance, amount of analgesia consumed and intra-procedure bleeding.

  4. Combining in vitro protein detection and in vivo antibody detection identifies potential vaccine targets against Staphylococcus aureus during osteomyelitis.

    Science.gov (United States)

    den Reijer, P Martijn; Sandker, Marjan; Snijders, Susan V; Tavakol, Mehri; Hendrickx, Antoni P A; van Wamel, Willem J B

    2017-02-01

    Currently, little is known about the in vivo human immune response against Staphylococcus aureus during a biofilm-associated infection, such as osteomyelitis, and how this relates to protein production in biofilms in vitro. Therefore, we characterized IgG responses in 10 patients with chronic osteomyelitis against 50 proteins of S. aureus, analyzed the presence of these proteins in biofilms of the infecting isolates on polystyrene (PS) and human bone in vitro, and explored the relation between in vivo and in vitro data. IgG levels against 15 different proteins were significantly increased in patients compared to healthy controls. Using a novel competitive Luminex-based assay, eight of these proteins [alpha toxin, Staphylococcus aureus formyl peptide receptor-like 1 inhibitor (FlipR), glucosaminidase, iron-responsive surface determinants A and H, the putative ABC transporter SACOL0688, staphylococcal complement inhibitor (SCIN), and serine-aspartate repeat-containing protein E (SdrE)] were also detected in a majority of the infecting isolates during biofilm formation in vitro. However, 4 other proteins were detected in only a minority of isolates in vitro while, vice versa, 7 proteins were detected in multiple isolates in vitro but not associated with significantly increased IgG levels in patients. Detection of proteins was largely confirmed using a transcriptomic approach. Our data provide further insights into potential therapeutic targets, such as for vaccination, to reduce S. aureus virulence and biofilm formation. At the same time, our data suggest that either in vitro or immunological in vivo data alone should be interpreted cautiously and that combined studies are necessary to identify potential targets.

  5. Expression of nucleolar-related proteins in porcine preimplantation embryos produced in vivo and in vitro

    DEFF Research Database (Denmark)

    Bjerregaard, Bolette; Wrenzycki, Christine; Strejcek, Frantisek

    2004-01-01

    embryonic mRNA transcription. Localization of proteins involved in the rRNA transcription (upstream binding factor [UBF], topoisomerase I, RNA polymerase I [RNA Pol I], and the RNA Pol I-associated factor PAF53) and processing (fibrillarin, nucleophosmin, and nucleolin) was assessed by immunocytochemistry....... Moreover, immunolocalization of RNA Pol I, but not of UBF, and the mRNA expression of PAF53 and UBF were significantly reduced or absent after culture with alpha-amanitin, indicating that RNA Pol I, PAF53, and presumably, UBF are derived from de novo embryonic transcription. Embryonic genomic activation...... was delayed in porcine embryos produced in vitro compared to the in vivo-derived counterparts with respect to mRNAs encoding PAF53 and UBF. Moreover, differences existed in the mRNA expression patterns of pRb between in vivo- and in vitro-developed embryos. These findings show, to our knowledge for the first...

  6. Effects of androgen-binding protein (ABP) on spermatid Tnp1 gene expression in vitro.

    Science.gov (United States)

    Della-Maria, Julie; Gerard, Anne; Franck, Patricia; Gerard, Hubert

    2002-12-30

    In vitro studies were designed to determine whether Sertoli cell-delivered ABP could act on spermatogenetic events, whether such an action could occur via a paracrine or a juxtacrine pathway and whether sex steroids could be involved in this action. ABP delivery to germ cells was achieved using an in vitro model based on recombinant rat ABP-producing mouse Sertoli cells cocultivated with rat spermatids. Using semi-quantitative RT-PCR, the expression of the Tnp 1 gene encoding the Transition Protein 1, involved in the histone to protamine replacement during spermatid nuclear transformation, was analyzed. Our results provide clear evidence that Sertoli cell-derived ABP acts on spermatids by modifying the TP1 mRNA level. This outcome, strictly requiring juxtacrine conditions, is obtained in the absence of sex steroid hormones. To our knowledge this is the first evidence of an effect of ABP itself on male germ cells. Copyright 2002 Elsevier Science Ireland Ltd.

  7. In Vitro Assembly and Stabilization of Dengue and Zika Virus Envelope Protein Homo-Dimers.

    Science.gov (United States)

    Metz, Stefan W; Gallichotte, Emily N; Brackbill, Alex; Premkumar, Lakshmanane; Miley, Michael J; Baric, Ralph; de Silva, Aravinda M

    2017-07-03

    Zika virus (ZIKV) and the 4 dengue virus (DENV) serotypes are mosquito-borne Flaviviruses that are associated with severe neuronal and hemorrhagic syndromes. The mature flavivirus infectious virion has 90 envelope (E) protein homo-dimers that pack tightly to form a smooth protein coat with icosahedral symmetry. Human antibodies that strongly neutralize ZIKV and DENVs recognize complex quaternary structure epitopes displayed on E-homo-dimers and higher order structures. The ZIKV and DENV E protein expressed as a soluble protein is mainly a monomer that does not display quaternary epitopes, which may explain the modest success with soluble recombinant E (sRecE) as a vaccine and diagnostic antigen. New strategies are needed to design recombinant immunogens that display these critical immune targets. Here we present two novel methods for building or stabilizing in vitro E-protein homo-dimers that display quaternary epitopes. In the first approach we immobilize sRecE to enable subsequent dimer generation. As an alternate method, we describe the use of human mAbs to stabilize homo-dimers in solution. The ability to produce recombinant E protein dimers displaying quaternary structure epitopes is an important advance with applications in flavivirus diagnostics and vaccine development.

  8. Identification of archaeal proteins that affect the exosome function in vitro

    Directory of Open Access Journals (Sweden)

    Palhano Fernando L

    2010-05-01

    Full Text Available Abstract Background The archaeal exosome is formed by a hexameric RNase PH ring and three RNA binding subunits and has been shown to bind and degrade RNA in vitro. Despite extensive studies on the eukaryotic exosome and on the proteins interacting with this complex, little information is yet available on the identification and function of archaeal exosome regulatory factors. Results Here, we show that the proteins PaSBDS and PaNip7, which bind preferentially to poly-A and AU-rich RNAs, respectively, affect the Pyrococcus abyssi exosome activity in vitro. PaSBDS inhibits slightly degradation of a poly-rA substrate, while PaNip7 strongly inhibits the degradation of poly-A and poly-AU by the exosome. The exosome inhibition by PaNip7 appears to depend at least partially on its interaction with RNA, since mutants of PaNip7 that no longer bind RNA, inhibit the exosome less strongly. We also show that FITC-labeled PaNip7 associates with the exosome in the absence of substrate RNA. Conclusions Given the high structural homology between the archaeal and eukaryotic proteins, the effect of archaeal Nip7 and SBDS on the exosome provides a model for an evolutionarily conserved exosome control mechanism.

  9. In-vitro starch hydrolysis of chitosan incorporating whey protein and wheat starch composite gels

    Directory of Open Access Journals (Sweden)

    Natasha Yang

    2017-10-01

    Full Text Available The study examined the influence of chitosan, incorporated into whey protein and wheat starch thermo gels, on the in-vitro hydrolysis of the polysaccharide. Gels were subjected to the following external conditions containing α-amylase at constant incubation temperature of 37 °C: In the first procedure, they were immersed in phosphate buffer (0.05 M and maintained at pH 6.9 throughout the entire digestion. In the second instance, they were introduced into a salt solution, with pH and total volume adjusted at times in sync with the human gastrointestinal tract. Results indicate that low and medium molecular weight chitosan, in combination with whey protein, were effective at enhancing the protective barrier against starch degradation. Less maltose was liberated from gels containing medium molecular weight chitosan, as opposed to the low molecular weight counterpart, and results compare favorably with the outcome of the in-vitro digestion of binary whey protein and wheat starch composites. Keywords: Food science

  10. Sortase-Mediated Ligation of Purely Artificial Building Blocks

    Directory of Open Access Journals (Sweden)

    Xiaolin Dai

    2018-02-01

    Full Text Available Sortase A (SrtA from Staphylococcus aureus has been often used for ligating a protein with other natural or synthetic compounds in recent years. Here we show that SrtA-mediated ligation (SML is universally applicable for the linkage of two purely artificial building blocks. Silica nanoparticles (NPs, poly(ethylene glycol and poly(N-isopropyl acrylamide are chosen as synthetic building blocks. As a proof of concept, NP–polymer, NP–NP, and polymer–polymer structures are formed by SrtA catalysis. Therefore, the building blocks are equipped with the recognition sequence needed for SrtA reaction—the conserved peptide LPETG—and a pentaglycine motif. The successful formation of the reaction products is shown by means of transmission electron microscopy (TEM, matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF MS, and dynamic light scattering (DLS. The sortase catalyzed linkage of artificial building blocks sets the stage for the development of a new approach to link synthetic structures in cases where their synthesis by established chemical methods is complicated.

  11. Impact of germination time and type of illumination on carotenoidcontent, protein solubility and in vitro protein digestibility of chickpea(Cicer arietinum L.) sprouts.

    Science.gov (United States)

    Khattak, Amal Badshah; Zeb, Aurang; Bibi, Nizakat

    2008-08-15

    Sprouts have been reported to be nutritionally superior to their respective seeds with higher levels of nutrients and lower amounts of antinutrients. Significant differences occur in these nutrients and antinutrients with germination under different types of illumination. This paper reports the impact of germination conditions on changes in β-carotene content, protein solubility and in vitro protein digestibility of chickpea sprouts. The influence of germination time, type of illumination and their interaction on β-carotene content, protein solubility and in vitro protein digestibility of chickpea sprouts was highly significant (p<0.01). Highest value for β-carotene were observed for sprouts germinated under yellow light for 72h (131.74mg 100g(-1)) and lowest for blue light group after 120h germination. Sprouts of irradiated group had overall higher content of β-carotene throughout germination period. Protein solubility was also higher for sprouts of irradiated group and green illumination group after 120h germination. Sprouts of irradiated group had highest value for% in vitro protein digestibility after 96h germination followed by the same group after 120h germination. It is inferred from the study that irradiation of chickpea seed prior to germination improved the β-carotene content, protein solubility and in vitro protein digestibility of chickpea sprouts. Copyright © 2008 Elsevier Ltd. All rights reserved.

  12. In vitro protein degradation of 38 sainfoin accessions and its relationship to tannin content by different assays.

    Science.gov (United States)

    Lorenz, Martin M; Hayot Carbonero, Christine; Smith, Lydia; Udén, Peter

    2012-05-23

    This study compared 38 sainfoin and 2 Lotus accessions to their respective tannin contents, N buffer solubility, and in vitro protein degradation. Tannin contents were measured by a protein precipitation method using either bovine serum albumin or Rubisco and by the colorimetric HCl/butanol method. Precipitation of bovine serum albumin and Rubisco was highly correlated (R(2) = 0.939). Correlations between the protein precipitation variants and the HCl/butanol method were relatively low (R(2) < 0.6). Protein degradation was measured at 4 h of incubation in an inhibited in vitro system and could not be explained by any of the tannin assays (R(2) < 0.03) and only partially by N buffer solubility (R(2) ≤ 0.433). Decisive factors other than the quantity of tannins or their ability to precipitate proteins must be considered. Resistance of soluble protein toward degradation can possibly be caused by tannin protein binding.

  13. In-vitro starch hydrolysis of chitosan incorporating whey protein and wheat starch composite gels

    OpenAIRE

    Natasha Yang; John Ashton; Elisabeth Gorczyca; Stefan Kasapis

    2017-01-01

    The study examined the influence of chitosan, incorporated into whey protein and wheat starch thermo gels, on the in-vitro hydrolysis of the polysaccharide. Gels were subjected to the following external conditions containing α-amylase at constant incubation temperature of 37 °C: In the first procedure, they were immersed in phosphate buffer (0.05 M) and maintained at pH 6.9 throughout the entire digestion. In the second instance, they were introduced into a salt solution, with pH and total ...

  14. Effect of radiation processing on antinutrients, in-vitro protein digestibility and protein efficiency ratio bioassay of legume seeds

    Energy Technology Data Exchange (ETDEWEB)

    El-Niely, Hania F.G. [Food Irradiation Research Department, National Center for Radiation Research and Technology, Atomic Energy Authority, P.O. Box 29, Nasr City, Cairo (Egypt)]. E-mail: elniely@hotmail.com

    2007-06-15

    The effects of irradiation (dose levels of 5, 7.5 and 10 kGy) on nutritive characteristics of peas (Pisum satinum L), cowpeas (Vigna unguiculata L.Walp), lentils (Lens culinaris Med), kidneybeans (Phaseolus vulgaris L), and chickpeas (Cicer arietinum L) were examined. Analyses included proximate composition, levels of anti-nutrients (phytic acid, tannins), available lysine (AL), in vitro protein digestibility (IVPD), and protein efficiency ratio (PER) in the growing rat. The results showed that moisture, crude protein, crude fat, crude fiber, and ash were unchanged by the irradiation. Radiation processing significantly (p<0.05) reduced the levels of phytic acid (PA), tannins (TN), and AL. IVPD and PER were significantly enhanced in a dose-dependent manner, relative to unirradiated control samples, for all legumes. The data sets for each legume exhibited high correlation coefficients between radiation dose and PA, TN, AL, IVPD, and PER. These results demonstrate the benefits of irradiation on the nutritional properties of these legumes.

  15. DNA splicing by directed ligation (SDL).

    Science.gov (United States)

    Berlin, Y A

    1999-01-01

    Splicing by directed ligation (SDL) is a method of in-phase joining of PCR-generated DNA fragments that is based on a pre-designed combination of class IIS restriction endonuclease recognition and cleavage sites. Since these enzymes cleave outside of their recognition sites, the resulting sticky end can have any desired sequence, and the site itself can be removed and does not appear in the final spliced DNA product. SDL is based on the addition of class IIS recognition sites onto primers used to amplify DNA sequences. Cleavage of the PCR products results in elimination of the recognition site-containing flanking sequences and leaves the DNA fragments crowned with protruding ends. With careful design of the sticky ends, several segments can be ligated together in a predetermined order in a single reaction. SDL requires fewer rounds of amplification than overlap extension methods, and is particularly useful for creating a series of recombinants that differ in one segment.

  16. A comparative study of protein synthesis in in vitro systems: from the prokaryotic reconstituted to the eukaryotic extract-based

    Directory of Open Access Journals (Sweden)

    Hillebrecht Jason R

    2008-07-01

    Full Text Available Abstract Background Cell-free protein synthesis is not only a rapid and high throughput technology to obtain proteins from their genes, but also provides an in vitro platform to study protein translation and folding. A detailed comparison of in vitro protein synthesis in different cell-free systems may provide insights to their biological differences and guidelines for their applications. Results Protein synthesis was investigated in vitro in a reconstituted prokaryotic system, a S30 extract-based system and a eukaryotic system. Compared to the S30 system, protein synthesis in the reconstituted system resulted in a reduced yield, and was more cold-sensitive. Supplementing the reconstituted system with fractions from a size-exclusion separation of the S30 extract significantly increased the yield and activity, to a level close to that of the S30 system. Though protein synthesis in both prokaryotic and eukaryotic systems showed no significant differences for eukaryotic reporter proteins, drastic differences were observed when an artificial fusion protein was synthesized in vitro. The prokaryotic systems failed to synthesize and correctly fold a significant amount of the full-length fusion protein, even when supplemented with the eukaryotic lysate. The active full-length fusion protein was synthesized only in the eukaryotic system. Conclusion The reconstituted bacterial system is sufficient but not efficient in protein synthesis. The S30 system by comparison contains additional cellular factors capable of enhancing protein translation and folding. The eukaryotic translation machinery may have evolved from its prokaryotic counterpart in order to translate more complex (difficult-to-translate templates into active proteins.

  17. Synthesis of l- and d-Ubiquitin by One-Pot Ligation and Metal-Free Desulfurization.

    Science.gov (United States)

    Huang, Yi-Chao; Chen, Chen-Chen; Gao, Shuai; Wang, Ye-Hai; Xiao, Hua; Wang, Feng; Tian, Chang-Lin; Li, Yi-Ming

    2016-05-23

    Native chemical ligation combined with desulfurization has become a powerful strategy for the chemical synthesis of proteins. Here we describe the use of a new thiol additive, methyl thioglycolate, to accomplish one-pot native chemical ligation and metal-free desulfurization for chemical protein synthesis. This one-pot strategy was used to prepare ubiquitin from two or three peptide segments. Circular dichroism spectroscopy and racemic protein X-ray crystallography confirmed the correct folding of ubiquitin. Our results demonstrate that proteins synthesized chemically by streamlined 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis coupled with a one-pot ligation-desulfurization strategy can supply useful molecules with sufficient purity for crystallographic studies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Proteomic analysis identifies interleukin 11 regulated plasma membrane proteins in human endometrial epithelial cells in vitro

    Science.gov (United States)

    2011-01-01

    Background During the peri-implantation period, the embryo adheres to an adequately prepared or receptive endometrial surface epithelium. Abnormal embryo adhesion to the endometrium results in embryo implantation failure and infertility. Endometrial epithelial cell plasma membrane proteins critical in regulating adhesion may potentially be infertility biomarkers or targets for treating infertility. Interleukin (IL) 11 regulates human endometrial epithelial cells (hEEC) adhesion. Its production is abnormal in women with infertility. The objective of the study was to identify IL11 regulated plasma membrane proteins in hEEC in vitro using a proteomic approach. Methods Using a 2D-differential in-gel electrophoresis (DIGE) electrophoresis combined with LCMS/MS mass spectrometry approach, we identified 20 unique plasma membrane proteins differentially regulated by IL11 in ECC-1 cells, a hEEC derived cell line. Two IL11 regulated proteins with known roles in cell adhesion, annexin A2 (ANXA2) and flotillin-1 (FLOT1), were validated by Western blot and immunocytochemistry in hEEC lines (ECC-1 and an additional cell line, Ishikawa) and primary hEEC. Flotilin-1 was further validated by immunohistochemistry in human endometrium throughout the menstrual cycle (n = 6-8/cycle). Results 2D-DIGE analysis identified 4 spots that were significantly different between control and IL11 treated group. Of these 4 spots, there were 20 proteins that were identified with LCMS/MS. Two proteins; ANXA2 and FLOT1 were chosen for further analyses and have found to be significantly up-regulated following IL11 treatment. Western blot analysis showed a 2-fold and a 2.5-fold increase of ANXA2 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. Similarly, a 1.8-fold and a 2.3/2.4-fold increase was also observed for FLOT1 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. In vitro, IL11 induced stronger ANXA2 expression on cell surface of primary hEEC and ECC-1 whilst

  19. Proteomic analysis identifies interleukin 11 regulated plasma membrane proteins in human endometrial epithelial cells in vitro

    Directory of Open Access Journals (Sweden)

    Stanton Peter G

    2011-05-01

    Full Text Available Abstract Background During the peri-implantation period, the embryo adheres to an adequately prepared or receptive endometrial surface epithelium. Abnormal embryo adhesion to the endometrium results in embryo implantation failure and infertility. Endometrial epithelial cell plasma membrane proteins critical in regulating adhesion may potentially be infertility biomarkers or targets for treating infertility. Interleukin (IL 11 regulates human endometrial epithelial cells (hEEC adhesion. Its production is abnormal in women with infertility. The objective of the study was to identify IL11 regulated plasma membrane proteins in hEEC in vitro using a proteomic approach. Methods Using a 2D-differential in-gel electrophoresis (DIGE electrophoresis combined with LCMS/MS mass spectrometry approach, we identified 20 unique plasma membrane proteins differentially regulated by IL11 in ECC-1 cells, a hEEC derived cell line. Two IL11 regulated proteins with known roles in cell adhesion, annexin A2 (ANXA2 and flotillin-1 (FLOT1, were validated by Western blot and immunocytochemistry in hEEC lines (ECC-1 and an additional cell line, Ishikawa and primary hEEC. Flotilin-1 was further validated by immunohistochemistry in human endometrium throughout the menstrual cycle (n = 6-8/cycle. Results 2D-DIGE analysis identified 4 spots that were significantly different between control and IL11 treated group. Of these 4 spots, there were 20 proteins that were identified with LCMS/MS. Two proteins; ANXA2 and FLOT1 were chosen for further analyses and have found to be significantly up-regulated following IL11 treatment. Western blot analysis showed a 2-fold and a 2.5-fold increase of ANXA2 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. Similarly, a 1.8-fold and a 2.3/2.4-fold increase was also observed for FLOT1 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. In vitro, IL11 induced stronger ANXA2 expression on cell surface of primary h

  20. INHIBITION OF IN VITRO FERTILIZATION IN THE HAMSTER BY ANTIBODIES RAISED AGAINST THE RAT SPERM PROTEIN SP22

    Science.gov (United States)

    INHIBITION OF IN VITRO FERTILIZATION IN THE HAMSTER BY ANTIBODIES RAISED AGAINST THE RAT SPERM PROTEIN SP22. SC Jeffay*, SD Perreault, KL Bobseine*, JE Welch*, GR Klinefelter, US EPA, Research Triangle Park, NC. SP22, a rat sperm membrane protein that is highly-correlated w...

  1. Source of protein supplementation during in vitro culture does not affect the quality of resulting blastocysts in the domestic cat.

    Science.gov (United States)

    Nestle, E; Graves-Herring, J; Keefer, C; Comizzoli, P

    2012-12-01

    The objective of this study was to assess and compare the quality of cat blastocysts produced in vitro using commercial blastocyst growth media supplemented with different sources of proteins (serum protein substitute from in vitro maturation through embryo development vs 4 mg/ml of bovine serum albumin for maturation and 5% foetal calf serum for fertilization and embryo development). Impact was specifically examined on the proportion of blastocyst formation, total number of blastomeres, proportion of inner cell mass and expression of pluripotency marker proteins NANOG and OCT-4. Blastocyst formation per total cleaved embryos was similar (p > 0.05) regardless of the protein supplementation. There were no differences (p > 0.05) between culture conditions regarding average number of blastomeres and proportion of inner cell mass in each embryo. Presence of OCT-4 protein was detected in nuclei of both trophectoderm and inner cell mass region, with a stronger signal in the latter regardless of the culture medium. NANOG protein also was present in the inner cell mass regardless of the in vitro culture condition. We therefore demonstrated that serum protein substitute was as good as semi-defined protein sources for the production of good-quality blastocysts and embryonic stem cells. In addition, a single defined medium could be successfully used for cat oocyte maturation, in vitro fertilization and embryo development. © 2012 Blackwell Verlag GmbH.

  2. Seed protein yield from some Crotalaria spp and in vitro nutritional quality of that from C. juncea.

    Science.gov (United States)

    Pandey, V N; Srivastava, A K

    1990-07-01

    Seed Protein Concentrates (SPC) of 6 species of Crotalaria were extracted and the extractabilities of SPC, total N and protein N determined. SPC of high yielding species (C. Juncea) was analyzed for the contents of amino acids, ash, phosphorus, sugar, starch, fibre and calories, as well as for in vitro digestibility. Results indicate the promising potential of C. juncea for SPC yield.

  3. Lipid digestion of protein stabilized emulsions investigated in a dynamic in vitro gastro-intestinal model system

    NARCIS (Netherlands)

    Helbig, A.; Silletti, E.; Aken, G.A. van; Oosterveld, A.; Minekus, M.; Hamer, R.J.; Gruppen, H.

    2013-01-01

    This study investigated the effect of gastric passage of protein stabilized emulsions, i.e., whey protein isolate (WPI) and lysozyme, under dynamic in vitro conditions on both the gastric and intestinal lipolysis. Emulsions were prepared at neutral pH to enable an opposite surface charge.

  4. Lipid Digestion of Protein Stabilized Emulsions Investigated in a Dynamic In Vitro Gastro-Intestinal Model System

    NARCIS (Netherlands)

    Helbig, A.; Silletti, E.; Aken, van G.A.; Oosterveld, A.; Minekus, M.; Hamer, R.J.; Gruppen, H.

    2013-01-01

    This study investigated the effect of gastric passage of protein stabilized emulsions, i.e., whey protein isolate (WPI) and lysozyme, under dynamic in vitro conditions on both the gastric and intestinal lipolysis. Emulsions were prepared at neutral pH to enable an opposite surface charge.

  5. Shear bond strength comparison between two orthodontic adhesives and self-ligating and conventional brackets.

    Science.gov (United States)

    Northrup, Rodney G; Berzins, David W; Bradley, Thomas Gerard; Schuckit, William

    2007-07-01

    To evaluate and compare the shear bond strengths of two adhesives using two types of brackets: a conventional and a self-ligating bracket system. Sixty extracted human premolars were collected. The premolars were randomly divided into three groups of 20 teeth. All three groups were direct bonded. Groups 1 and 2 used light-cured adhesive and primer (Transbond XT) with a conventional (Orthos) and a self-ligating bracket (Damon 2), respectively. Group 3 used a light-cured primer (Orthosolo) and a light-cured adhesive (Blūgloo) with a self-ligating bracket (Damon 2). The specimens were stored in distilled water at 37 degrees C for 40 +/- 2 hours, after which they were debonded and inspected for Adhesive Remnant Index (ARI) scoring. The mean shear bond strength was 15.2 MPa for group 1, 23.2 MPa for group 2, and 24.8 MPa for group 3. A one-way analysis of variance and post hoc Tukey test showed significant differences in bond strength (P .05) between groups 2 and 3. A Weibull analysis demonstrated that all three groups provided sufficient bond strength with over 90% survival rate at normal masticatory and orthodontic force levels. A Kruskal-Wallis test showed no significant difference (P > .05) in ARI scores among all three groups. All three groups demonstrated clinically acceptable bond strength. The Damon 2 self-ligating bracket exhibited satisfactory in vitro bond strength with both adhesive systems used.

  6. [Effect of enamel matrix protein on periodontal cells in an in vitro wound healing model].

    Science.gov (United States)

    Yin, Yuan-zheng; Shen, Chi-jing; Song, Zhong-chen; Zhang, Xiu-li

    2007-06-01

    The aim of this study was to evaluate the influence of enamel matrix protein (EMP) on wound filling of periodontal ligament cells (PDLC) and gingival fibroblasts (GF), using an in vitro model of wound healing. In vitro wound models were mechanically created in subconfluent cultures of human GF and PDLC, by removing a 7mm wide band of the cell layer respectively. Wounded cultures were then incubated for a time periods up to 2,6,9 days in a media containing 10% fetal bovine serum (FBS), stimulated with EMP (100microg/ml) simultaneously, negative controls were those cultured only with media containing 10% FBS. Slides were fixed, stained with crystal violet and cell filling area within the wound boundaries was quantified by computer assisted histomorphometry. Statistical analysis was performed by SAS6.12 software package to determine the differences between the time points and groups. in the control group, there was difference between GF and PDLC in filling wound in vitro over 9 days of healing period. The difference was significant (P0.05) between the filling rate of two types of periodontal cells at the 6th, 9th day after wound creating. GF has a significantly greater ability to fill a wound than PDLC. However, EMP appears to exert an influence on cells that is compatible with improved wound healing, especially in that of PDLC.

  7. Effect of surface modification on the In vitro protein adsorption and cell cytotoxicity of vinorelbine nanoparticles

    Directory of Open Access Journals (Sweden)

    Nandhakumar Sathyamoorthy

    2017-01-01

    Full Text Available Context: Nanocarriers possessing long-circulating abilities could take advantage of the pathophysiology of tumor vasculature to achieve spatial placement. To attain such qualities, the drug carriers should possess suitable physicochemical properties such as size and surface hydrophilicity. Aim: The aim of this study was to prepare poly(ε-caprolactone nanoparticles (NPs loaded with vinorelbine bitartrate (VB and to modify its steric properties using polyethylene glycol and poloxamer. Furthermore, the influence of surface modification of NPs on their physicochemical and cell interactive properties was evaluated. Materials and Methods: NPs were prepared by double emulsion solvent extraction–evaporation technique. The prepared NPs were evaluated for their physicochemical properties, in vitro protein adsorption and cell cytotoxicity. Results and Discussion: The NPs were <250 nm with an entrapment efficiency ranging between 40% and 52%. The zeta potential of the NPs varied from −7.52 mV to −1.27 mV depending on the surface modification. The in vitro release studies exhibited a biphasic pattern with an initial burst release followed by controlled release of the drug over 72 h. The protein adsorption studies revealed that the ability to resist protein adsorption was influenced by the concentration of surface-modifying agents and the amount of proteins available for interaction. The surface-modified NPs produced cell cytotoxicity comparable to free VB at higher concentrations owing to sustained release of the drug into the cellular environment. Conclusion: The results emphasize that surface modification of nanocarriers is an essential and effective tool to dodge opsonization and phagocytosis in the physiological milieu.

  8. In vivo and in vitro protein digestibility of formulated feeds for Artemesia longinaris (Crustacea, Penaeidae

    Directory of Open Access Journals (Sweden)

    Analía Verónica Fernández Gimenez

    2009-12-01

    Full Text Available This study was undertaken to determine the in vivo crude protein apparent digestibility in the prawn Artemesia longinaris, using feeds with 0.25% of chromic oxide and animal (fish meal, meat and bone meal and squid protein concentrate and plant (soybean meal ingredients. Three replicate groups of prawn were fed and the feces were collected. The rate of protein hydrolysis was measured in vitro using midgut gland enzyme extract from the prawns fed the respective feeds and was compared with those found with enzyme extract of wild prawn. The in vivo apparent digestibility coefficients showed significant differences among the feeds (PO objetivo do presente trabalho foi determinar a digestibilidade aparente in vivo da proteína bruta de ingredientes de origem animal (farinhas de peixe, osso e carne e concentrado de proteína de lula e ingredientes vegetais (farinha de soja em camarões Artemesia longinaris utilizando rações contendo 0,25% de óxido de cromo. Três grupos de camarões, utilizados como replicatas, foram alimentados e as fezes coletadas. A velocidade de hidrólise da proteína de cada ração foi medida in vitro utilizando extrato enzimático da glândula do intestino médio dos camarões alimentados com a ração correspondente e foi comparado com aqueles obtidos com o extrato enzimático de camarões selvagens. Os coeficientes de digestibilidade aparente in vivo mostraram diferenças significativas entre as rações testadas (P<0,05. A farinha de peixe apresentou a maior digestibilidade (92%, enquanto valores intermediários de digestibilidade (83% foram encontrados para a farinha de carne e ossos. A ração contendo farinha de soja e concentrado de proteína de lula resultou em menor digestibilidade (63%. Não houve diferença significativa entre os valores de digestibilidade in vitro para as rações testadas. Estes resultados indicam a limitação inerente dos ensaios enzimáticos in vitro, os quais poderiam ser complementados com

  9. In vitro prion protein conversion suggests risk of bighorn sheep (Ovis canadensis) to transmissible spongiform encephalopathies

    Science.gov (United States)

    Johnson, Christopher J.; Morawski, A.R.; Carlson, C.M.; Chang, H.

    2013-01-01

    Background: Transmissible spongiform encephalopathies (TSEs) affect both domestic sheep (scrapie) and captive and free-ranging cervids (chronic wasting disease; CWD). The geographical range of bighorn sheep (Ovis canadensis; BHS) overlaps with states or provinces that have contained scrapie-positive sheep or goats and areas with present epizootics of CWD in cervids. No TSEs have been documented in BHS, but the susceptibility of this species to TSEs remains unknown. Results: We acquired a library of BHS tissues and found no evidence of preexisting TSEs in these animals. The prion protein gene (Prnp) in all BHS in our library was identical to scrapie-susceptible domestic sheep (A136R 154Q171). Using an in vitro prion protein conversion assay, which has been previously used to assess TSE species barriers and, in our study appears to recollect known species barriers in mice, we assessed the potential transmissibility of TSEs to BHS. As expected based upon Prnp genotype, we observed BHS prion protein conversion by classical scrapie agent and evidence for a species barrier between transmissible mink encephalopathy (TME) and BHS. Interestingly, our data suggest that the species barrier of BHS to white-tailed deer or wapiti CWD agents is likely low. We also used protein misfolding cyclic amplification to confirm that CWD, but not TME, can template prion protein misfolding in A136R 154Q171genotype sheep. Conclusions: Our results indicate the in vitro conversion assay used in our study does mimic the species barrier of mice to the TSE agents that we tested. Based on Prnp genotype and results from conversion assays, BHS are likely to be susceptible to infection by classical scrapie. Despite mismatches in amino acids thought to modulate prion protein conversion, our data indicate that A136R154Q171 genotype sheep prion protein is misfolded by CWD agent, suggesting that these animals could be susceptible to CWD. Further investigation of TSE transmissibility to BHS, including

  10. Bioactivity of Cod and Chicken Protein Hydrolysates 
before and after in vitro Gastrointestinal Digestion.

    Science.gov (United States)

    Jamnik, Polona; Istenič, Katja; Koštomaj, Tatjana; Wulff, Tune; Geirsdóttir, Margrét; Almgren, Annette; Jónsdóttir, Rósa; Kristinsson, Hordur G; Undeland, Ingrid

    2017-09-01

    Bioactivity of cod (Gadus morhua) and chicken (Gallus domesticus) protein hydrolysates before and after in vitro gastrointestinal (GI) digestion was investigated using yeast Saccharomyces cerevisiae as a model organism. Both hydrolysates were exposed to in vitro GI digestion prior to cellular exposure to simulate digestion conditions in the human body and therefore investigate the role of modulations in the GI tract on the cell response. The effect of digested and undigested hydrolysates on intracellular oxidation, cellular metabolic energy and proteome level was investigated. No difference in the effect on intracellular oxidation activity was obtained between cod and chicken hydrolysates, while higher affect on intracellular oxidation was provided by digested hydrolysates, with relative values of intracellular oxidation of cod of (70.2±0.8) and chicken of (74.5±1.4) % than by undigested ones, where values of cod and chicken were (95.5±1.2) and (90.5±0.7) %, respectively. Neither species nor digestion had any effect on cellular metabolic energy. At proteome level, digested hydrolysates gave again significantly stronger responses than undigested counterparts; cod peptides here also gave somewhat stronger response than chicken peptides. The knowledge of the action of food protein hydrolysates and their digests within live cells, also at proteome level, is important for further validation of their activity in higher eukaryotes to develop new functional food ingredients, such as in this case chicken and cod muscle-derived peptides.

  11. The effects of ovalbumin as a protein source during the in vitro production of bovine embryos

    Directory of Open Access Journals (Sweden)

    Tatiane Almeida Drummond Tetzner

    2011-10-01

    Full Text Available Embryo quality is influenced by the culture conditions that affect in vitro maturation (IVM, fertilization (IVF and culture (IVC rates. The present study investigated the feasibility of producing bovine embryos after the replacement of fetal calf serum (FCS and bovine serum albumin (BSA by ovalbumin (OVA. The IVM and IVC medium were supplemented with 10% FCS, 4 mg/mL BSA, or 4 mg/mL OVA. The IVF medium was supplemented with 6 mg/mL BSA or OVA. For IVM, supplementation with FCS, BSA, and OVA did not affect nuclear maturation or cortical granule migration. Higher rates of formation of two pronuclei were obtained when FCS was employed for IVM (79.97%, regardless of the supplement used for IVF, and when BSA was used for IVF (59.4%, regardless of the supplement used for IVM. Supplementation with OVA for IVM+IVC (20.40% and for IVF (22.15% was inferior to supplementation with FCS for IVM+IVC (30.47% and with BSA for IVF (28.91% for blastocyst development. Hatching rates were lower using OVA for IVM+IVC (23.02% and for IVF (28.93% compared with FCS and BSA under the same conditions (40.78 and 34.82%, respectively and BSA for IVF (36.82%. Supplementation with OVA for IVM+IVC and IVF resulted in reduced inner cell mass, trophectoderm cells and total blastocyst cell numbers (17.29, 37.88, and 55.17, respectively. In conclusion, OVA is a protein source for bovine in vitro embryo production, although the quantity and quality of bovine blastocysts using only ovalbumin in the entire in vitro production process are lower than those obtained in the presence of FCS and BSA, when used as supplements in any step of bovine in vitro embryo production.

  12. 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.

  13. Tracking the fate of pasta (T. Durum semolina) immunogenic proteins by in vitro simulated digestion.

    Science.gov (United States)

    Mamone, Gianfranco; Nitride, Chiara; Picariello, Gianluca; Addeo, Francesco; Ferranti, Pasquale; Mackie, Alan

    2015-03-18

    The aim of the present study was to identify and characterize the celiacogenic/immunogenic proteins and peptides released during digestion of pasta (Triticum durum semolina). Cooked pasta was digested using a harmonized in vitro static model of oral-gastro-duodenal digestion. The course of pasta protein digestion was monitored by SDS-PAGE, and gluten proteins were specifically analyzed by Western blot using sera of celiac patients. Among the allergens, nonspecific lipid-transfer protein was highly resistant to gastro-duodenal hydrolysis, while other digestion-stable allergens such as α-amylase/trypsin inhibitors were not detected being totally released in the pasta cooking water. To simulate the final stage of intestinal degradation, the gastro-duodenal digesta were incubated with porcine jejunal brush-border membrane hydrolases. Sixty-one peptides surviving the brush-border membrane peptidases were identified by liquid chromatography-mass spectrometry, including several gluten-derived sequences encrypting different motifs responsible for the induction of celiac disease. These results provide new insights into the persistence of wheat-derived peptides during digestion of cooked pasta samples.

  14. HUMAN SERUM ALBUMIN AND PROLACTIN INDUCIBLE PROTEIN COMPLEX ENHANCES SPERM CAPACITATION IN VITRO

    Directory of Open Access Journals (Sweden)

    Anil Kumar Tomar

    2016-06-01

    Full Text Available Prolactin inducible protein (PIP is a 17 kDa protein expressed in human body fluids. It interacts with several other proteins including fibrinogen, IgG, actin, CD4, ZAG, etc. and their association contributes to multifaceted molecular functions of PIP in diverse classes of biological processes. The roles of PIP are reported in immunoregulation, tumor progression, apoptosis and fertility. Recently, PIP was purified in complex with human serum albumin (HSA from human semen. Here, we have reported comparative expression analysis of this complex in human seminal plasma samples of various categories of infertility conditions (oligozoospermia, azoospermia and asthenozoospermia and fertile controls (normozoospermia. We have also evaluated sperm capacitation and acrosome reaction in presence of varying concentrations of this complex. Comparative expression analysis was performed under same experimental conditions by co-immunoprecipitation followed by western blot analysis and results highlighted that HSA-PIP complex is down-regulated in azoospermia as depicted by the intensity of protein bands on the blot. Assessment of sperm functions in presence or absence of this complex revealed that this complex acts as an inducer of in vitro sperm capacitation. It was observed that 87% sperm were capacitated after 4h incubation with HSA-PIP complex (100 µg/mL in comparison to 60.33% in +ve control. On the basis of our findings, we conclude that this complex might be a positive regulator of sperm motility and capacitation in vivo.

  15. Merging in-silico and in vitro salivary protein complex partners using the STRING database: A tutorial.

    Science.gov (United States)

    Crosara, Karla Tonelli Bicalho; Moffa, Eduardo Buozi; Xiao, Yizhi; Siqueira, Walter Luiz

    2018-01-16

    Protein-protein interaction is a common physiological mechanism for protection and actions of proteins in an organism. The identification and characterization of protein-protein interactions in different organisms is necessary to better understand their physiology and to determine their efficacy. In a previous in vitro study using mass spectrometry, we identified 43 proteins that interact with histatin 1. Six previously documented interactors were confirmed and 37 novel partners were identified. In this tutorial, we aimed to demonstrate the usefulness of the STRING database for studying protein-protein interactions. We used an in-silico approach along with the STRING database (http://string-db.org/) and successfully performed a fast simulation of a novel constructed histatin 1 protein-protein network, including both the previously known and the predicted interactors, along with our newly identified interactors. Our study highlights the advantages and importance of applying bioinformatics tools to merge in-silico tactics with experimental in vitro findings for rapid advancement of our knowledge about protein-protein interactions. Our findings also indicate that bioinformatics tools such as the STRING protein network database can help predict potential interactions between proteins and thus serve as a guide for future steps in our exploration of the Human Interactome. Our study highlights the usefulness of the STRING protein database for studying protein-protein interactions. The STRING database can collect and integrate data about known and predicted protein-protein associations from many organisms, including both direct (physical) and indirect (functional) interactions, in an easy-to-use interface. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Hamster oviductin regulates tyrosine phosphorylation of sperm proteins during in vitro capacitation.

    Science.gov (United States)

    Saccary, Laurelle; She, Yi-Min; Oko, Richard; Kan, Frederick W K

    2013-08-01

    Oviductin or OVGP1, also known as oviduct-specific glycoprotein, has been shown to enhance sperm capacitation in addition to its other beneficial effects on fertilization and early embryo development. We hypothesized that estrus stage-specific hamster oviductin (eHamOVGP1) can potentiate the enhancement of tyrosine phosphorylation of sperm proteins during capacitation. Immunofluorescent staining and confocal microscopy as well as immunocytochemistry and surface replica technique localized tyrosine-phosphorylated proteins to the equatorial segment and midpiece after incubation of hamster sperm in capacitation medium in the presence or absence of eHamOVGP1. Increase of tyrosine phosphorylation level in the equatorial segment occurred as early as 5 min after incubation in the presence of eHamOVGP1. Immunostaining for eHamOVGP1 further increased upon prolonged incubation of sperm in medium containing the glycoprotein. Regardless of the presence or absence of eHamOVGP1, phosphotyrosine expression was observed along the tail, particularly at the midpiece. Western blotting of NP40-extracted sperm proteins (25, 37, and 44 kDa) and NP40-non-extractable sperm proteins (70, 83, 90 kDa) showed increased immunolabeling intensity after 5, 60, 120, and 180 min of capacitation in the presence of eHamOVGP1. Mass spectrometric analysis identified several proteins of functions known to be involved in metabolic pathways responsible for enhancement of tyrosine phosphorylation in its presence. The present investigation provides evidence that eHamOVGP1 regulates the expression of protein tyrosine phosphorylation in sperm capacitated in vitro, further supporting an important role of the presence of OVGP1 in the oviductal milieu during the process of fertilization.

  17. A high-throughput, in-vitro assay for Bacillus thuringiensis insecticidal proteins.

    Science.gov (United States)

    Izumi Willcoxon, Michi; Dennis, Jaclyn R; Lau, Sabina I; Xie, Weiping; You, You; Leng, Song; Fong, Ryan C; Yamamoto, Takashi

    2016-01-10

    A high-throughput, in-vitro assay for Bacillus thuringiensis (Bt) insecticidal proteins designated as Cry was developed and evaluated for screening a large number of Cry protein variants produced by DNA shuffling. This automation-amenable assay exploits an insect cell line expressing a single receptor of Bt Cry proteins. The Cry toxin used to develop this assay is a variant of the Cry1Ab protein called IP1-88, which was produced previously by DNA shuffling. Cell mortality caused by the activated Bt Cry toxin was determined by chemical cell viability assay in 96/384-well microtiter plates utilizing CellTiter 96(®) obtained from Promega. A widely-accepted mode-of-action theory of certain Bt Cry proteins suggests that the activated toxin binds to one or more receptors and forms a pore through the insect gut epithelial cell apical membrane. A number of insect proteins such as cadherin-like protein (Cad), aminopeptidase-N (APN), alkaline phosphatase (ALP) and ABC transporter (ABCC) have been identified as the receptors of Bt Cry toxins. In this study, Bt Cry toxin receptors Ostrinia nubilalis (European corn borer) cadherin-like protein (On-Cad) and aminopeptidase-N 1 and 3 (On-APN1, On-APN3) and Spodoptera frugiperda (fall armyworm) cadherin-like protein (Sf-Cad) were cloned in an insect cell line, Sf21, and a mammalian cell line, Expi293F. It was observed by ligand blotting and immunofluorescence microscopy that trypsin-activated IP1-88 bound to On-Cad and On-APN1, but not Sf-Cad or On-APN3. In contrast, IP1-88 bound only to APN1 in BBMV (Brush Border Membrane Vesicles) prepared from the third and fourth-instar O. nubilalis larval midgut. The sensitivity of the recombinant cells to the toxin was then tested. IP1-88 showed no toxicity to non-recombinant Sf21 and Expi293F. Toxicity was observed only when the On-Cad gene was cloned and expressed. Sf-Cad and On-APN1 were not able to make those cells sensitive to the toxin. Since the expression of On-Cad alone was

  18. Interfacial Bioorthogonal Chemistry for Biomaterials Synthesis and Patterning and Development of Catalytic Method for "Turning-On" the Tetrazine Ligation

    Science.gov (United States)

    Zhang, Han

    The bioorthogonal trans-cyclooctene-tetrazine ligation has emerged into a powerful tool in the field of biomedical research. The development and the versatile applications of tetrazine ligation was made possible by the advancement of trans-cyclooctene synthesis. Based on the previous art of photoisomerization methods in Fox group, I carried out the first practical photosynthesis of trans-cycloheptene derivatives that were stabilized as silver(I) complexes form, as well as the photoisomerization of silicon-containing hetero- trans-cycloheptene derivatives. The reactivity of both the trans-cycloheptene silver(I) complexes and the hetero-trans-cycloheptene derivatives were investigated. Based on the rapid trans-cyclooctene-tetrazine ligation, first example of interfacial crosslinking will be described in Chapter 2. Bioocompatible hyaluronic acid-based hydrogel microspheres and channels were generated in a diffusion controlled fashion. These hydrogels can be covalently tagged with 3D resolution without the help of any external stimulus or triggers. An in vitro tumor model was achieved by 3D encapsulation and culture of LNCaP prostate cancer cells. Also included in Chapter 2 will be a novel interfacial polymerization strategy developed for the synthesis of hybrid multiblock copolymer. Meter-long copolymer fibers were pulled out of interface of two immiscible solutions. The unique modular approach enables the facile incorporation of functional peptides into the copolymer to fine-tune its biological properties. A fibronectin-derived peptide was successfully introduced onto the fibers during the polymerization and dramatically promoted the attachment and alignment of fibroblasts and myoepithelial-like cells. In Chapter 3, a novel method to activate rapid bioorthogonal reactivity catalytically will be described. This was achieved by catalytic conversion of an unreactive, latent dihydrotetrazine to reaction-ready tetrazine functionality. Series of long wavelength

  19. In vitro protein digestibility and physico-chemical properties of flours and protein concentrates from two varieties of lentil (Lens culinaris).

    Science.gov (United States)

    Barbana, Chockry; Boye, Joyce Irene

    2013-02-01

    The chemical composition of whole lentil flours and lentil protein concentrates prepared by alkaline extraction and iso-electric precipitation from Blaze and Laird varieties of lentil were studied. The protein composition of the flours and concentrates, determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and size-exclusion high-performance liquid chromatography (SE-HPLC) showed that the extracted proteins were composed mainly of globulins and albumins. Trypsin inhibitor activity ranged between 0.94 and 1.94 trypsin inhibitor units (TIU) mg(-1) for the flours, but was markedly lower in the protein concentrates ranging between 0.17 and 0.66 TIU mg(-1). In vitro protein digestibility ranged between 75.90 and 77.05% for the flours, whereas significantly (P lentil protein concentrates is higher than that of the flours, however, both lentil flours and protein concentrates contain useful proteins that could serve as value-added ingredients in food formulations.

  20. RX-207, a Small Molecule Inhibitor of Protein Interaction with Glycosaminoglycans (SMIGs), Reduces Experimentally Induced Inflammation and Increases Survival Rate in Cecal Ligation and Puncture (CLP)-Induced Sepsis

    Czech Academy of Sciences Publication Activity Database

    Juhás, Štefan; Harris, N.; Ilková, G.; Rehák, P.; Zsila, F.; Kogan, F. Y.; Lahmy, O.; Zhuk, R.; Gregor, P.; Koppel, J.

    2018-01-01

    Roč. 41, č. 1 (2018), s. 307-314 ISSN 0360-3997 Institutional support: RVO:67985904 Keywords : heparin binding protein * glycosaminoglycan * neutrophil Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.955, year: 2016

  1. The importance of protein binding for the in vitro-in vivo extrapolation (IVIVE)-example of ibuprofen, a highly protein-bound substance.

    Science.gov (United States)

    Mielke, H; Di Consiglio, E; Kreutz, R; Partosch, F; Testai, E; Gundert-Remy, U

    2017-04-01

    A physiologically based human kinetic model (PBHKM) was used to predict the in vivo ibuprofen dose leading to the same concentration-time profile as measured in cultured human hepatic cells (Truisi et al. in Toxicol Lett 233(2):172-186, 2015). We parameterized the PBHKM with data from an in vivo study. Tissue partition coefficients were calculated by an algorithm and also derived from the experimental in vitro data for the liver. The predicted concentration-time profile in plasma was in excellent agreement with human experimental data when the liver partition coefficient was calculated by the algorithm (3.01) demonstrating values in line with findings obtained from human postmortem tissues. The results were less adequate when the liver partition coefficient was based on the experimental in vitro data (11.1). The in vivo doses necessary to reach the in vitro concentrations in the liver cells were 3610 mg using the best fitting model with a liver partition coefficient of 3.01 compared to 2840 mg with the in vitro liver partition coefficient of 11.1. We found that this difference is possibly attributable to the difference between protein binding in vivo (99.9 %) and in vitro (nearly zero) as the partition coefficient is highly dependent on protein binding. Hence, the fraction freely diffusible in the liver tissue is several times higher in vitro than in vivo. In consequence, when extrapolating from in vitro to in vivo liver toxicity, it is important to consider non-intended in vitro/in vivo differences in the tissue concentration which may occur due to a low protein content of the medium.

  2. Amino acid composition, available lysine content and in vitro protein digestibility of selected tropical crop seeds.

    Science.gov (United States)

    Petzke, K J; Ezeagu, I E; Proll, J; Akinsoyinu, A O; Metges, C C

    1997-01-01

    As the search for alternative sources of food to alleviate hunger continues, this study was undertaken to determine nitrogen and amino acid content, chemical score, protein digestibility corrected amino acid score, available lysine and in vitro digestibility of 8 lesser known, wild tropical seeds, gathered in Nigeria. Results were contrasted with a tropical soybean variety (Glycine max, TGX 1660-15F). The investigated seeds were Millettia thonningii, Gliricidia sepium, Lonchocarpus sericeus, Albizia zygia, Daneillia ogea and Afzella bella from the family of Leguminosae, Diospyros mespiliformis (Ebenaceae) and Entandrophragma angolense (Meliaceae). The crude protein content, based on nitrogen determination, was found to be lower in the wild seeds compared to soybean, which was partly due to the relatively high content of non-protein nitrogen. With reference to amino acid requirement and digestibility in most seed samples, lysine, followed by sulphur amino acids and threonine, were the limiting amino acids. It was concluded, that these less familiar wild seed plants may be used as valuable food or feed complements. However, further investigation is necessary to elucidate potential toxic and antinutritional factors.

  3. Engineering multifunctional protein nanoparticles by in vitro disassembling and reassembling of heterologous building blocks

    Science.gov (United States)

    Unzueta, Ugutz; Serna, Naroa; Sánchez-García, Laura; Roldán, Mónica; Sánchez-Chardi, Alejandro; Mangues, Ramón; Villaverde, Antonio; Vázquez, Esther

    2017-12-01

    The engineering of protein self-assembling at the nanoscale allows the generation of functional and biocompatible materials, which can be produced by easy biological fabrication. The combination of cationic and histidine-rich stretches in fusion proteins promotes oligomerization as stable protein-only regular nanoparticles that are composed by a moderate number of building blocks. Among other applications, these materials are highly appealing as tools in targeted drug delivery once empowered with peptidic ligands of cell surface receptors. In this context, we have dissected here this simple technological platform regarding the controlled disassembling and reassembling of the composing building blocks. By applying high salt and imidazole in combination, nanoparticles are disassembled in a process that is fully reversible upon removal of the disrupting agents. By taking this approach, we accomplish here the in vitro generation of hybrid nanoparticles formed by heterologous building blocks. This fact demonstrates the capability to generate multifunctional and/or multiparatopic or multispecific materials usable in nanomedical applications.

  4. G-protein-coupled receptor 137 accelerates proliferation of urinary bladder cancer cells in vitro.

    Science.gov (United States)

    Du, Yiheng; Bi, Wenhuan; Zhang, Fei; Wu, Wenbo; Xia, Shujie; Liu, Haitao

    2015-01-01

    Urinary bladder cancer is a worldwide concern because of its level of incidence and recurrence. To search an effective therapeutic strategy for urinary bladder cancer, it is important to identify proteins involved in tumorigenesis that could serve as potential targets for diagnosis and treatment. G-protein-coupled receptors (GPRs) constitute a large protein family of receptors that sense molecules outside the cell and activate signal transduction pathways and cellular responses inside the cell. GPR137 is a newly discovered human gene encoding orphan GPRs. In this study, we aimed to investigate the physiological role of GPR137 in urinary bladder cancer. The effect of GPR137 on cell growth was examined via an RNA interference (RNAi) lentivirus system in two human urinary bladder cancer cell lines BT5637 and T24. Lentivirus-mediated RNAi could specifically suppressed GPR137 expression in vitro, resulting in alleviated cell viability and impaired colony formation, as well as blocks G0/G1 and S phases of the cell cycle. These results suggested GPR137 as an essential player in urinary bladder cancer cell growth, and it may serve as a potential target for gene therapy in the treatment of urinary bladder cancer. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

  5. Free Fatty Acid Concentration and Carboxy methyl cellulase Activity of Some Formulas of Protected Fat-proteins Tested In Vitro

    Directory of Open Access Journals (Sweden)

    Lilis Hartati

    2015-05-01

    Full Text Available The aim of this study was to determine the levels of free fatty acids and carboxymethylcellulase activity (cmc-ase activity of some protected fat-proteins base on in vitro Tilley and Terry method. Two sources of fat, i.e. crude palm oil and fish oil and three sources of protein i.e. skim milk, soybean flour and soybean meal were used in the formulation of protected fat-protein, and thus there were six treatment combinations. The filtrate from the in vitro test was analyzed for the levels of free fatty acids and  cmcase activity. The result of this research indicates that different combinations of feed materials and fat give different content of free fatty acid in first stage and second stage in vitro, with the best results in the combination treatment of skim milk and palm oil that give the lowest result of  free fatty acid concentration in fisrt stage in vitro (0.168% and the highest result free fatty acid concentration in second stage in vitro ( 4.312% . The activity of CMC-ase was not influenced by different  sources of fat and protein. It can be concluded was that the protection of the combination between skim milk and CPO gives the highest protection results.

  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. Whole Genome Shotgun Sequencing Shows Selection on Leptospira Regulatory Proteins during in vitro Culture Attenuation

    Science.gov (United States)

    Lehmann, Jason S.; Corey, Victoria C.; Ricaldi, Jessica N.; Vinetz, Joseph M.; Winzeler, Elizabeth A.; Matthias, Michael A.

    2016-01-01

    Leptospirosis is the most common zoonotic disease worldwide with an estimated 500,000 severe cases reported annually, and case fatality rates of 12–25%, due primarily to acute kidney and lung injuries. Despite its prevalence, the molecular mechanisms underlying leptospirosis pathogenesis remain poorly understood. To identify virulence-related genes in Leptospira interrogans, we delineated cumulative genome changes that occurred during serial in vitro passage of a highly virulent strain of L. interrogans serovar Lai into a nearly avirulent isogenic derivative. Comparison of protein coding and computationally predicted noncoding RNA (ncRNA) genes between these two polyclonal strains identified 15 nonsynonymous single nucleotide variant (nsSNV) alleles that increased in frequency and 19 that decreased, whereas no changes in allelic frequency were observed among the ncRNA genes. Some of the nsSNV alleles were in six genes shown previously to be transcriptionally upregulated during exposure to in vivo-like conditions. Five of these nsSNVs were in evolutionarily conserved positions in genes related to signal transduction and metabolism. Frequency changes of minor nsSNV alleles identified in this study likely contributed to the loss of virulence during serial in vitro culture. The identification of new virulence-associated genes should spur additional experimental inquiry into their potential role in Leptospira pathogenesis. PMID:26711524

  8. HMGN2 protein inhibits the growth of infected T24 cells in vitro.

    Science.gov (United States)

    Wei, Danfeng; Zhang, Ping; Zhou, Min; Feng, Yun; Chen, Qianming

    2014-01-01

    Natural killer (NK) cells and cytolytic T lymphocytes (CTL) have been implicated as important effectors of antitumor defense. High mobility group nucleosomal-binding domain 2 (HMGN2) may be one of the effector molecules of CTL and NK cells. The antitumor effect and mechanism of HMGN2 was investigated in this study. HMGN2 was isolated and purified from the human monocyte cell line THP-1 and then characterized by Tricine-SDS-PAGE, western blot, and mass spectrum determination. Confluent T24 cells were incubated with Klebsiella pneumoniae for 2 h, after which the extracellular bacteria were killed by the addition of gentamicin. The cells then were treated with a variety of concentrations of HMGN2. The effect of HMGN2 on the proliferation of T24 cells was analyzed with MTT, Hoechst and flow cytometry assays. Cell growth assay results demonstrated that HMGN2 significantly inhibited the growth of T24 bladder cancer cell lines infected by K. pneumoniae. Furthermore, results of the Hoechst and flow cytometry assays indicated that HMGN2 may promote apoptosis in this experimental model. These results suggest HMGN2 could inhibit the growth of the infected human bladder cancer cells in vitro. HMGN2 protein could inhibit the growth of infected T24 cells in vitro, and the anti-tumor action of HMGN2 was due to induce apoptosis.

  9. Development and in vitro characterization of insulin loaded whey protein and alginate microparticles.

    Science.gov (United States)

    Déat-Lainé, Emmanuelle; Hoffart, Valérie; Cardot, Jean-Michel; Subirade, Muriel; Beyssac, Eric

    2012-12-15

    Insulin was encapsulated into microparticles (MP) made of denaturized whey proteins (WP) and alginate (ALG) using an extrusion/cold gelation process with calcium ions. High encapsulation efficiency of 85% was obtained. Influence of insulin on polymeric viscosity and on microparticle behavior was evaluated. Insulin seemed to interact with WP chains by non covalent binding and steric hindrance. This influence was balanced by ALG addition. Nevertheless, insulin was released rapidly by diffusion at both acidic and intestinal dissolution media. Despite this fast in vitro release, WP/ALG MP showed an important enzymatic inhibition effect on trypsin and alpha-chymotrypsin. Thus, WP/ALG MP contributed to an effective insulin protection towards enzymatic degradation. The aforementioned results suggested that WP based microparticles are a promising carrier for improving oral delivery of insulin. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. The HubBLe trial: haemorrhoidal artery ligation (HAL versus rubber band ligation (RBL for haemorrhoids

    Directory of Open Access Journals (Sweden)

    Tiernan Jim

    2012-10-01

    Full Text Available Abstract Background Haemorrhoids (piles are a very common condition seen in surgical clinics. After exclusion of more sinister causes of haemorrhoidal symptoms (rectal bleeding, perianal irritation and prolapse, the best option for treatment depends upon persistence and severity of the symptoms. Minor symptoms often respond to conservative treatment such as dietary fibre and reassurance. For more severe symptoms treatment such as rubber band ligation may be therapeutic and is a very commonly performed procedure in the surgical outpatient setting. Surgery is usually reserved for those who have more severe symptoms, as well as those who do not respond to non-operative therapy; surgical techniques include haemorrhoidectomy and haemorrhoidopexy. More recently, haemorrhoidal artery ligation has been introduced as a minimally invasive, non destructive surgical option. There are substantial data in the literature concerning efficacy and safety of 'rubber band ligation including multiple comparisons with other interventions, though there are no studies comparing it to haemorrhoidal artery ligation. A recent overview has been carried out by the National Institute for Health and Clinical Excellence which concludes that current evidence shows haemorrhoidal artery ligation to be a safe alternative to haemorrhoidectomy and haemorrhoidopexy though it also highlights the lack of good quality data as evidence for the advantages of the technique. Methods/design The aim of this study is to establish the clinical effectiveness and cost effectiveness of haemorrhoidal artery ligation compared with conventional rubber band ligation in the treatment of people with symptomatic second or third degree (Grade II or Grade III haemorrhoids. Design: A multi-centre, parallel group randomised controlled trial. Outcomes: The primary outcome is patient-reported symptom recurrence twelve months following the intervention. Secondary outcome measures relate to symptoms

  11. The HubBLe trial: haemorrhoidal artery ligation (HAL) versus rubber band ligation (RBL) for haemorrhoids.

    Science.gov (United States)

    Tiernan, Jim; Hind, Daniel; Watson, Angus; Wailoo, Allan J; Bradburn, Michael; Shephard, Neil; Biggs, Katie; Brown, Steven

    2012-10-25

    Haemorrhoids (piles) are a very common condition seen in surgical clinics. After exclusion of more sinister causes of haemorrhoidal symptoms (rectal bleeding, perianal irritation and prolapse), the best option for treatment depends upon persistence and severity of the symptoms. Minor symptoms often respond to conservative treatment such as dietary fibre and reassurance. For more severe symptoms treatment such as rubber band ligation may be therapeutic and is a very commonly performed procedure in the surgical outpatient setting. Surgery is usually reserved for those who have more severe symptoms, as well as those who do not respond to non-operative therapy; surgical techniques include haemorrhoidectomy and haemorrhoidopexy. More recently, haemorrhoidal artery ligation has been introduced as a minimally invasive, non destructive surgical option.There are substantial data in the literature concerning efficacy and safety of 'rubber band ligation including multiple comparisons with other interventions, though there are no studies comparing it to haemorrhoidal artery ligation. A recent overview has been carried out by the National Institute for Health and Clinical Excellence which concludes that current evidence shows haemorrhoidal artery ligation to be a safe alternative to haemorrhoidectomy and haemorrhoidopexy though it also highlights the lack of good quality data as evidence for the advantages of the technique. The aim of this study is to establish the clinical effectiveness and cost effectiveness of haemorrhoidal artery ligation compared with conventional rubber band ligation in the treatment of people with symptomatic second or third degree (Grade II or Grade III) haemorrhoids. A multi-centre, parallel group randomised controlled trial. The primary outcome is patient-reported symptom recurrence twelve months following the intervention. Secondary outcome measures relate to symptoms, complications, health resource use, health related quality of life and cost

  12. Denaturation and in Vitro Gastric Digestion of Heat-Treated Quinoa Protein Isolates Obtained at Various Extraction pH.

    Science.gov (United States)

    Ruiz, Geraldine Avila; Opazo-Navarrete, Mauricio; Meurs, Marlon; Minor, Marcel; Sala, Guido; van Boekel, Martinus; Stieger, Markus; Janssen, Anja E M

    The aim of this study was to determine the influence of heat processing on denaturation and digestibility properties of protein isolates obtained from sweet quinoa (Chenopodium quinoa Willd) at various extraction pH values (8, 9, 10 and 11). Pretreatment of suspensions of protein isolates at 60, 90 and 120 °C for 30 min led to protein denaturation and aggregation, which was enhanced at higher treatment temperatures. The in vitro gastric digestibility measured during 6 h was lower for protein extracts pre-treated at 90 and 120 °C compared to 60 °C. The digestibility decreased with increasing extraction pH, which could be ascribed to protein aggregation. Protein digestibility of the quinoa protein isolates was higher compared to wholemeal quinoa flour. We conclude that an interactive effect of processing temperature and extraction pH on in vitro gastric digestibility of quinoa protein isolates obtained at various extraction pH is observed. This gives a first indication of how the nutritional value of quinoa protein could be influenced by heat processing, protein extraction conditions and other grain components.

  13. Atrial natriuretic peptide-Fc, ANP-Fc, fusion proteins: semisynthesis, in vitro activity and pharmacokinetics in rats.

    Science.gov (United States)

    Mezo, Adam R; McDonnell, Kevin A; Low, Susan C; Song, Jeff; Reidy, Tom J; Lu, Qi; Amari, John V; Hoehn, Todd; Peters, Robert T; Dumont, Jennifer; Bitonti, Alan J

    2012-03-21

    Atrial natriuretic peptide (ANP) may be a useful molecule for the treatment of cardiovascular diseases due to its potent natriuretic effects. In an effort to prolong the short in vivo half-life of ANP, fusions of the peptide to the Fc domain of IgG were generated using a semisynthetic methodology. Synthetic ANP peptides were synthesized with thioesters at either the N- or C-termini of the peptide and subsequently linked to the N-terminus of recombinantly expressed Fc using native chemical ligation. The linker length between the ANP and Fc moieties was varied among 2, 11, or 16 amino acids. In addition, either one ("monomeric") or two ("dimeric") ANP peptides were linked to Fc to study whether this modification had an effect on in vitro activity and/or in vivo half-life. The various constructs were studied for in vitro activity using a cell-based cGMP assay. The ANP-Fc fusion constructs were between 16- and ∼375-fold weaker than unconjugated ANP in this assay, and a trend was observed where the most potent conjugates were those with longer linkers and in the dimeric configuration. The pharmacokinetics of several constructs were assessed in rats, and the half-life of the ANP-Fc's were found to be approximately 2 orders of magnitude longer than that of the unconjugated peptide. There was no significant difference in terminal half-life between the monomeric and dimeric constructs (2.8-5.5 h), but a trend was observed where the C(max) of the monomeric constructs was approximately 3-fold higher than that of the dimeric constructs, although the origin of this effect is not understood. These novel ANP-Fc fusion constructs hold promise for future therapeutic application in the treatment of cardiovascular diseases.

  14. In vitro crude protein digestibility of Tenebrio molitor and Hermetia illucens insect meals and its correlation with chemical composition traits

    Directory of Open Access Journals (Sweden)

    Stefania Marono

    2015-07-01

    Full Text Available The aims of this study were to evaluate the correlation between in vitro crude protein digestibility coefficients of insect meals from Tenebrio molitor (TI and Hermetia illucens (HI and their chemical composition traits as well as to develop regression equations able to estimate the in vitro crude protein digestibility (CPd from proximate analysis of insect meals. Twelve samples of insect meals (6 from TM larvae, TM 1-6 and 6 from HI larvae, HI 1-6 were obtained from different producers and analysed for chemical composition and in vitro crude protein digestibility by a two-step enzymatic method (digestion with pepsin and trypsin-enriched pancreatin. For both insect meal samples, CPd was negatively correlated to ADF and chitin contents, while just for HI there was a positive correlation (P<0.01 between CP percentage of the samples and CPd. For both insect meals the former variable chosen in the stepwise analysis was the chitin, explaining the 79.45% of CPd variability for Tenebrio molitor samples and the 98.30% for Hermetia illucens. In the second step, the amount of protein linked to ADF was added in the model for T. molitor and CP for H. illucens samples. The coefficients chitin is the main constituent of insect body able to affect the crude protein digestibility of Tenebrio molitor and Hermetia illucens larvae meals estimated by an in vitro enzymatic method.

  15. Content of antinutrients and in vitro protein digestibility of the African yambean, pigeon and cowpea.

    Science.gov (United States)

    Ene-Obong, H N

    1995-10-01

    The protein, trypsin inhibitor (TI), tannin, phytate, phytic acid phosphorus and in vitro protein digestibility (IVPD) of cultivars of the African yambean (Sphenostylis stenocarpa) - AYB, pigeon pea (Cajanus cajan) - PP and cowpea (Vigna unguiculata) - CP were determined. The protein content of CP (24-28.0%) was higher than those of PP (21-22.5%) and AYB (21-22.5%). The cream and speckled AYB contained more TI (30.9 and 25.3 mg/g) than PP (7.5-14.1 mg/g) and CP (9.8-20.5 mg/g). Apart from the white CP cultivar, they contained more tannin (1.24-1.42 mg/g) than PP (0.14-0.97 mg/g) and AYB (0.71-1.17 mg/g). Phytate was lowest in the AYB (6.30-7.49 mg/g) than PP (8.31-11.31 mg/g) and CP (8.40-9.92 mg/g). Phytic acid contributed 67-74% of the phosphorous in the AYB, 66-75% in PP and 54-59% in CP. The IVPD of the AYB (73.3 + or - 0.7%) was significantly lower (p <0.05) than those of PP (76.34 + or - 0.2%) and CP (77.8 + or - 0.4%). There was a significant negative correlation between TI and IVPD (r = -0.63, p<0.05). There was no significant correlation between IVPD and phytate and tannin contents. There was a positive correlation between protein content and IVPD (r = 0.69**) for the legumes under study. These legumes may pose no serious problems to populations consuming them especially when heat treatment is applied before consumption.

  16. Short- and long-term outcomes of the absence of protein during bovine blastocyst formation in vitro.

    Science.gov (United States)

    Murillo-Ríos, A; Maillo, V; Muñoz, M; Gutiérrez-Adán, A; Carrocera, S; Martín-González, D; Fernandez-Buznego, A; Gómez, E

    2017-06-01

    In cattle, individual in vitro embryo culture after Day 6 benefits development, allowing non-invasive analysis of culture medium. However, undefined supplements in culture reduce analytical reliability. In this study we assayed the short- and long-term performance of embryos after bovine serum albumin removal over a 24-h period in individual culture. The absence of protein decreased embryo development and cell counts in the inner cell mass without affecting blastocyst sex ratio. However, the absence of protein produced embryos with an improved tendency to survive vitrification after 24h in culture (P=0.07). After transfer to recipients, birth rates of embryos that had been cultured with protein tended to decrease (Pembryos cultured with or without protein. In fresh blastocysts cultured without protein, gene expression analysis showed higher abundance (Pembryos cultured without protein (P<0.01). The absence of protein results in fewer blastocysts but improved long-term viability after cryopreservation.

  17. In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160

    OpenAIRE

    Wagner, Stefan; Chiosea, Simion; Ivshina, Maria; Nickerson, Jeffrey A.

    2004-01-01

    We present a new in vitro system for characterizing the binding and mobility of enhanced green fluorescent protein (EGFP)–labeled nuclear proteins by fluorescence recovery after photobleaching in digitonin-permeabilized cells. This assay reveals that SRm160, a splicing coactivator and component of the exon junction complex (EJC) involved in RNA export, has an adenosine triphosphate (ATP)–dependent mobility. Endogenous SRm160, lacking the EGFP moiety, could also be released from sites at splic...

  18. A rare myelin protein zero (MPZ variant alters enhancer activity in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Anthony Antonellis

    2010-12-01

    Full Text Available Myelin protein zero (MPZ is a critical structural component of myelin in the peripheral nervous system. The MPZ gene is regulated, in part, by the transcription factors SOX10 and EGR2. Mutations in MPZ, SOX10, and EGR2 have been implicated in demyelinating peripheral neuropathies, suggesting that components of this transcriptional network are candidates for harboring disease-causing mutations (or otherwise functional variants that affect MPZ expression.We utilized a combination of multi-species sequence comparisons, transcription factor-binding site predictions, targeted human DNA re-sequencing, and in vitro and in vivo enhancer assays to study human non-coding MPZ variants.Our efforts revealed a variant within the first intron of MPZ that resides within a previously described SOX10 binding site is associated with decreased enhancer activity, and alters binding of nuclear proteins. Additionally, the genomic segment harboring this variant directs tissue-relevant reporter gene expression in zebrafish.This is the first reported MPZ variant within a cis-acting transcriptional regulatory element. While we were unable to implicate this variant in disease onset, our data suggests that similar non-coding sequences should be screened for mutations in patients with neurological disease. Furthermore, our multi-faceted approach for examining the functional significance of non-coding variants can be readily generalized to study other loci important for myelin structure and function.

  19. In vitro Activity and Function of B7-H4-Ig Fusion Protein

    DEFF Research Database (Denmark)

    Rasmussen, Susanne B; Kosicki, Michael; Svendsen, Signe Goul

    2013-01-01

    was assayed for its effects in allogeneic mixed lymphocyte culture (MLC) systems. Soluble B7- H4-Ig had no significant effect in the MLC, but with a tendency to promote allogeneic response. Immobilized, but not soluble B7-H4-Ig inhibited plastic bound anti-CD3 mediated activation of T cells. This inhibition......B7-H4 has been shown to inhibit T cell proliferation, cytokine production and cell cycle in vitro. B7-H4 deficient mice develop exacerbated disease in the mouse models of Rheumatoid Arthritis (RA), Type 1 Diabetes (T1D) and Experimental Autoimmune Encephalomyelitis (EAE). On the other hand, B7-H4......-Ig fusion protein has been documented to assuage the symptoms in mouse models of RA, T1D, and multiple sclerosis in vivo. In the present study, B7-H4-Ig bound to the majority of human peripheral blood monocytes and NK cells, but not to either normal or activated T cells. B7-H4-Ig fusion protein...

  20. Assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay

    Science.gov (United States)

    Johnson, Christopher J.; Carlson, Christina M.; Morawski, Aaron R.; Manthei, Alyson; Cashman, Neil R.

    2015-01-01

    Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitroprion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host’s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host’s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

  1. Ligation of major histocompatibility complex class I antigens (MHC-I) prevents apoptosis induced by Fas or SAPK/JNK activation in T-lymphoma cells

    DEFF Research Database (Denmark)

    Lamberth, K; Claesson, M H

    2001-01-01

    Early apoptosis in Jurkat T-lymphoma cells was induced by agonistic anti-Fas Ab or by anisomycin which activates the stress kinases SAPK/JNK. Apoptosis was inhibited by ligation of major histocompatibility complex class I antigens (MHC-I). MHC-I ligation induced upregulation of the anti......-apoptotic Bcl-2 protein and stabilized the mitochondrial membrane potential (Deltapsim). MHC-I ligation also prevented downregulation of Bcl-2 and destabilization of Deltapsim induced by anti-Fas Ab treatment or anisomycin exposure. Studies on three different Jurkat cell mutants deficient for src p56(lck), ZAP...

  2. Chicken hepatic metabolism in vitro. Protein and energy relations in the broiler chicken--VI. Effect of dietary protein and energy restrictions on in vitro carbohydrate and lipid metabolism and metabolic hormone profiles.

    Science.gov (United States)

    Rosebrough, R W; McMurtry, J P; Mitchell, A D; Steele, N C

    1988-01-01

    1. Ross male broiler chicks growing from 14 to 28 days of age were fed 14 and 20% protein diets (4 kcal day-1/body wt0.66) or 20 and 28% protein diets (2.8 kcal day-1/body wt0.66) in a 2 x 2 factorial arrangement to determine the effects of protein and energy intakes on in vitro lipogenesis (IVL) and net glucose production (NGP). Plasma concentrations of insulin, glucagon, thyroid hormones (T3 and T4) and somatomedin-C (Sm-C) were estimated by radioimmunoassay. 2. There was a significant (P less than 0.05) decrease in IVL in the chicks given the higher daily protein intake. 3. The higher protein intake increased (P less than 0.05) NGP while the lower energy intake decreased (P less than 0.05) NGP. 4. Insulin, both thyroid hormones and Sm-C were affected by dietary energy and protein intakes.

  3. A Low Protein Diet Alters Bone Material Level Properties and the Response to In Vitro Repeated Mechanical Loading

    Directory of Open Access Journals (Sweden)

    Victor Dubois-Ferrière

    2014-01-01

    Full Text Available Low protein intake is associated with an alteration of bone microstructure and material level properties. However, it remains unknown whether these alterations of bone tissue could influence the response to repeated mechanical loading. The authors investigated the in vitro effect of repeated loading on bone strength in humeri collected from 20 6-month-old female rats pair-fed with a control (15% casein or an isocaloric low protein (2.5% casein diet for 10 weeks. Bone specimens were cyclically loaded in three-point bending under load control for 2000 cycles. Humeri were then monotonically loaded to failure. The load-displacement curve of the in vitro cyclically loaded humerus was compared to the contralateral noncyclically loaded humerus and the influence of both protein diets. Material level properties were also evaluated through a nanoindentation test. Cyclic loading decreased postyield load and plastic deflection in rats fed a low protein diet, but not in those on a regular diet. Bone material level properties were altered in rats fed a low protein diet. This suggests that bone biomechanical alterations consequent to cyclic loading are more likely to occur in rats fed a low protein diet than in control animals subjected to the same in vitro cyclic loading regimen.

  4. In-Vitro dual inhibition of protein glycation, and oxidation by some Arabian plants.

    Science.gov (United States)

    Siddiqui, Maqsood A; Rasheed, Saima; Saquib, Quaiser; Al-Khedhairy, Abdulaziz A; Al-Said, Mansour S; Musarrat, Javed; Choudhary, Muhammad Iqbal

    2016-08-05

    Diabetes mellitus is a metabolic disorder of epidemic proportion, projected to become the major cause of morbidity and mortality in the world in future. Despite extensive research in understanding this disease at molecular level, and the discovery of new drugs, diabetes and its complications remain largely untreated. Many of the late diabetic complications are associated with the glycation of proteins in the body. Natural flora has long been a rich source for therapeutic agents, especially against diabetes. The present study deals with the anti-glycation properties of some medicinally important plants of Arabian region. Twenty-six medicinal plants, commonly found in different regions of Arabian Peninsula, were evaluated for their protein anti-glycation activity by using BSA-MG glycation assay in-vitro. The extracts were incubated with BSA and MG at 37 °C for 9 days, each sample was then examined for the presence of fluorescence (λex 330 nm, and λem 420 nm), which represent the extent of protein glycation. Antioxidant activity was evaluated by using 1,1-diphenyl- 2-picrylhydrazyl (DPPH), iron chelation, and superoxide radical scavenging asaays. The data revealed that out of 26 medicinal plants, five plants viz. Sida cordifolia, Plumbago zeylanica, Tribulus terrestris, Glycyrrhiza glabra, and Rosa indica were active against the in-vitro protein glycation with IC50 values between 0.408- 1.690 mg/mL. Among the active plants, Glycyrrhiza glabra L. was found to be the most potent (IC50 = 0.408 ± 0.027 mg/mL), followed by Rosa indica (IC50 = 0.596 ± 0.0179 mg/mL), and Sida cordifolia L. (IC50 = 0.63 ± 0.009 mg/mL). The antioxidant potential of these plant extracts were also determined by using DPPH (2,2-diphenyl-1-picrylhydrazyl), iron chelation, and superoxide anion radical scavenging assays. Among five plants, Sida cordifolia exhibited a potent anti-oxidant activity in both DPPH and superoxide anion radical scavenging assays (IC50

  5. In vitro and in vivo protein phosphorylation in Avena sativa L. coleoptiles: effects of Ca2+, calmodulin antagonists, and auxin

    Science.gov (United States)

    Veluthambi, K.; Poovaiah, B. W.

    1986-01-01

    In vitro and in vivo protein phosphorylations in oat (Avena sativa L.) coleoptile segments were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by two-dimensional gel electrophoresis. In vitro phosphorylation of several polypeptides was distinctly promoted at 1 to 15 micromolar free Ca2+ concentrations. Ca2(+)-stimulated phosphorylation was markedly reduced by trifluoperazine, chlorpromazine, and naphthalene sulfonamide (W7). Two polypeptides were phosphorylated both under in vitro and in vivo conditions, but the patterns of phosphorylation of several other polypeptides were different under the two conditions indicating that the in vivo phosphorylation pattern of proteins is not truly reflected by in vitro phosphorylation studies. Trifluoperazine, W7, or ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) + calcium ionophore A23187 treatments resulted in reduced levels of in vivo protein phosphorylation of both control and auxin-treated coleoptile segments. Analysis by two-dimensional electrophoresis following in vivo phosphorylation revealed auxin-dependent changes of certain polypeptides. A general inhibition of phosphorylation by calmodulin antagonists suggested that both control and auxin-treated coleoptiles exhibited Ca2+, and calmodulin-dependent protein phosphorylation in vivo.

  6. Secondary Structure and Subunit Composition of Soy Protein In Vitro Digested by Pepsin and Its Relation with Digestibility

    Directory of Open Access Journals (Sweden)

    Yong Yang

    2016-01-01

    Full Text Available In the present study, in vitro digestibility and structure of soybean protein isolates (SPIs prepared from five soybean varieties were investigated in simulated gastric fluid (SGF, using FT-IR microspectroscopy and SDS-PAGE. The result indicated that β-conformations were prone to be hydrolyzed by pepsin preferentially and transformed to unordered structure during in vitro digestion, followed by the digestion of α-helix and unordered structure. A negative linear correlation coefficient was found between the β-conformation contents of five SPIs and their in vitro digestibility values. The intensities of the protein bands corresponding to 7S and 11S fractions were decreased and many peptide bands appeared at 11~15 kDa during enzymatic hydrolysis. β-conglycinin was poorly hydrolyzed with pepsin, especially the β-7S subunit. On the other hand, basic polypeptides of glycinin degraded slower than acidic polypeptides and represented a large proportion of the residual protein after digestion. 11S-A3 of all SPIs disappeared after 1 h digestion. Moreover, a significant negative linear correlation coefficient (r=-0.89 was found between the β-7S contents of five SPIs and their in vitro digestibility values. These results are useful for further studies of the functional properties and bioactive properties of these varieties and laid theoretical foundations for the development of the specific functional soy protein isolate.

  7. Expression of the Immediate-Early Gene-Encoded Protein Egr-1 ("zif268") during in Vitro Classical Conditioning

    Science.gov (United States)

    Mokin, Maxim; Keifer, Joyce

    2005-01-01

    Expression of the immediate-early genes (IEGs) has been shown to be induced by activity-dependent synaptic plasticity or behavioral training and is thought to play an important role in long-term memory. In the present study, we examined the induction and expression of the IEG-encoded protein Egr-1 during an in vitro neural correlate of eyeblink…

  8. Wnt-induced secreted protein 1/CCN4 in liver fibrosis both in vitro and in vivo.

    Science.gov (United States)

    Jian, Yi-Cheng; Wang, Jin-Jun; Dong, Shuang; Hu, Jun-Wei; Hu, Li-Juan; Yang, Gen-Mei; Zheng, Ya-Xin; Xiong, Wu-Jun

    2014-01-01

    Wnt-induced secreted protein-1 (WISP-1/CCN4) is a member of the CCN family growth factors, and its role in liver fibrosis is largely unknown. For in vitro, hepatic stellate cells (HSCs) were isolated from Sprague-Dawley rats. Expression of WISP-1 during progressive activation of cultured rat HSCs was analyzed by qRT-PCR. The effects of TNF-a and TGF-beta1 on WISP-1 expression were analyzed in stellate cell lines HSC-T6 and LX-2. The effect of exogenous WISP-1 protein on LX-2 proliferation was examined. For in vivo, expressions of WISP-1 in fibrotic liver of a carbon tetrachloride (CCl4)-induced fibrosis rat model were analyzed by qRT-PCR and immunohistochemistry. In vitro, WISP-1 was increasingly expressed during progressive activation of cultured rat HSCs. WISP-1 was significantly induced in HSC-T6 cells by TNF-a and in LX-2 cells by TGF-beta1. Recombinant WISP-1 protein promoted LX-2 proliferation in a dose-dependent manner. In vivo, both mRNA and protein expression levels of WISP-1 were increased significantly in experimental hepatic fibrosis model. Our results showed the upregulation of WISP-1 in both in vitro and in vivo liver fibrosis models, and WISP-1 stimulated the proliferation of HSCs in vitro. These results may be helpful to elucidate the exact role of WISP-1 in liver fibrogenesis.

  9. "In Vitro" Synthesis and Activity of Reporter Proteins in an "Escherichia coli" S30 Extract System: An Undergraduate Experiment

    Science.gov (United States)

    Higgins, Pamela J.

    2005-01-01

    This undergraduate laboratory experiment integrates multiple techniques ("in vitro" synthesis, enzyme assays, Western blotting) to determine the production and detection sensitivity of two common reporter proteins (beta-galactosidase and luciferase) within an "Escherichia coli" S30 transcription/translation extract. Comparison of the data suggests…

  10. In vitro rumen fermentation and effect of protein fractions of canola meals on methane production

    Directory of Open Access Journals (Sweden)

    Jacinto Efrén Ramirez-Bribiesca

    Full Text Available ABSTRACT: Canola provides oil for human consumption and a by-product from the extraction of its oil canola meal (CM, is used as a good quality protein and lipid supplement for ruminants. The aim of this study was to determine the chemical composition and fermentation profile associated with the potential for methane production of three types of CM: solvent-extracted CM of Brassica napus (CMBN, B. juncea (CMBJ and cold press-extracted CM from B. napus (CPCM used to feed ruminants. The protein content of canola meal ranged from 36 to 41 %. The acid and neutral detergent insoluble in nitrogen ranged from 1 to 2 % and 4 to 5 %, respectively. The lipid content of canola meal samples ranged from 2 % (CMBN, CMBJ to 14 % (CPCM. In vitro, dry matter disappearance of CMBJ was higher than that of other types of CM at 4 h, 24 h and 48 h. Gas production from CMBJ was greater than that of CPCM and CMBN at 24 h and 48 h of incubation. CPCM produced lower methane than CMBJ at 48 h of incubation. CPCM produced a higher (p < 0.05 molar proportion of propionate from 12 h to 48 h of incubation. In conclusion, this study found that CPCM could be a good source of protein (mainly PB2 and lipids in the feed of ruminants; it will decrease methane production and enhance propionate production. There was correlation between less methane production, and the content of lipids and PB2 in the three types of CM studied.

  11. Nuclear actin and protein 4.1: Essential interactions during nuclear assembly in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Krauss, Sharon Wald; Chen, Cynthia; Penman, Sheldon; Heald, Rebecca

    2003-06-11

    Structural protein 4.1, which has crucial interactions within the spectin-actin lattice of the human red cell membrane skeleton, also is widely distributed at diverse intracellular sites in nucleated cells. We previously showed that 4.1 is essential for assembly of functional nuclei in vitro and that the capacity of 4.1 to bind actin is required. Here we report that 4.1 and actin colocalize in mammalian cell nuclei using fluorescence microscopy and, by higher resolution cell whole mount electron microscopy, are associated on nuclear filaments. We also devised a cell-free assay using Xenopus egg extract containing fluorescent actin to follow actin during nuclear assembly. By directly imaging actin under non-perturbing conditions, the total nuclear actin population is retained and is visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As the nuclear lamina assembled, but preceding DNA synthesis, a discrete actin network formed throughout the nucleus. Protein 4.1 epitopes also were detected when actin began to accumulate in nuclei, producing a diffuse coincident pattern. As nuclei matured, actin was detected both coincident with and also independent of 4.1 epitopes. To test whether acquisition of nuclear actin is required for nuclear assembly, the actin inhibitor latrunculin A was added to Xenopus egg extracts during nuclear assembly. Latrunculin A strongly perturbed nuclear assembly and produced distorted nuclear structures containing neither actin nor protein 4.1. Our results suggest that actin as well as 4.1 is necessary for nuclear assembly and that 4.1-actin interactions may be critical.

  12. Laparoscopic-assisted percutaneous internal ring ligation in children

    African Journals Online (AJOL)

    Laparoscopic-assisted percutaneous internal ring ligation in children. Mostafa A. Hamad a,b. , Mohamed A. Osman a,b and Mahmoud Abdelhamed a,b. Aim To evaluate the feasibility and safety of laparoscopic ligation of internal ring in congenital inguinal hernia in children. Patients and methods Laparoscopic ...

  13. Esophageal variceal ligation in the secondary prevention of variceal ...

    African Journals Online (AJOL)

    Introduction: Long-term outcome of patients after band ligation have been poorly defined. Therefore, we conducted a long-term follow-up study to delineate the outcome of ligation in patients with portal hypertension in the Hassan II university hospital, Fes, Morocco. Methods: Over 118 months patients treated by endoscopic ...

  14. Tubal ligation and risk of ovarian cancer subtypes

    DEFF Research Database (Denmark)

    Sieh, Weiva; Salvador, Shannon; McGuire, Valerie

    2013-01-01

    Tubal ligation is a protective factor for ovarian cancer, but it is unknown whether this protection extends to all invasive histological subtypes or borderline tumors. We undertook an international collaborative study to examine the association between tubal ligation and ovarian cancer subtypes....

  15. Laparoscopic-assisted percutaneous internal ring ligation in children

    African Journals Online (AJOL)

    Aim To evaluate the feasibility and safety of laparoscopic ligation of internal ring in congenital inguinal hernia in children. Patients and methods Laparoscopic percutaneous ligation of internal inguinal ring has been performed on 97 children with 133 hernias. The age ranged between 6 months and 11.5 years.

  16. Oxo-ester mediated native chemical ligation: concept and applications.

    Science.gov (United States)

    Wan, Qian; Chen, Jin; Yuan, Yu; Danishefsky, Samuel J

    2008-11-26

    A direct oxo-ester peptide ligation method has been developed. Through the use of an activated C-terminal para nitrophenyl ester (1), it is possible to achieve direct cysteine ligations (1 + 2 --> 4). Peptide substrates incorporating bulky C-terminal amino acids (1) can be accommodated with high reaction efficiency.

  17. Crystal structure and redox properties of a novel cyanobacterial heme protein with a His/Cys heme axial ligation and a Per-Arnt-Sim (PAS)-like domain.

    Science.gov (United States)

    Motomura, Taiki; Suga, Michihiro; Hienerwadel, Rainer; Nakagawa, Akiko; Lai, Thanh-Lan; Nitschke, Wolfgang; Kuma, Takahiro; Sugiura, Miwa; Boussac, Alain; Shen, Jian-Ren

    2017-06-09

    Photosystem II catalyzes light-induced water oxidation leading to the generation of dioxygen indispensable for sustaining aerobic life on Earth. The Photosystem II reaction center is composed of D1 and D2 proteins encoded by psbA and psbD genes, respectively. In cyanobacteria, different psbA genes are present in the genome. The thermophilic cyanobacterium Thermosynechococcus elongatus contains three psbA genes: psbA1, psbA2, and psbA3, and a new c-type heme protein, Tll0287, was found to be expressed in a strain expressing the psbA2 gene only, but the structure and function of Tll0287 are unknown. Here we solved the crystal structure of Tll0287 at a 2.0 Å resolution. The overall structure of Tll0287 was found to be similar to some kinases and sensor proteins with a Per-Arnt-Sim-like domain rather than to other c-type cytochromes. The fifth and sixth axial ligands for the heme were Cys and His, instead of the His/Met or His/His ligand pairs observed for most of the c-type hemes. The redox potential, E½, of Tll0287 was -255 ± 20 mV versus normal hydrogen electrode at pH values above 7.5. Below this pH value, the E½ increased by ≈57 mV/pH unit at 15 °C, suggesting the involvement of a protonatable group with a pKred = 7.2 ± 0.3. Possible functions of Tll0287 as a redox sensor under microaerobic conditions or a cytochrome subunit of an H2S-oxidizing system are discussed in view of the environmental conditions in which psbA2 is expressed, as well as phylogenetic analysis, structural, and sequence homologies. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Axial Ligation and Redox Changes at the Cobalt Ion in Cobalamin Bound to Corrinoid Iron-Sulfur Protein (CoFeSP or in Solution Characterized by XAS and DFT.

    Directory of Open Access Journals (Sweden)

    Peer Schrapers

    Full Text Available A cobalamin (Cbl cofactor in corrinoid iron-sulfur protein (CoFeSP is the primary methyl group donor and acceptor in biological carbon oxide conversion along the reductive acetyl-CoA pathway. Changes of the axial coordination of the cobalt ion within the corrin macrocycle upon redox transitions in aqua-, methyl-, and cyano-Cbl bound to CoFeSP or in solution were studied using X-ray absorption spectroscopy (XAS at the Co K-edge in combination with density functional theory (DFT calculations, supported by metal content and cobalt redox level quantification with further spectroscopic methods. Calculation of the highly variable pre-edge X-ray absorption features due to core-to-valence (ctv electronic transitions, XANES shape analysis, and cobalt-ligand bond lengths determination from EXAFS has yielded models for the molecular and electronic structures of the cobalt sites. This suggested the absence of a ligand at cobalt in CoFeSP in α-position where the dimethylbenzimidazole (dmb base of the cofactor is bound in Cbl in solution. As main species, (dmbCoIII(OH2, (dmbCoII(OH2, and (dmbCoIII(CH3 sites for solution Cbl and CoIII(OH2, CoII(OH2, and CoIII(CH3 sites in CoFeSP-Cbl were identified. Our data support binding of a serine residue from the reductive-activator protein (RACo of CoFeSP to the cobalt ion in the CoFeSP-RACo protein complex that stabilizes Co(II. The absence of an α-ligand at cobalt not only tunes the redox potential of the cobalamin cofactor into the physiological range, but is also important for CoFeSP reactivation.

  19. Appearance of dentin gamma-carboxyglutamic acid-containing proteins in developing rat molars in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Finkelman, R.D.; Butler, W.T.

    1985-07-01

    An in vitro model of mineralization was devised in order to study the developmental appearance of dentin gamma-carboxyglutamic acid-containing proteins (DGPs) in relation to the onset of mineralization. Maxillary third molars from 11-day-old rats were cultured with or without fetal calf serum (FCS) as modified from Navia et al. Molars were incubated without radiolabel, or with either /sup 45/CaCl/sub 2/ (5 microCi/ml) for 24 hr at various stages of a ten-day culture period or (/sup 3/H)-leucine (10 microCi/ml) for 24 hr at the eighth day of culture. Molars were lyophilized and extracted with 10% formic acid overnight at 4 degrees C. DGPs in extracts were detected by immunologic and chromatographic techniques; DGPs in molar sections were detected by immunolocalization using indirect immunofluorescence. Molar development was evaluated histologically using the Von Kossa staining technique. Molars cultured with FCS showed histologic evidence for mineralized dentin and enamel and a significant increase in /sup 45/Ca uptake after the sixth day in vitro. Eleven-day-old molars in vivo and molars cultured without FCS showed no evidence of the presence of mineralized tissues. (/sup 3/H)-Leucine-labeled DGPs were isolated and identified by affinity and reversed-phase high-performance liquid chromatography and by gel electrophoresis from both mineralized and unmineralized molars. DGP antigens were localized immunohistochemically using rabbit anti-rat antibodies raised against a highly purified DGP preparation. In the unmineralized molar, antigenicity was seen in odontoblasts but not in predentin matrix, preodontoblasts, or in any other cell type. Antigens in the mineralized molar were localized to odontoblasts and dentin.

  20. Phosphorylation of yeast phosphatidylserine synthase in vivo and in vitro by cyclic AMP-dependent protein kinase.

    OpenAIRE

    Kinney, A J; Carman, G M

    1988-01-01

    Evidence is presented that demonstrates that phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) from Saccharomyces cerevisiae is phosphorylated in vivo and in vitro by cAMP-dependent protein kinase. Phosphatidylserine synthase activity in cell extracts was reduced in the bcy1 mutant (which has high cAMP-dependent protein kinase activity) and elevated in the cyr1 mutant (which has low cAMP-dependent protein kinase activity) when compared with wild-ty...

  1. In vivo versus in vitro protein abundance analysis of Shigella dysenteriae type 1 reveals changes in the expression of proteins involved in virulence, stress and energy metabolism

    Directory of Open Access Journals (Sweden)

    Donohue-Rolfe Arthur

    2011-06-01

    Full Text Available Abstract Background Shigella dysenteriae serotype 1 (SD1 causes the most severe form of epidemic bacillary dysentery. Quantitative proteome profiling of Shigella dysenteriae serotype 1 (SD1 in vitro (derived from LB cell cultures and in vivo (derived from gnotobiotic piglets was performed by 2D-LC-MS/MS and APEX, a label-free computationally modified spectral counting methodology. Results Overall, 1761 proteins were quantitated at a 5% FDR (false discovery rate, including 1480 and 1505 from in vitro and in vivo samples, respectively. Identification of 350 cytoplasmic membrane and outer membrane (OM proteins (38% of in silico predicted SD1 membrane proteome contributed to the most extensive survey of the Shigella membrane proteome reported so far. Differential protein abundance analysis using statistical tests revealed that SD1 cells switched to an anaerobic energy metabolism under in vivo conditions, resulting in an increase in fermentative, propanoate, butanoate and nitrate metabolism. Abundance increases of transcription activators FNR and Nar supported the notion of a switch from aerobic to anaerobic respiration in the host gut environment. High in vivo abundances of proteins involved in acid resistance (GadB, AdiA and mixed acid fermentation (PflA/PflB indicated bacterial survival responses to acid stress, while increased abundance of oxidative stress proteins (YfiD/YfiF/SodB implied that defense mechanisms against oxygen radicals were mobilized. Proteins involved in peptidoglycan turnover (MurB were increased, while β-barrel OM proteins (OmpA, OM lipoproteins (NlpD, chaperones involved in OM protein folding pathways (YraP, NlpB and lipopolysaccharide biosynthesis (Imp were decreased, suggesting unexpected modulations of the outer membrane/peptidoglycan layers in vivo. Several virulence proteins of the Mxi-Spa type III secretion system and invasion plasmid antigens (Ipa proteins required for invasion of colonic epithelial cells, and release

  2. In vivo versus in vitro protein abundance analysis of Shigella dysenteriae type 1 reveals changes in the expression of proteins involved in virulence, stress and energy metabolism.

    Science.gov (United States)

    Kuntumalla, Srilatha; Zhang, Quanshun; Braisted, John C; Fleischmann, Robert D; Peterson, Scott N; Donohue-Rolfe, Arthur; Tzipori, Saul; Pieper, Rembert

    2011-06-24

    Shigella dysenteriae serotype 1 (SD1) causes the most severe form of epidemic bacillary dysentery. Quantitative proteome profiling of Shigella dysenteriae serotype 1 (SD1) in vitro (derived from LB cell cultures) and in vivo (derived from gnotobiotic piglets) was performed by 2D-LC-MS/MS and APEX, a label-free computationally modified spectral counting methodology. Overall, 1761 proteins were quantitated at a 5% FDR (false discovery rate), including 1480 and 1505 from in vitro and in vivo samples, respectively. Identification of 350 cytoplasmic membrane and outer membrane (OM) proteins (38% of in silico predicted SD1 membrane proteome) contributed to the most extensive survey of the Shigella membrane proteome reported so far. Differential protein abundance analysis using statistical tests revealed that SD1 cells switched to an anaerobic energy metabolism under in vivo conditions, resulting in an increase in fermentative, propanoate, butanoate and nitrate metabolism. Abundance increases of transcription activators FNR and Nar supported the notion of a switch from aerobic to anaerobic respiration in the host gut environment. High in vivo abundances of proteins involved in acid resistance (GadB, AdiA) and mixed acid fermentation (PflA/PflB) indicated bacterial survival responses to acid stress, while increased abundance of oxidative stress proteins (YfiD/YfiF/SodB) implied that defense mechanisms against oxygen radicals were mobilized. Proteins involved in peptidoglycan turnover (MurB) were increased, while β-barrel OM proteins (OmpA), OM lipoproteins (NlpD), chaperones involved in OM protein folding pathways (YraP, NlpB) and lipopolysaccharide biosynthesis (Imp) were decreased, suggesting unexpected modulations of the outer membrane/peptidoglycan layers in vivo. Several virulence proteins of the Mxi-Spa type III secretion system and invasion plasmid antigens (Ipa proteins) required for invasion of colonic epithelial cells, and release of bacteria into the host cell

  3. Membrane integration of in vitro-translated gap junctional proteins: co- and post-translational mechanisms.

    Science.gov (United States)

    Zhang, J T; Chen, M; Foote, C I; Nicholson, B J

    1996-03-01

    Connexins (Cx) are protein components of gap junction channels that permit the passage of small molecules between neighboring cells. cDNAs of a large family of connexins have been isolated and sequenced. A gap junction channel consists of two connexons, one from each cell in contact, composed of six connexin subunits. It has been suggested by Musil and coworkers that the oligomerization of formation of a connexon occurs at the level of the trans-Golgi network. In the present study, we initiated an analysis of the early stages of protein synthesis and membrane insertion of Cx32 and Cx26, two connexins that we have demonstrated are co-expressed in the same junctions in hepatocytes. Using an in vitro transcription and a coupled cell-free translation and translocation system, we observed that both Cx32 and Cx26 could insert into microsome membranes co-translationally, producing a topological structure indistinguishable from that in isolated gap junctions. To our surprise, Cx26 could also insert into membranes post-translationally with a native orientation. This post-translational membrane insertion process is dependent on nucleotides but not their hydrolysis. Cx32, on the other hand, could not insert into membranes post-translationally. These disparate properties of Cx32 and Cx26 are not due to the significant difference in the lengths of their C-terminal domains, but rather to their internal amino acid sequences. These observations raise the possibility that there may be another pathway for Cx26 to insert into membranes in cells and this feature may be important for the regulation of its functions. These findings may also lead us to a new approach to reconstitution without detergent extraction.

  4. In vitro antioxidant activity of rice protein affected by alkaline degree and gastrointestinal protease digestion.

    Science.gov (United States)

    Liu, Ye; Wang, Zhengxuan; Li, Hui; Liang, Mingcai; Yang, Lin

    2016-12-01

    To elucidate whether and how alkali treatment, which is a common process for rice protein (RP) extraction, affects antioxidant activity of RP, the different degree of alkali (from 0.1% to 0.4% of NaOH) was used to extract RP (RP-1, RP-2, RP-3, RP-4). The antioxidant capacities of scavenging free radicals [2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid] diammonium salt, ABTS; 1,1-diphenyl-2-picrylhydrazyl, DPPH), chelating metals (iron, copper) and reducing power investigated in the hydrolysates of RPs (RP-1, RP-2, RP-3, RP-4) during in vitro pepsin-pancreatin digestion were effectively affected by alkali treatment. The present study demonstrated that the weakest antioxidant responses to ABTS radical-scavenging activity, DPPH radical-scavenging activity, iron chelating activity, copper chelating activity and reducing power were produced by RP-4 extracted by the highest alkali proportion (0.4% NaOH). The present study indicates that antioxidant capacity of RP could be more readily depressed by strict alkali degree and affected by gastrointestinal proteases. Results suggest that alkali extraction is a vital process to regulate the antioxidant activity of RP through modifying the compositions of amino acids, which are dependent on alkali magnitude. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  5. Rapid tyrosine phosphorylation of Lck following ligation of the tumor-associated cell surface molecule A6H

    DEFF Research Database (Denmark)

    Labuda, T; Gerwien, J; Ødum, Niels

    1999-01-01

    . In addition, A6H ligation induced an up-regulation of CD3-mediated phosphorylation of the 23 kDa high mol. wt form of TCR zeta and the zeta-associated protein, ZAP-70. Co-precipitation of Lck and ZAP-70 was only seen in T cells activated by combined A6H and anti-CD3 stimulation. In contrast, another Src...... family PTK, Fyn, was not affected by A6H ligation. In conclusion, we now demonstrate, for the first time, that A6H ligation triggers Lck phosphorylation, and that cross-talk between A6H and the TCR-CD3 complex involves Lck, ZAP-70 and the slow migrating isoform of TCR zeta. These results further suggests...

  6. In vitro detection of fatty liver infiltration in protein-depleted rats using proton nuclear magnetic resonance.

    Science.gov (United States)

    Jacobs, D O; Trerotola, S O; Settle, R G; Rolandelli, R H; Wolf, G L; Rombeau, J L

    1985-07-01

    To determine if NMR techniques might be used to detect hepatic steatosis secondary to protein malnutrition, the T1 and T2 relaxation times of liver tissue from rats subjected to long-term protein malnutrition were measured in vitro. The liver tissue from rats fed a protein-deficient rat chow (PD) for 37 days (N = 9) was characterized by increased proportion of fat (P less than 0.001) but decreased water and nitrogen contents (P less than 0.001) relative to controls (N = 9). Mean T1 times were significantly shorter and T2 times significantly longer in liver tissue from protein-depleted animals (P less than 0.001). There was no overlap of T2 times between the protein-depleted and control animals. The consistent changes in T2 that occur with fatty infiltration of the liver should be detectable by current NMR imagers.

  7. The importance of selecting a proper biological milieu for protein corona analysis in vitro: Human plasma versus human serum.

    Science.gov (United States)

    Mirshafiee, Vahid; Kim, Raehyun; Mahmoudi, Morteza; Kraft, Mary L

    2016-06-01

    Nanoparticle (NP) exposure to biological fluids in the body results in protein binding to the NP surface, which forms a protein coating that is called the "protein corona". To simplify studies of protein-NP interactions and protein corona formation, NPs are incubated with biological solutions, such as human serum or human plasma, and the effects of this exposure are characterized in vitro. Yet, how NP exposure to these two different biological milieus affects protein corona composition and cell response has not been investigated. Here, we explore the differences between the protein coronas that form when NPs are incubated in human serum versus human plasma. NP characterization indicated that NPs that were exposed to human plasma had higher amounts of proteins bound to their surfaces, and were slightly larger in size than those exposed to human serum. In addition, significant differences in corona composition were also detected with gel electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry, where a higher fraction of coagulation proteins and complement factors were found on the plasma-exposed NPs. Flow cytometry and confocal microscopy showed that the uptake of plasma-exposed NPs was higher than that of serum-exposed NPs by RAW 264.7 macrophage immune cells, but not by NIH 3T3 fibroblast cells. This difference is likely due to the elevated amounts of opsonins, such as fibrinogen, on the surfaces of the NPs exposed to plasma, but not serum, because these components trigger NP internalization by immune cells. As the human plasma better mimics the composition of the in vivo environment, namely blood, in vitro protein corona studies should employ human plasma, and not human serum, so the biological phenomena that is observed is more similar to that occurring in vivo. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Theory and simulation of explicit solvent effects on protein folding in vitro and in vivo

    Science.gov (United States)

    England, Jeremy L.

    The aim of this work is to develop theoretical tools for understanding what happens to water that is confined in amphipathic cavities, and for testing the consequences of this understanding for protein folding in vitro and in vivo. We begin in the first chapter with a brief review of the theoretical and simulation literature on the hydrophobic effect and the aqueous solvation of charged species that also puts forward a simple theoretical framework within which various solvation phenomena reported in past studies may be unified. Subsequently, in the second chapter we also review past computational and theoretical work on the specific question of how chaperonin complexes assist the folding of their substrates. With the context set, we turn in Chapter 3 to the case of an open system with water trapped between hydrophobic plates that experiences a uniform electric field normal to and between the plates. Classic bulk theory of electrostriction in polarizable fluids tells us that the electric field should cause an increase in local water density as it rises, yet some simulations have suggested the opposite. We present a mean-field Potts model we have developed to explain this discrepancy, and show how such a simple, coarse-grained lattice description can capture the fundamental consequences of the fact that external electric fields can frustrate the hydrogen bond network in confined water. Chapter 4 continues to pursue the issue of solvent evacuation between hydrophobic plates, but focuses on the impact of chemical denaturants on hydrophobic effects using molecular dynamics simulations of hydrophobic dewetting. We find that while urea and guanidinium have similar qualitative effects at the bulk level, they seem to differ in the microscopic mechanism by which they denature proteins, although both inhibit the onset of dewetting. Lastly, Chapters 5 and 6 examine the potential importance of solvent-mediated forces to protein folding in vivo. Chapter 5 develops a Landau

  9. In vitro anti-inflammatory and antioxidant activities and protein quality of high hydrostatic pressure treated squids (Todarodes pacificus).

    Science.gov (United States)

    Zhang, Yifeng; Dai, Bona; Deng, Yun; Zhao, Yanyun

    2016-07-15

    This study investigated the in vitro anti-inflammatory and antioxidant properties, protein quality, and other related characteristics obtained by the single-cycle and two-cycle high hydrostatic pressure (HHP at 200, 400 and 600 MPa) treatment of squids (Todarodes pacificus). The soluble protein nitrogen content and in vitro protein digestibility increased significantly (p<0.05) after all HHP treatments, and the two-cycle 600 MPa HHP treatments yielded the highest values, 7.59% and 84.42%, respectively. The estimated protein efficiency ratios, and antioxidant and anti-inflammatory properties of squids significantly increased by all HHP treatments. (1)H nuclear magnetic resonance (NMR) showed that the main spectral changes associated to the anti-inflammatory properties of proteins following HHP treatment were in the range of 3.00-3.19 and 3.60-3.79 ppm. This indicates that the HHP treatments modified the protein and functional properties of squids and gave the relevant chemical shifts in NMR signals, either migrated or disappeared. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Effects of heat treatment on protein feeds evaluated in vitro by the method of estimating utilisable crude protein at the duodenum.

    Science.gov (United States)

    Vaga, M; Hetta, M; Huhtanen, P

    2017-12-01

    The effect of heat treatment on the protein value of field beans, lupins and peas was studied using an in vitro method. Protein feeds were subjected to heat treatment for 30, 60 and 90 min in forced air oven at 120, 140 and 160 °C and in autoclave at 105, 120 and 135 °C. The heat-treated protein feeds were incubated in buffered rumen fluid together with grass silage and barley in complete isonitrogenous diets. The gas production (GP) was recorded continuously, and ammonia-N (NH3 -N) concentrations were determined during the in vitro incubation at 8, 24 and 48 h and used to determine the utilisable crude protein (uCP) at the duodenum at 16 h of incubation (uCP16 ). Heat treatments decreased the concentration of soluble crude protein and increased neutral detergent insoluble CP (NDICP) in all protein feeds compared to untreated. Inclusion of protein feeds to basal diet showed no increase in the uCP16 in untreated field bean diet and only a small numerical increase in the uCP16 concentrations from 160 g/kg dry matter (DM) to 166 and 172 g/kg DM in untreated lupine and pea diets, respectively, indicating high degradability of untreated feeds. Increasing the time and temperature of the heat treatment linearly increased the uCP16 concentrations in field bean and pea diets, but not in lupin diets. Autoclave treatment was more effective in decreasing uCP16 than oven treatment despite the lower temperatures used. However, the combination of highest temperatures and treatment time in autoclave increased acid detergent insoluble CP (ADICP) concentrations in protein feeds, indicating protein damage and decreased intestinal digestibility. Determining in vitro uCP and ADICP shows to be a promising method for evaluating protein value in heat-treated animal feeds. Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH.

  11. Formatting and ligating biopolymers using adjustable nanoconfinement

    Science.gov (United States)

    Berard, Daniel J.; Shayegan, Marjan; Michaud, Francois; Henkin, Gil; Scott, Shane; Leslie, Sabrina

    2016-07-01

    Sensitive visualization and conformational control of long, delicate biopolymers present critical challenges to emerging biotechnologies and biophysical studies. Next-generation nanofluidic manipulation platforms strive to maintain the structural integrity of genomic DNA prior to analysis but can face challenges in device clogging, molecular breakage, and single-label detection. We address these challenges by integrating the Convex Lens-induced Confinement (CLiC) technique with a suite of nanotopographies embedded within thin-glass nanofluidic chambers. We gently load DNA polymers into open-face nanogrooves in linear, concentric circular, and ring array formats and perform imaging with single-fluorophore sensitivity. We use ring-shaped nanogrooves to access and visualize confinement-enhanced self-ligation of long DNA polymers. We use concentric circular nanogrooves to enable hour-long observations of polymers at constant confinement in a geometry which eliminates the confinement gradient which causes drift and can alter molecular conformations and interactions. Taken together, this work opens doors to myriad biophysical studies and biotechnologies which operate on the nanoscale.

  12. Synthesis and Labeling of RNA In Vitro

    Science.gov (United States)

    Huang, Chao; Yu, Yi-Tao

    2013-01-01

    This unit discusses several methods for generating large amounts of uniformly labeled, end-labeled, and site-specifically labeled RNAs in vitro. The methods involve a number of experimental procedures, including RNA transcription, 5′ dephosphorylation and rephosphorylation, 3′ terminal nucleotide addition (via ligation), site-specific RNase H cleavage directed by 2′-O-methyl RNA-DNA chimeras, and 2-piece splint ligation. The applications of these RNA radiolabeling approaches are also discussed. PMID:23547015

  13. In vitro and in vivo interactions of selected nanoparticles with rodent serum proteins and their consequences in biokinetics

    Directory of Open Access Journals (Sweden)

    Wolfgang G. Kreyling

    2014-10-01

    Full Text Available When particles incorporated within a mammalian organism come into contact with body fluids they will bind to soluble proteins or those within cellular membranes forming what is called a protein corona. This binding process is very complex and highly dynamic due to the plethora of proteins with different affinities and fractions in different body fluids and the large variation of compounds and structures of the particle surface. Interestingly, in the case of nanoparticles (NP this protein corona is well suited to provide a guiding vehicle of translocation within body fluids and across membranes. This NP translocation may subsequently lead to accumulation in various organs and tissues and their respective cell types that are not expected to accumulate such tiny foreign bodies. Because of this unprecedented NP accumulation, potentially adverse biological responses in tissues and cells cannot be neglected a priori but require thorough investigations. Therefore, we studied the interactions and protein binding kinetics of blood serum proteins with a number of engineered NP as a function of their physicochemical properties. Here we show by in vitro incubation tests that the binding capacity of different engineered NP (polystyrene, elemental carbon for selected serum proteins depends strongly on the NP size and the properties of engineered surface modifications. In the following attempt, we studied systematically the effect of the size (5, 15, 80 nm of gold spheres (AuNP, surface-modified with the same ionic ligand; as well as 5 nm AuNP with five different surface modifications on the binding to serum proteins by using proteomics analyses. We found that the binding of numerous serum proteins depended strongly on the physicochemical properties of the AuNP. These in vitro results helped us substantially in the interpretation of our numerous in vivo biokinetics studies performed in rodents using the same NP. These had shown that not only the

  14. In vitro pepsin resistance of proteins: Effect of non-reduced SDS-PAGE analysis on fragment observation

    Directory of Open Access Journals (Sweden)

    Juliette Pickles

    2014-01-01

    Full Text Available The introduction of novel proteins to food products carries with it the need to assess the potential allergenicity of such materials. Resistance to in vitro pepsin digestion is one parameter considered in such a risk assessment using a weight of evidence approach; however, the methodology used to investigate this has not been fully standardised. In vitro pepsin resistance assays typically involve SDS-PAGE performed under reducing conditions, with limited published data available on the assay using non-reducing conditions despite the need to consider non-reducing analysis techniques having been highlighted by regulatory bodies such as the European Food Safety Authority (EFSA. The purpose of the work reported here was to investigate the applicability of (and additional insight provided by non-reducing analyses, by digesting a set of proteins using a ring-trial validated method, with analysis by SDS-PAGE under both reducing and non-reducing conditions. In silico prediction of digest fragments was also investigated. Significant differences were observed between results under reduced and non-reduced conditions for proteins in which disulphide bonds have a major role in protein structure, such as ribulose 1,5-diphosphate carboxylase (RUBISCO and bovine serum albumin. For proteins with no or few disulphide bonds, no significant differences were seen in the results. Structural information such as disulphide bond numbers and positions should be considered during experimental design, as a non-reduced approach may be appropriate for some proteins. The in silico approach was a useful tool to suggest potential digest fragments, however the predictions were not always confirmed in vitro and should be considered a guide only.

  15. Single daily dosing of antibiotics: importance of in vitro killing rate, serum half-life, and protein binding.

    OpenAIRE

    Potel, G.; Chau, N P; Pangon, B; Fantin, B; Vallois, J M; Faurisson, F; Carbon, C.

    1991-01-01

    The relative importance of pharmacokinetic and pharmacodynamic parameters for the feasibility of a single daily dose (SDD) of antibiotics remains to be established. Therefore, we studied the relationship between in vitro bacteriological parameters (MIC, MBC, and killing rate [KR], defined as the reduction in the inoculum within 3 h), pharmacokinetic parameters (t1/2 and protein binding [PB], and in vivo antibacterial effect of a single antibiotic dose in an experimental rabbit model of Escher...

  16. Distinct roles for nucleic acid in vitro assembly of purified Mason-Pfizer monkey virus CANC proteins

    Czech Academy of Sciences Publication Activity Database

    Ulbrich, P.; Haubová, Š.; Nermut, M. V.; Hunter, E.; Rumlová, Michaela; Ruml, Tomáš

    2006-01-01

    Roč. 80, č. 14 (2006), s. 7089-7099 ISSN 0022-538X R&D Projects: GA AV ČR(CZ) IAA4055304; GA ČR(CZ) GP203/03/P094; GA MŠk 1M0520 Institutional research plan: CEZ:AV0Z40550506 Keywords : M-PMV * CANC proteins * HIV -1 * in vitro assembly Subject RIV: CE - Biochemistry Impact factor: 5.341, year: 2006

  17. Cross-Linked Collagen Triple Helices by Oxime Ligation.

    Science.gov (United States)

    Hentzen, Nina B; Smeenk, Linde E J; Witek, Jagna; Riniker, Sereina; Wennemers, Helma

    2017-09-13

    Covalent cross-links are crucial for the folding and stability of triple-helical collagen, the most abundant protein in nature. Cross-linking is also an attractive strategy for the development of synthetic collagen-based biocompatible materials. Nature uses interchain disulfide bridges to stabilize collagen trimers. However, their implementation into synthetic collagen is difficult and requires the replacement of the canonical amino acids (4R)-hydroxyproline and proline by cysteine or homocysteine, which reduces the preorganization and thereby stability of collagen triple helices. We therefore explored alternative covalent cross-links that allow for connecting triple-helical collagen via proline residues. Here, we present collagen model peptides that are cross-linked by oxime bonds between 4-aminooxyproline (Aop) and 4-oxoacetamidoproline placed in coplanar Xaa and Yaa positions of neighboring strands. The covalently connected strands folded into hyperstable collagen triple helices (Tm ≈ 80 °C). The design of the cross-links was guided by an analysis of the conformational properties of Aop, studies on the stability and functionalization of Aop-containing collagen triple helices, and molecular dynamics simulations. The studies also show that the aminooxy group exerts a stereoelectronic effect comparable to fluorine and introduce oxime ligation as a tool for the functionalization of synthetic collagen.

  18. THE EFFECT OF NITROGEN FERTILIZATION OF MAIZE ON PROTEIN CONCENTRATION AND IN VITRO FEMENTABILITY OF GRAIN

    Directory of Open Access Journals (Sweden)

    D BABNIK

    2002-12-01

    Full Text Available The effect of nitrogen fertilization of maize on fermentability of maize grain in the rumen was studied by means of in vitro method based on the measurement of gas produced during the incubation of samples with rumen liquor. Gas production was recorded continuously up to 72 h incubation time and cumulative gas production was described by the Gompertz equation Y=A*exp(-exp(-d*(t-tm. Seven treatments, one of them unfertilized and others fertilized with 100 to 250 kg N ha–1, were compared. Grain yield and concentration of crude protein (CP in grain increased linearly with nitrogen fertilization. Grain yield increased for 25 kg dry matter (DM ha–1 and CP concentration for 0.13 g kg–1 DM per each additional kg of N. Concentration of CP in grain, which varied from 83 to 115 g kg–1 DM, was closely related to the dynamics of gas production. The maximal gas production rate (MPR was negatively related to CP concentration in the grain (R2 = 0.53; p < 0.10 and the time of MPR (tm was positively related to the amount of added N (R2 = 0.74; p < 0.05 and concentration of CP in the grain (R2 = 0.88; p < 0.01. It is likely that intensive N fertilization of maize limits ruminal digestion of maize starch. Due to the shift of starch digestion from the rumen to lower gastrointestinal tract better utilization of energy can be expected in maize grain of extensively fertilized maize than in the grain of maize, in which supply of N is sub-optimal.

  19. Recombinant Probiotic Expressing Listeria Adhesion Protein Attenuates Listeria monocytogenes Virulence In Vitro

    Science.gov (United States)

    Koo, Ok Kyung; Amalaradjou, Mary Anne Roshni; Bhunia, Arun K.

    2012-01-01

    Background Listeria monocytogenes, an intracellular foodborne pathogen, infects immunocompromised hosts. The primary route of transmission is through contaminated food. In the gastrointestinal tract, it traverses the epithelial barrier through intracellular or paracellular routes. Strategies to prevent L. monocytogenes entry can potentially minimize infection in high-risk populations. Listeria adhesion protein (LAP) aids L. monocytogenes in crossing epithelial barriers via the paracellular route. The use of recombinant probiotic bacteria expressing LAP would aid targeted clearance of Listeria from the gut and protect high-risk populations from infection. Methodology/Principal Findings The objective was to investigate the ability of probiotic bacteria or LAP-expressing recombinant probiotic Lactobacillus paracasei (LbpLAP) to prevent L. monocytogenes adhesion, invasion, and transwell-based transepithelial translocation in a Caco-2 cell culture model. Several wild type probiotic bacteria showed strong adhesion to Caco-2 cells but none effectively prevented L. monocytogenes infection. Pre-exposure to LbpLAP for 1, 4, 15, or 24 h significantly (Pmonocytogenes in Caco-2 cells, whereas pre-exposure to parental Lb. paracasei had no significant effect. Similarly, LbpLAP pre-exposure reduced L. monocytogenes translocation by as much as 46% after 24 h. LbpLAP also prevented L. monocytogenes-mediated cell damage and compromise of tight junction integrity. Furthermore, LbpLAP cells reduced L. monocytogenes-mediated cell cytotoxicity by 99.8% after 1 h and 79% after 24 h. Conclusions/Significance Wild type probiotic bacteria were unable to prevent L. monocytogenes infection in vitro. In contrast, LbpLAP blocked adhesion, invasion, and translocation of L. monocytogenes by interacting with host cell receptor Hsp60, thereby protecting cells from infection. These data show promise for the use of recombinant probiotics in preventing L. monocytogenes infection in high

  20. Esophageal variceal ligation for hemostasis of acute variceal bleeding

    African Journals Online (AJOL)

    Esophageal variceal ligation for hemostasis of acute variceal bleeding: efficacy and safety. Mounia Lahbabi, Mounia Elyousfi, Nouredine Aqodad, Mohammed Elabkari, Ihssane Mellouki, Sidi Adil Ibrahimi, Dafr Allah Benaja ...

  1. Ligation bias in Illumina next-generation DNA libraries

    DEFF Research Database (Denmark)

    Seguin-Orlando, Andaine; Schubert, Mikkel; Clary, Joel

    2013-01-01

    -products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate...... that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting...... of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected...

  2. Elastic band ligation of hemorrhoids using flexible gastroscope

    Directory of Open Access Journals (Sweden)

    Hadi Abd Zaid Al-Khattabi

    2017-03-01

    Conclusion High success rate, cost effectiveness and the simplicity of rubber band ligation as an outpatient procedure promote its use as the frst line of treatment for frst, second and early third degree hemorrhoids.

  3. Effect of processing on the in vitro and in vivo protein quality of red and green lentils (Lens culinaris).

    Science.gov (United States)

    Nosworthy, Matthew G; Medina, Gerardo; Franczyk, Adam J; Neufeld, Jason; Appah, Paulyn; Utioh, Alphonsus; Frohlich, Peter; House, James D

    2018-02-01

    In order to determine the effect of extrusion, baking and cooking on the protein quality of red and green lentils, a rodent bioassay was conducted and compared to an in vitro method of protein quality determination. On average, the Protein Digestibility-Corrected Amino Acid Score of red lentils (55.0) was higher than that of green lentils (50.8). Extruded lentil flour had higher scores (63.01 red, 57.09 green) than either cooked (57.40 red, 52.92 green) or baked (53.84 red, 47.14 green) flours. The average Digestible Indispensable Amino Acid Score of red lentils (0.54) was higher than green lentils (0.49). The Protein Efficiency Ratio of the extruded lentil flours (1.30 red, 1.34 green) was higher than that of the baked flour (0.98 red, 1.09 green). A correlation was found between in vivo and in vitro methods of determining protein digestibility (R2=0.8934). This work could influence selection of processing method during product development. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. In Vitro and In Vivo Investigation of the Potential of Amorphous Microporous Silica as a Protein Delivery Vehicle

    Directory of Open Access Journals (Sweden)

    Amol Chaudhari

    2013-01-01

    Full Text Available Delivering growth factors (GFs at bone/implant interface needs to be optimized to achieve faster osseointegration. Amorphous microporous silica (AMS has a potential to be used as a carrier and delivery platform for GFs. In this work, adsorption (loading and release (delivery mechanism of a model protein, bovine serum albumin (BSA, from AMS was investigated in vitro as well as in vivo. In general, strong BSA adsorption to AMS was observed. The interaction was stronger at lower pH owing to favorable electrostatic interaction. In vitro evaluation of BSA release revealed a peculiar release profile, involving a burst release followed by a 6 h period without appreciable BSA release and a further slower release later. Experimental data supporting this observation are discussed. Apart from understanding protein/biomaterial (BSA/AMS interaction, determination of in vivo protein release is an essential aspect of the evaluation of a protein delivery system. In this regard micropositron emission tomography (μ-PET was used in an exploratory experiment to determine in vivo BSA release profile from AMS. Results suggest stronger in vivo retention of BSA when adsorbed on AMS. This study highlights the possible use of AMS as a controlled protein delivery platform which may facilitate osseointegration.

  5. Discrimination of in vitro and in vivo digestion products of meat proteins from pork, beef, chicken, and fish

    Science.gov (United States)

    Wen, Siying; Zhou, Guanghong; Song, Shangxin; Xu, Xinglian; Voglmeir, Josef; Liu, Li; Zhao, Fan; Li, Mengjie; Li, Li; Yu, Xiaobo; Bai, Yun

    2015-01-01

    In vitro digestion products of proteins were compared among beef, pork, chicken, and fish. Gastric and jejunal contents from the rats fed these meat proteins were also compared. Cooked pork, beef, chicken, and fish were homogenized and incubated with pepsin alone or followed by trypsin. The digestion products with molecular weights of less than 3000 Da were identified with MALDI‐TOF‐MS and nano‐LC‐MS/MS. Gastric and jejunal contents obtained from the rats fed the four meat proteins for 7 days were also analyzed. After pepsin digestion, pork, and beef samples had a greater number of fragments in similarity than chicken and fish samples, but the in vitro digestibility was the greatest (p pork and the smallest for beef samples. After trypsin digestion, the species differences were less pronounced (p > 0.05). A total of 822 and 659 peptides were identified from the in vitro and in vivo digestion products, respectively. Our results could interpret for the differences in physiological functions after the ingestion of different species of meat. PMID:26227428

  6. Nitrate simultaneously enhances lipid and protein accumulation in developing yellow lupin cotyledons cultured in vitro, but not under field conditions.

    Science.gov (United States)

    Borek, Sławomir; Kalemba, Ewa Marzena; Pukacka, Stanisława; Pietrowska-Borek, Małgorzata; Stawiński, Stanisław; Ratajczak, Lech

    2017-09-01

    The research was conducted on yellow lupin (Lupinus luteus L.) mature seeds, developing cotyledons, developing pods, and seedlings. The main storage compound in yellow lupin seeds is protein, whose content may reach up to 45%. Oil content in seeds of yellow lupin is about 6%. In such protein-storing seeds there is a strong negative relationship between accumulation of storage lipid and protein. An increase in protein content causes a decrease in lipid level, and vice versa. However, simultaneous increase in lipid and protein content is possible in developing lupin cotyledons (the main storage organs of lupin seeds) cultured in vitro. Such an effect was obtained by feeding the cotyledons with nitrate (35mM). The same positive relationship in storage lipid and protein accumulation was also obtained in developing lupin pods fed with nitrate (35mM), detached from the mother plant, and maintained under quasi in vitro conditions. Fertilization of lupin plants with nitrate under field conditions (40 or 80kgNha-1 applied before sowing, at the nodulation stage or at the flowering and pod formation stage) did not cause significant changes in lipid and protein contents in mature seeds. Experiments performed on lupin seedlings cultivated hydroponically showed that nitrate added to the medium was accumulated mainly in roots, and at a remarkably lower level in shoots. We hypothesize that the lack of stimulatory effect of nitrate on storage lipid and protein accumulation in seeds under field conditions is due to inefficient transport of nitrate from the root to developing pods in lupin plants. This causes that the level of nitrate inside the developing lupin seeds is not elevated under field conditions. Copyright © 2017 Elsevier GmbH. All rights reserved.

  7. [Rubber band ligation in treatment of hemorrhoids: our experience].

    Science.gov (United States)

    Gaj, F; Biviano, I; Sportelli, G; Candeloro, L

    2015-01-01

    Hemorrhoids are a very common condition. The treatment depends upon persistence and severity of symptoms. For hemorrhoids of II and III grade the rubber band ligation may be therapeutic. Our aim is to report the outcomes of rubber band ligation of hemorrhoids, with a follow up of 6 months. A total of 50 patients underwent rubber band ligation for symptomatic hemorrhoids (grade II and III) without prolapse, between June 2012 and June 2014. All patients underwent plug test to rule out presence of rectal mucosal prolapse and were classified according to PATE classification (1). Each hemorrhoid was ligated with one rubber band through a ligator. All patients were evaluated immediately at the end of the procedure, after ten days and six months after the treatment. Patient's demographic and operative data were collected and analyzed. The mean patients age was 47.6±12.3 years (range 24-72). All procedures were performed without complications. Before rubber band ligation, 42 patients had rectal bleeding, 38 had perineal discomfort and 27 had itching. Ten days after the treatment, 12 patients presented self-limited rectal bleeding, but 10 of these had more hemorrhoids and underwent a second rubber band ligation. No patients complained perineal discomfort, and 8 patients had itching; 78% and 16% of patients respectively, experienced feeling of a foreign body inside the canal anal and anal pain. After 6 months, only 13 patients were occasionally symptomatic: 4 patients had rectal bleeding, 2 had perineal discomfort and 4 had itching. Three more patients presented both perineal discomfort and hitching. None had the feeling of a foreign body in anal canal or anal pain. Rubber band ligation is an efficacious, cost-effective and simple treatment for the second and third degree hemorrhoids without rectal mucosal prolapsed. In our hands, no severe complications developed and minor complications could be handled with ease.

  8. Identification of candidate protein markers of Bovine Parainfluenza Virus Type 3 infection using an in vitro model.

    Science.gov (United States)

    Gray, Darren W; Welsh, Michael D; Doherty, Simon; Mooney, Mark H

    2017-05-01

    Bovine Parainfluenza Virus Type 3 (BPI3V) infections are often asymptomatic, causing respiratory tissue damage and immunosuppression, predisposing animals to severe bacterial pneumonia, the leading cause of Bovine Respiratory Disease (BRD) mortality. As with many pathogens, routine BPI3V serology does not indicate the presence of damaged respiratory tissue or active infection. In vitro proteomic marker screening using disease relevant cell models could help identify markers of infection and tissue damage that are also detectable during in vivo infections. This study utilised a proteomic approach to investigate in vitro cellular responses during BPI3V infection to enhance the current understanding of intracellular host-virus interactions and identify putative markers of in vivo infection. Through 2D gel electrophoresis proteomic analysis, BPI3V Phosphoprotein P and host T-complex Protein 1 subunit theta were found to be accumulated at the latter stages of infection within bovine fibroblasts. These proteins were subsequently detected using targeted multiple reaction monitoring (MRM) mass spectrometry in the plasma of animals challenged with BPI3V, with differential protein level profiles observed dependant on animal vaccination status. Potential mechanisms by which BPI3V overcomes host cellular immune response mechanisms allowing for replication and production of viral proteins were also revealed. Assessment of circulating protein marker levels identified through an in vitro approach as described may enable more effective diagnosis of active viral infection and diseased or damaged respiratory tissue in animals and allow for more effective utilisation of preventative therapeutic interventions prior to bacterial disease onset and significantly aid the management and control of BRD. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Development of a vivo rabbit ligated intestinal Loop Model for HCMV infection.

    Science.gov (United States)

    Tang, Jin; Wu, Qiaoxing; Tang, Xinming; Shi, Ruihan; Suo, Jingxia; Huang, Guangping; An, Junqing; Wang, Jingyuan; Yang, Jinling; Hao, Wenzhuo; She, Ruiping; Suo, Xun

    2016-01-01

    Human Cytomegalovirus (HCMV) infections can be found throughout the body, especially in epithelial tissue. Animal model was established by inoculation of HCMV (strain AD-169) or coinoculation with Hepatitis E virus (HEV) into the ligated sacculus rotundus and vermiform appendix in living rabbits. The specimens were collected from animals sacrificed 1 and a half hours after infection. The virus was found to be capable of reproducing in these specimens through RT-PCR and Western-blot. Severe inflammation damage was found in HCMV-infected tissue. The viral protein could be detected in high amounts in the mucosal epithelium and lamina propria by immunohistochemistry and immunofluorescense. Moreover, there are strong positive signals in lymphocytes, macrophages, and lymphoid follicles. Quantitative statistics indicate that lymphocytes among epithlium cells increased significantly in viral infection groups. The results showed that HCMV or HEV + HCMV can efficiently infect in rabbits by vivo ligated intestine loop inoculation. The present study successfully developed an infective model in vivo rabbit ligated intestinal Loop for HCMV pathogenesis study. This rabbit model can be helpful for understanding modulation of the gut immune system with HCMV infection.

  10. Lipid Raft is required for PSGL-1 ligation induced HL-60 cell adhesion on ICAM-1.

    Directory of Open Access Journals (Sweden)

    Tingshuang Xu

    Full Text Available P-selectin glycoprotein ligand-1 (PSGL-1 and integrins are adhesion molecules that play critical roles in host defense and innate immunity. PSGL-1 mediates leukocyte rolling and primes leukocytes for integrin-mediated adhesion. However, the mechanism that PSGL-1 as a rolling receptor in regulating integrin activation has not been well characterized. Here, we investigate the function of lipid raft in regulating PSGL-1 induced β2 integrin-mediated HL-60 cells adhesion. PSGL-1 ligation with antibody enhances the β2 integrin activation and β2 integrin-dependent adhesion to ICAM-1. Importantly, with the treatment of methyl-β-cyclodextrin (MβCD, we confirm the role of lipid raft in regulating the activation of β2 integrin. Furthermore, we find that the protein level of PSGL-1 decreased in raft fractions in MβCD treated cells. PSGL-1 ligation induces the recruitment of spleen tyrosine kinase (Syk, a tyrosine kinase and Vav1 (the pivotal downstream effector of Syk signaling pathway involved in cytoskeleton regulation to lipid raft. Inhibition of Syk activity with pharmacologic inhibitor strongly reduces HL-60 cells adhesion, implicating Syk is crucial for PSGL-1 mediated β2 integrin activation. Taken together, we report that ligation of PSGL-1 on HL-60 cells activates β2 integrin, for which lipid raft integrity and Syk activation are responsible. These findings have shed new light on the mechanisms that connect leukocyte initial rolling with subsequent adhesion.

  11. Multiplex, quantitative, ligation-dependent probe amplification for determination of allergens in food.

    Science.gov (United States)

    Mustorp, Stina L; Drømtorp, Signe M; Holck, Askild L

    2011-05-25

    Legislation requires labeling of foods containing allergenic ingredients. Here, we present a robust 10-plex quantitative and sensitive ligation-dependent probe amplification method, the allergen-multiplex ligation-dependent probe amplification (MLPA) method, for specific detection of eight allergens: sesame, soy, hazelnut, peanut, lupine, gluten, mustard, and celery. Ligated probes were amplified by polymerase chain reaction (PCR), and amplicons were detected using capillary electrophoresis. Quantitative results were obtained by comparing signals with an internal positive control. The limit of detection varied from approximately 5 to 400 gene copies, depending on the allergen. The method was tested using different foods spiked with mustard, celery, soy, or lupine flour in the 1-0.001% range. Depending on the allergen, sensitivities were similar or better than those obtained with qPCR. The allergen-MLPA method is modular and can be adapted by adding probe pairs for other allergens. The DNA-based allergen-MLPA method will constitute a complementary method to the traditional protein-based methods.

  12. Axially Ligated Phthalocyanine Conductors with Magnetic Moments

    Directory of Open Access Journals (Sweden)

    Tamotsu Inabe

    2017-04-01

    Full Text Available This mini-review describes electrical conductivity, magnetic properties, and magnetotransport properties of one-dimensional partially oxidized salts composed of axially ligated phthalocyanines, TPP[M(Pc(CN2]2 (TPP = tetraphenylphosphonium, Pc = phthalocyaninato, with M of Fe (d5, S = 1/2 and Cr (d3, S = 3/2. These salts are isomorphous, and π–π interactions in the crystal, that becomes the origin of the charge carriers, are nearly the same. Both the Fe and Cr salts show carrier localization and charge disproportionation which is enhanced by the interaction between local magnetic moments and conduction π-electrons (π–d interaction. However, the magnetic properties are slightly different between them. M = Fe has been found to show unique anisotropic magnetic properties and antiferromagnetic short-range magnetic order between the d-spins. On the other hand, for M = Cr, its magnetic moment is isotropic. Temperature dependence of the magnetic susceptibility shows typical Curie–Weiss behavior with negative Weiss temperature, but the exchange interaction is complicated. Both M = Fe and M = Cr show large negative magnetoresistance, reflecting the difference in the anisotropy. The magnetoresistance ratio (MR is larger in the Fe system than in the Cr system in the low magnetic field range, but MR in the Cr system exceeds that in the Fe system when the magnetic field becomes higher than 15 T. We discuss the mechanism of the giant negative magnetoresistance with reference to the d–d, π–d, and π–π interactions.

  13. Endoscopic variceal ligation-induced ulcer bleeding

    Science.gov (United States)

    Cho, Eunae; Jun, Chung Hwan; Cho, Sung Bum; Park, Chang Hwan; Kim, Hyun Soo; Choi, Sung Kyu; Rew, Jong Sun

    2017-01-01

    Abstract This study was aimed to determine the risk factors of endoscopic variceal ligation-(EVL) induced ulcer bleeding. The prevalence of EVL-induced ulcer bleeding is reported to be 3.6%. However, there are only limited reports of this serious complication, and the risk factors and the treatment methods are not well established. A total of 430 patients who had undergone EVL in Chonnam National University Hospital from January 2014 to October 2016 were studied. EVL was performed for prophylaxis or acute hemorrhage. The patients were classified into 2 groups: a bleeding group (n = 33) and a non-bleeding group (n = 397). The patients who had endoscopically confirmed EVL-induced ulcer bleeding were included in the bleeding group. EVL-induced ulcer bleeding occurred in 7.7% (n = 33) of the patients. In a multivariate analysis, model for end-stage liver disease (MELD) score >10 (odds ratio [OR]: 3.42, 95% confidence interval [CI]: 1.10–10.64), concomitant GV F3 (OR: 14.1, 95% CI: 2.84–71.43), and detachment of o-ring bands on follow-up endoscopy (OR: 8.06, 95% CI: 2.55–25.64) were independent predictive factors of EVL-induced ulcer bleeding. Various endoscopic modalities were attempted for hemostasis (EVL in 8 cases [24.2%], endoscopic variceal obturation [EVO] with cyanoacrylate in 6 cases [18.2%], argon plasma coagulation [APC] in 1 case (3%), Sengstaken–Blakemore (SB) tube in 3 cases [9.1%]), and proton pump inhibitor therapy only in 15 cases (45.5%). MELD score >10, concomitant GV F3, and detachment of o-ring bands on follow-up endoscopy are risk factors for EVL-induced ulcer bleeding. PMID:28614248

  14. Histamine receptors expressed in circulating progenitor cells have reciprocal actions in ligation-induced arteriosclerosis.

    Science.gov (United States)

    Yamada, Sohsuke; Wang, Ke-Yong; Tanimoto, Akihide; Guo, Xin; Nabeshima, Atsunori; Watanabe, Takeshi; Sasaguri, Yasuyuki

    2013-09-01

    Histamine is synthesized as a low-molecular-weight amine from L-histidine by histidine decarboxylase (HDC). Recently, we demonstrated that carotid artery-ligated HDC gene-deficient mice (HDC(-/-)) showed less neointimal formation than wild-type (WT) mice, indicating that histamine participates in the process of arteriosclerosis. However, little is known about the roles of histamine-specific receptors (HHRs) in arteriosclerosis. To define the roles of HHRs in arteriosclerosis, we investigated intimal remodeling in ligated carotid arteries of HHR-deficient mice (H1R(-/-) or H2R(-/-)). Quantitative analysis showed that H1R(-/-) mice had significantly less arteriosclerogenesis, whereas H2R(-/-) mice had more, as compared with WT mice. Bone marrow transplantation from H1R(-/-) or H2R(-/-) to WT mice confirmed the above observation. Furthermore, the increased expression of monocyte chemoattractant protein (MCP-1), platelet-derived growth factor (PDGF), adhesion molecules and liver X receptor (LXR)-related inflammatory signaling factors, including Toll-like receptor (TLR3), interleukin-1 receptor (IL-1R) and tumor necrosis factor receptor (TNF-R), was consistent with the arteriosclerotic phenotype of H2R(-/-) mice. Peripheral progenitor cells in H2R(-/-) mice accelerate ligation-induced arteriosclerosis through their regulation of MCP-1, PDGF, adhesion molecules and LXR-related inflammatory signaling factors. In contrast, peripheral progenitor cells act to suppress arteriosclerosis in H1R(-/-) mice, indicating that HHRs reciprocally regulate inflammation in the ligation-induced arteriosclerosis. © 2013 The Authors. Pathology International © 2013 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.

  15. A Quantitative Description of the Binding States and In Vitro Function of Antitermination Protein N of Bacteriophage λ

    Science.gov (United States)

    Conant, Clarke R.; Van Gilst, Marc R.; Weitzel, Stephen E.; Rees, William A.; von Hippel, Peter H.

    2008-01-01

    The N protein of bacteriophage λ activates transcription of genes that lie downstream of termination sequences by suppressing transcription termination. N binds to specific (boxB) and non-specific sites on the transcript RNA and contacts RNA polymerase via cis-RNA looping, resulting in “antitermination” of transcription. To find the effect of N–boxB binding on antitermination, we quantitatively relate binding measurements made in isolation to in vitro antitermination activity. We measure binding of N to boxB RNA, non-specific single-stranded RNA, and non-specific double-stranded DNA fluorimetrically, and use an equilibrium model to describe quantitatively the binding of N to nucleic acids of Escherichia coli transcription elongation complexes. We then test the model by comparison with in vitro N antitermination activity measured in reactions containing these same elongation complexes. We find that binding of N protein to the nucleic acid components of transcription elongation complexes can quantitatively predict antitermination activity, suggesting that antitermination in vitro is determined by a nucleic acid binding equilibrium with one molecule of N protein per RNA transcript being sufficient for antitermination. Elongation complexes contain numerous overlapping non-specific RNA and DNA-binding sites for N; the large number of sites compensates for the low N binding affinity, so multiple N proteins are expected to bind to elongation complexes. The occupancy/activity of these proteins is described by a binomial distribution of proteins on transcripts containing multiple non-specific sites. The contribution of specific (boxB) binding to activity also depends on this distribution. Specificity is not measured accurately by measurements made in the presence and in the absence of boxB. We find that antitermination is inhibited by non-productive binding of N to non-specific sites on template DNA, and that NusA protein covers RNA sites on the transcript, limiting

  16. Recombinant GM-CSF/IL-3 fusion protein: its effect on in vitro human megakaryocytopoiesis.

    Science.gov (United States)

    Bruno, E; Briddell, R A; Cooper, R J; Brandt, J E; Hoffman, R

    1992-05-01

    An evaluation of the effectiveness of a genetically engineered recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin 3 (IL-3) fusion protein (FP) as a means of delivering cytokine combinations to megakaryocyte (MK) progenitor cells was performed, utilizing a serum-depleted clonal assay system and a long-term bone marrow culture system. The effects of the FP, alone and in combination with a variety of other cytokines, on the primitive MK progenitor cell, the megakaryocyte burst-forming unit (BFU-MK), and the more differentiated megakaryocyte colony-forming unit (CFU-MK) were assessed. Subpopulations of bone marrow cells (CD34+ DR- for BFU-MK and CD34+ DR+ for CFU-MK) served as sources of these two classes of MK progenitor cells. The FP was equivalent to a combination of optimal concentrations of GM-CSF and IL-3 in promoting both the number and size of BFU-MK-derived colonies. The GM-CSF/IL-3 combination, however, promoted the formation of far greater CFU-MK-derived colonies than did the FP alone. The size of MK colonies formed in the presence of the FP or GM-CSF/IL-3 was similar. The ability of the FP to stimulate BFU-MK- but not CFU-MK-derived colony formation was also further augmented by the addition of interleukin 1 alpha (IL-1 alpha). The addition of c-kit ligand (KL) increased both FP-stimulated CFU-MK- and BFU-MK-derived colony numbers but only BFU-MK-derived colony size. In addition, the FP alone sustained long-term megakaryocytopoiesis in vitro to a level equivalent to that of the GM-CSF/IL-3 combination and was superior in this regard to either GM-CSF or IL-3 alone. These data indicate that FP is capable of supporting various stages of human megakaryocytopoiesis. We conclude that such genetically engineered molecules as the FP may prove to be effective means of pharmacologically delivering the biological effects of specific cytokine combinations.

  17. Trypanothione reductase: a target protein for a combined in vitro and in silico screening approach.

    Science.gov (United States)

    Beig, Mathias; Oellien, Frank; Garoff, Linnéa; Noack, Sandra; Krauth-Siegel, R Luise; Selzer, Paul M

    2015-01-01

    With the goal to identify novel trypanothione reductase (TR) inhibitors, we performed a combination of in vitro and in silico screening approaches. Starting from a highly diverse compound set of 2,816 compounds, 21 novel TR inhibiting compounds could be identified in the initial in vitro screening campaign against T. cruzi TR. All 21 in vitro hits were used in a subsequent similarity search-based in silico screening on a database containing 200,000 physically available compounds. The similarity search resulted in a data set containing 1,204 potential TR inhibitors, which was subjected to a second in vitro screening campaign leading to 61 additional active compounds. This corresponds to an approximately 10-fold enrichment compared to the initial pure in vitro screening. In total, 82 novel TR inhibitors with activities down to the nM range could be identified proving the validity of our combined in vitro/in silico approach. Moreover, the four most active compounds, showing IC50 values of <1 μM, were selected for determining the inhibitor constant. In first on parasites assays, three compounds inhibited the proliferation of bloodstream T. brucei cell line 449 with EC50 values down to 2 μM.

  18. Activation of 5' adenosine monophosphate-activated protein kinase blocks cumulus cell expansion through inhibition of protein synthesis during in vitro maturation in Swine.

    Science.gov (United States)

    Santiquet, Nicolas; Sasseville, Maxime; Laforest, Martin; Guillemette, Christine; Gilchrist, Robert B; Richard, François J

    2014-08-01

    The serine/threonine kinase 5' adenosine monophosphate-activated protein kinase (AMPK), a heterotrimeric protein known as a metabolic switch, is involved in oocyte nuclear maturation in mice, cattle, and swine. The present study analyzed AMPK activation in cumulus cell expansion during in vitro maturation (IVM) of porcine cumulus-oocyte complexes (COC). 5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) is a well-known activator of AMPK. It inhibited oocyte meiotic resumption in COC. Moreover, cumulus cell expansion did not occur in the presence of AICAR, demonstrating its marked impact on cumulus cells. Activation of AMPK was supported by AICAR-mediated phosphorylation of alpha AMPK subunits. Furthermore, the presence of AICAR increased glucose uptake, a classical response to activation of this metabolic switch in response to depleted cellular energy levels. Neither nuclear maturation nor cumulus expansion was reversed by glucosamine, an alternative substrate in hyaluronic acid synthesis, through the hexosamine biosynthetic pathway, which ruled out possible depletion of substrates. Both increased gap junction communication and phosphodiesterase activity in COC are dependent on protein synthesis during the initial hours of IVM; however, both were inhibited in the presence of AICAR, which supports the finding that activation of AMPK by AICAR mediated inhibition of protein synthesis. Moreover, this protein synthesis inhibition was equivalent to that of the well-known protein synthesis inhibitor cycloheximide, as observed on cumulus expansion and protein concentration. Finally, the phosphorylation level of selected kinases was investigated. The pattern of raptor phosphorylation is supportive of activation of AMPK-mediated inhibition of protein synthesis. In conclusion, AICAR-mediated AMPK activation in porcine COC inhibited cumulus cell expansion and protein synthesis. These results bring new considerations to the importance of this kinase in ovarian

  19. The adsorption of α-amylase on barley proteins affects the in vitro digestion of starch in barley flour.

    Science.gov (United States)

    Yu, Wenwen; Zou, Wei; Dhital, Sushil; Wu, Peng; Gidley, Michael J; Fox, Glen P; Gilbert, Robert G

    2018-02-15

    The conversion of barley starch to sugars is a complex enzymic process. Most previous work concerned the biotechnical aspect of in situ barley enzymes. However, the interactions among the macromolecular substrates and their effects on enzymic catalysis has been little examined. Here, we explore the mechanisms whereby interactions of protein and starch in barley flour affect the kinetics of enzymatic hydrolysis of starch in an in vitro system, using digestion rate data and structural analysis by confocal microscopy. The degradation kinetics of both uncooked barley flour and of purified starches are found to be two-step sequential processes. Barley proteins, especially the water-soluble component, are found to retard the digestion of starch degraded by α-amylase: the enzyme binds with water-insoluble protein and with starch granules, leading to reduced starch hydrolysis. These findings are of potential industrial value in both the brewing and food industries. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Different in vitro and in vivo activity of low Mr phosphotyrosine protein phosphatase on epidermal growth factor receptor.

    Science.gov (United States)

    Rigacci, S; Marzocchini, R; Bucciantini, M; Berti, A

    1998-09-29

    Low Mr phosphotyrosine protein phosphatase is a cytosolic enzyme which dephosphorylates platelet-derived growth factor and insulin receptor in vivo, thus reducing cellular mitogenic response to such growth factors. Following cell stimulation with platelet-derived growth factor the phosphatase undergoes a redistribution from the citosol to the Triton X-100-insoluble fraction where its activity upon the growth factor receptor is intense. Previous research uncovered evidence that low Mr phosphotyrosine protein phosphatase dephosphorylates the epidermal growth factor receptor in vitro. Here we demonstrate that in vivo the enzyme is not active on the phosphorylated epidermal growth factor receptor and it does not influence the mitogenic response of cells. Since the enzyme distribution is not affected by epidermal growth factor stimulation, involvement of a recruitment mechanism in the definition of low Mr phosphotyrosine protein phosphatase substrate specificity is hypothesized. Copyright 1998 Academic Press.

  1. Genome wide binding (ChIP-Seq) of murine Bapx1 and Sox9 proteins in vivo and in vitro.

    Science.gov (United States)

    Chatterjee, Sumantra; Kraus, Petra; Sivakamasundari, V; Yap, Sook Peng; Kumar, Vibhor; Prabhakar, Shyam; Lufkin, Thomas

    2016-12-01

    This work pertains to GEO submission GSE36672, in vivo and in vitro genome wide binding (ChIP-Seq) of Bapx1/Nkx3.2 and Sox9 proteins. We have previously shown that data from a genome wide binding assay combined with transcriptional profiling is an insightful means to divulge the mechanisms directing cell type specification and the generation of tissues and subsequent organs [1]. Our earlier work identified the role of the DNA-binding homeodomain containing protein Bapx1/Nkx3.2 in midgestation murine embryos. Microarray analysis of EGFP-tagged cells (both wildtype and null) was integrated using ChIP-Seq analysis of Bapx1/Nkx3.2 and Sox9 DNA-binding proteins in living tissue.

  2. Buffalo Cheese Whey Proteins, Identification of a 24 kDa Protein and Characterization of Their Hydrolysates: In Vitro Gastrointestinal Digestion.

    Science.gov (United States)

    Bassan, Juliana C; Goulart, Antonio J; Nasser, Ana L M; Bezerra, Thaís M S; Garrido, Saulo S; Rustiguel, Cynthia B; Guimarães, Luis H S; Monti, Rubens

    2015-01-01

    Milk whey proteins are well known for their high biological value and versatile functional properties, characteristics that allow its wide use in the food and pharmaceutical industries. In this work, a 24 kDa protein from buffalo cheese whey was analyzed by mass spectrometry and presented homology with Bos taurus beta-lactoglobulin. In addition, the proteins present in buffalo cheese whey were hydrolyzed with pepsin and with different combinations of trypsin, chymotrypsin and carboxypeptidase-A. When the TNBS method was used the obtained hydrolysates presented DH of 55 and 62% for H1 and H2, respectively. Otherwise for the OPA method the DH was 27 and 43% for H1 and H2, respectively. The total antioxidant activities of the H1 and H2 samples with and without previous enzymatic hydrolysis, determined by DPPH using diphenyl-p-picrylhydrazyl radical, was 4.9 and 12 mM of Trolox equivalents (TE) for H2 and H2Dint, respectively. The increased concentrations for H1 and H2 samples were approximately 99% and 75%, respectively. The in vitro gastrointestinal digestion efficiency for the samples that were first hydrolyzed was higher compared with samples not submitted to previous hydrolysis. After in vitro gastrointestinal digestion, several amino acids were released in higher concentrations, and most of which were essential amino acids. These results suggest that buffalo cheese whey is a better source of bioavailable amino acids than bovine cheese whey.

  3. PENGARUH TINGKAT YOGURT DAN WAKTU FERMENTASI TERHADAP KECERNAAN IN VITRO BAHAN KERING, BAHAN ORGANIK, PROTEIN, DAN SERAT KASAR KULIT NANAS FERMENTASI

    Directory of Open Access Journals (Sweden)

    Nurhayati (Nurhayati

    2014-10-01

    Full Text Available Penelitian dilaksanakan untuk melihat pengaruh tingkat yogurt dan waktu fermentasi terhadap kecernaan in vitro bahan kering, bahan organik, protein, dan serat kasar kulit nanas fermentasi. Penelitian dilakukan menggunakan Rancangan Acak Lengkap pola faktorial dengan 2 faktor yaitu tingkat yogurt (0, 3, 6, dan 9 ml/kg dan lama waktu fermentasi (24, 48, dan 72 jam yang diulang sebanyak 5 kali. Bahan yang digunakan adalah kulit nanas, plain yogurt yang mengandung bakteri Lactobacillus delbrueckii subsp. bulgaricus dan Streptococcus salivarius subsp. thermophilus, bahan kimia yang digunakan untuk analisis proksimat protein dan serat kasar kulit nanas fermentasi, larutan saliva buatan McDougall dan cairan rumen. Hasil penelitian menunjukkan perbedaan pengaruh yang nyata (P<0,05 tingkat yogurt terhadap kecernaan in vitro bahan kering, bahan organik, dan protein kasar tetapi tidak berbeda nyata terhadap kecernaan serat kasar. Waktu fermentasi berpengaruh nyata (P<0,05 terhadap kecernaan bahan kering, bahan organik, protein kasar, dan serat kasar secara in vitro. Interaksi tingkat yogurt dengan waktu fermentasi memberikan perbedaan pengaruh yang nyata (P<0,05 terhadap kecernaan bahan kering, bahan organik dan protein kasar tetapi tidak memberikan perbedaan pengaruh nyata terhadap kecernaan serat kasar. Kesimpulan dari penelitian ini bahwa tingkat yogurt 6 ml/kg dan waktu fermentasi 72 jam dapat meningkatkan kecernaan in vitro bahan kering, bahan organik, dan protein kasar serta menurunkan kecernaan in vitro serat kasar kulit nanas fermentasi. (Kata kunci: Fermentasi, Kecernaan in vitro, Kulit nanas, Yogurt

  4. In vitro transcription translation system : a versatile tool in the search for missing proteins

    NARCIS (Netherlands)

    Horvatovich, Péter; Vegvari, Akos; Saul, Justin; Park, Jin G; Qiu, Ji; Syring, Micheal; Pirrotte, Patrick; Petritis, Konstantinos; Tegeler, Tony J; Aziz, Meraj; Fuentes, Manuel; Diez, Paula; Gonzalez-Gonzalez, Maria; Ibarrola, Nieves; Droste, Conrad; De Las Rivas, Javier; Gil, Concha; Clemente, Felipe; Hernáez, María Luisa; Corrales, Fernando Jose; Nilsson, Carol L; Berven, Frode S; Bischoff, Rainer; Fehniger, Thomas E; LaBaer, Joshua; Marko-Varga, György

    2015-01-01

    Approximately 18% of all human genes purported to encode proteins have not been directly evidenced at the protein level, according to the validation criteria established by neXtProt, and are considered as "missing" proteins. One of the goals of the Chromosome-Centric Human Proteome Project (C-HPP)

  5. Effect of dentine matrix proteins on human endometrial adult stem-like cells: in vitro regeneration of odontoblasts cells.

    Science.gov (United States)

    Tabatabaei, Fahimeh S; Ai, Jafar; Jafarzadeh Kashi, Tahereh S; Khazaei, Mozaffar; Kajbafzadeh, Abdol-Mohammad; Ghanbari, Zinat

    2013-07-01

    Human endometrium has enormous regenerative capacity due to the presence of endometrial stem cells. The present study sought to assess the possibility of differentiation of these cells into odontoblast-like cells by in vitro induction. Endometrial stem-like cells were obtained using enzymatic digestion of the biopsy samples of the endometrium after hysterectomy and cultured in Dulbecco's Modified Eagle's Medium (DMEM) which contained dentine non-collagenous proteins (dNCPs). The results were evaluated using the following assays: analysis of morphology, enzymatic assay for measuring alkaline phosphatase activity, Alizarin Red staining to detect in vitro formation of mineralized nodules, immunofluorescence for detection of dentine sialoprotein (DSP) and dentine-matrix protein (DMP1), and Western blotting for DMP1 expression. Following induction, endometrial stem-like cells demonstrated high alkaline phosphatase activity, and expression of DSP and DMP1 confirmed the odontoblast phenotype. DMP1 level increased in samples treated with dNCPs. Study results indicated that odontoblastic differentiation of endometrial stem cells can be induced by extracellular matrix proteins (e.g. dNCPs). The capacity of endometrial stem-like cells to differentiate into odontoblast-like cells under specific conditions gives new insights into the mechanism of odontogenesis and highlights the potential of such approaches for further research on dental tissue regeneration. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Recombinant Expressed Vector pET32a (+ S Constructed by Ligation Independent Cloning

    Directory of Open Access Journals (Sweden)

    Yu Wang

    2014-10-01

    Full Text Available The aim of this work was to develop a new method for constructing vectors, named ligation-independent cloning (LIC method. We constructed the S label expression vector and recombinant pET32a (+ S-phoN2 by LIC. The recombinant proteins were expressed in E. coli at a high level, and then the specificity of the recombinant proteins was identified by western blot. The target band was detected by S monoclonal antibody and Apyrase polyclonal antibodies but not Trx monoclonal antibody and HIS monoclonal antibody. Finally, we obtained protein Apyrase in E. coli (BL21, with a protein-only expression S tag. Collectively, our results demonstrated that LIC is effective for the construction of new vectors and recombinant plasmids. Free from the limitations of restriction enzyme sites and with a higher positive rate, LIC processes should find broad applications in molecular biology research.

  7. In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium.

    Science.gov (United States)

    Bouthier de la Tour, Claire; Mathieu, Martine; Meyer, Laura; Dupaigne, Pauline; Passot, Fanny; Servant, Pascale; Sommer, Suzanne; Le Cam, Eric; Confalonieri, Fabrice

    2017-01-01

    The bacterium Deinococcus radiodurans possesses a set of Deinococcus-specific genes highly induced after DNA damage. Among them, ddrC (dr0003) was recently re-annotated, found to be in the inverse orientation and called A2G07_00380. Here, we report the first in vivo and in vitro characterization of the corrected DdrC protein to better understand its function in irradiated cells. In vivo, the ΔddrC null mutant is sensitive to high doses of UV radiation and the ddrC deletion significantly increases UV-sensitivity of ΔuvrA or ΔuvsE mutant strains. We show that the expression of the DdrC protein is induced after γ-irradiation and is under the control of the regulators, DdrO and IrrE. DdrC is rapidly recruited into the nucleoid of the irradiated cells. In vitro, we show that DdrC is able to bind single- and double-stranded DNA with a preference for the single-stranded DNA but without sequence or shape specificity and protects DNA from various nuclease attacks. DdrC also condenses DNA and promotes circularization of linear DNA. Finally, we show that the purified protein exhibits a DNA strand annealing activity. Altogether, our results suggest that DdrC is a new DNA binding protein with pleiotropic activities. It might maintain the damaged DNA fragments end to end, thus limiting their dispersion and extensive degradation after exposure to ionizing radiation. DdrC might also be an accessory protein that participates in a single strand annealing pathway whose importance in DNA repair becomes apparent when DNA is heavily damaged.

  8. In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium.

    Directory of Open Access Journals (Sweden)

    Claire Bouthier de la Tour

    Full Text Available The bacterium Deinococcus radiodurans possesses a set of Deinococcus-specific genes highly induced after DNA damage. Among them, ddrC (dr0003 was recently re-annotated, found to be in the inverse orientation and called A2G07_00380. Here, we report the first in vivo and in vitro characterization of the corrected DdrC protein to better understand its function in irradiated cells. In vivo, the ΔddrC null mutant is sensitive to high doses of UV radiation and the ddrC deletion significantly increases UV-sensitivity of ΔuvrA or ΔuvsE mutant strains. We show that the expression of the DdrC protein is induced after γ-irradiation and is under the control of the regulators, DdrO and IrrE. DdrC is rapidly recruited into the nucleoid of the irradiated cells. In vitro, we show that DdrC is able to bind single- and double-stranded DNA with a preference for the single-stranded DNA but without sequence or shape specificity and protects DNA from various nuclease attacks. DdrC also condenses DNA and promotes circularization of linear DNA. Finally, we show that the purified protein exhibits a DNA strand annealing activity. Altogether, our results suggest that DdrC is a new DNA binding protein with pleiotropic activities. It might maintain the damaged DNA fragments end to end, thus limiting their dispersion and extensive degradation after exposure to ionizing radiation. DdrC might also be an accessory protein that participates in a single strand annealing pathway whose importance in DNA repair becomes apparent when DNA is heavily damaged.

  9. A comparative study of synthetic and semisynthetic approaches for ligating the epidermal growth factor to a bivalent scaffold.

    Science.gov (United States)

    Gell, Anna Lena; Groysbeck, Nadja; Becker, Christian F W; Conibear, Anne C

    2017-12-01

    A prominent target of monoclonal antibodies as targeted therapies for cancer is the epidermal growth factor receptor, which is overexpressed on the surface of various cancer cell types. Its natural binder, the epidermal growth factor (EGF), is a 53 amino acid polypeptide. Anticancer synthetic targeted immune system engagers (ISErs) comprising two 'binder' peptides, which are attached to a scaffold conveying immune stimulating 'effector' properties, via monodisperse polyethylene glycol chains. So far, preparation of ISErs has been limited to the use of small peptides (8-20 amino acids) as binding functionalities, and they have been entirely synthesized by solid phase peptide synthesis. Here, we describe a synthetic and a semisynthetic approach for the preparation of an ISEr bearing two murine EGF molecules as binding entities (ISEr-EGF2 ). EGF was either synthesized in segments by solid phase peptide synthesis or expressed recombinantly and ligated to the scaffold by native chemical ligation. We report the successful generation of synthetic and semisynthetic ISEr-EGF2 as well as several challenges encountered during the synthesis and ligations. We demonstrate the application of native chemical ligation for the design of larger ISEr constructs, facilitating new objectives for the coupling of small binder peptides and larger proteins to multivalent ISEr scaffolds. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  10. In vitro screening for protein tyrosine phosphatase 1B and dipeptidyl peptidase IV inhibitors from selected Nigerian medicinal plants

    OpenAIRE

    Saidu, Yusuf; Muhammad, Suleiman Alhaji; Abbas, Abdullahi Yahaya; Onu, Andrew; Tsado, Ibrahim Mohammed; Muhammad, Luba

    2016-01-01

    Background/Aim: Protein tyrosine phosphatase 1B (PTP 1B) and dipeptidyl peptidase IV (DPP IV) have been identified as one of the drug targets for the treatment of Type-2 diabetes. This study was designed to screen for PTP 1B and DPP-IV inhibitors from some Nigerian medicinal plants. Materials and Methods: PTP 1B and DPP-IV drug discovery kits from Enzo Life Sciences were used to investigate in vitro inhibitory effect of crude methanolic extract of 10 plants; Mangifera indica, Moringa oleifera...

  11. Application of a pig ligated intestinal loop model for early Lawsonia intracellularis infection

    Directory of Open Access Journals (Sweden)

    Agerholm Jørgen S

    2010-02-01

    Full Text Available Abstract Background Porcine proliferative enteropathy in pigs is caused by the obligate, intracellular bacterium Lawsonia intracellularis. In vitro studies have shown close bacterium-cell interaction followed by cellular uptake of the bacterium within 3 h post inoculation (PI. However, knowledge of the initial in vivo interaction between porcine intestinal epithelium and the bacterium is limited. The aims of the present study were to evaluate the usefulness of a ligated small intestinal loop model to study L. intracellularis infections and to obtain information on the very early L. intracellularis-enterocyte interactions. Methods A ligated small intestinal loop model using three different L. intracellularis inocula was applied to 10-11-week-old pigs. The inocula were 1 wild type bacteria derived from overnight incubation of L. intracellularis bacteria from spontaneous disease, 2 crude vaccine bacteria (Enterisol® Ileitis Vet, and 3 vaccine bacteria propagated in cell culture. The bacteria-enterocyte interaction was visualised using immunohistochemistry on specimens derived 1, 3 and 6 h PI respectively. Results Although at a low level, close contact between bacteria and the enterocyte brush border including intracellular uptake of bacteria in mature enterocytes was seen at 3 and 6 h PI for the vaccine and the propagated vaccine inocula. Interaction between the wild-type bacteria and villus enterocytes was scarce and only seen at 6 h PI, where a few bacteria were found in close contact with the brush border. Conclusions The ligated intestinal loop model was useful with respect to maintaining an intact intestinal morphology for up to 6 h. Furthermore, the study demonstrated that L. intracellularis interacts with villus enterocytes within 3 to 6 h after inoculation into intestinal loops and that the bacterium, as shown for the vaccine bacteria, propagated as well as non-propagated, was able to invade mature enterocytes. Thus, the study demonstrates

  12. Use of model peptide reactions for the characterization of kinetically controlled ligation.

    Science.gov (United States)

    Lee, Joongoo; Kwon, Yoonjin; Pentelute, Brad L; Bang, Duhee

    2011-08-17

    Since the introduction of kinetically controlled ligation (KCL), a chemoselective reaction between a peptide-(α)thioarylester and a Cys-peptide-(α)thioalkylester, KCL has been utilized for the total chemical synthesis of large proteins (i.e., lysozyme and HIV-protease) by providing fully convergent synthetic routes. Although KCL has the potential to become an important chemistry for protein synthesis, the principle of KCL is not fully characterized. In particular, prior work on KCL has focused on the reactivity difference of the two different -(α)thioester forms-alkyl vs aryl. Another equally important feature of KCL, Xaa-Cys ligation sites, has not been investigated. The work reported here describes combinatorial KCL reactions using model peptides to dissect the interplay of the Xaa(1), Xaa(2), -(α)thioarylester, and -(α)thioalkylester. Results from these studies provide fundamental insights into the KCL reaction, and will lead to the optimal synthetic route for the routine chemical synthesis of large target protein molecules.

  13. Data in support of a harmine-derived beta-carboline in vitro effects in cancer cells through protein synthesis

    Directory of Open Access Journals (Sweden)

    Annelise Carvalho

    2017-06-01

    Full Text Available A harmine-derived beta-carboline, CM16, inhibits cancer cells growth through its effects on protein synthesis, as described in “A harmine-derived beta-carboline displays anti-cancer effects in vitro by targeting protein synthesis” (Carvalho et al., 2017[1]. This data article provides accompanying data on CM16 cytostatic evaluation in cancer cells as well as data related to its effects on transcription and translation. After confirming the cytostatic effect of CM16, we investigated its ability to arrest the cell cycle in the glioma Hs683 and SKMEL-28 melanoma cell lines but no modification was evidenced. According to the global protein synthesis inhibition induced by CM16 [1], transcription phase, a step prior to mRNA translation, evaluated by labelled nucleotide incorporation assay was not shown to be affected under CM16 treatment in the two cell lines. By contrast, mRNA translation and particularly the initiation step were shown to be targeted by CM16 in [1]. To further decipher those effects, we established herein a list of main actors in the protein synthesis process according to literature survey for comparative analysis of cell lines displaying different sensitivity levels to CM16. Finally, one of these proteins, PERK, a kinase regulating eIF2-α phosphorylation and thereby activity, was evaluated under treatment with CM16 in a cell-free system.

  14. Rapid inducible protein displacement in Plasmodium in vivo and in vitro using knocksideways technology [version 1; referees: 3 approved

    Directory of Open Access Journals (Sweden)

    Katie R. Hughes

    2017-03-01

    Full Text Available A deeper understanding of the biology of the Plasmodium parasite is essential in order to identify targets for interventions, with the ultimate aim of eliminating malaria. Determining the function(s of essential proteins in Plasmodium has, until recently, been hampered by the lack of efficient conditional systems to abrogate proteins. We report the adaptation of a conditional technology, knocksideways (KS, for use in Plasmodium berghei, which can potentially rapidly inactivate proteins of interest through relocalisation. The system is induced using rapamycin, which allows for KS both in vitro and in vivo and is effective more rapidly than any other reported system. KS utilises pairs of fluorescent tags that facilitate live imaging and allows for rapid confirmation of efficient protein redistribution on live parasites, allowing for streamlined workflows. We demonstrate the characteristics of the system using transgenically expressed cytoplasmic GFP and provide proof of principle by inducibly redistributing a number of proteins with different native, subcellular locations.  We also demonstrate that KS can be applied to both mammalian and insect stages of Plasmodium. KS expands the range of (conditional technologies for genetic manipulation of malaria parasites and offers the potential to be further developed for medium throughput phenotype screens.

  15. In vitro selection of optimal DNA substrates for T4 RNA ligase

    Science.gov (United States)

    Harada, Kazuo; Orgel, Leslie E.

    1993-01-01

    We have used in vitro selection techniques to characterize DNA sequences that are ligated efficiently by T4 RNA ligase. We find that the ensemble of selected sequences ligated about 10 times as efficiently as the random mixture of sequences used as the input for selection. Surprisingly, the majority of the selected sequences approximated a well-defined consensus sequence.

  16. Ribosome-inhibiting proteins from in vitro cultures of Phytolacca dodecandra.

    Science.gov (United States)

    Thomsen, S; Hansen, H S; Nyman, U

    1991-06-01

    Phytolacca dodecandra (L'Herit) grown in cell cultures was investigated for content of ribosome-inhibiting proteins, which was evaluated by measuring inhibition of protein synthesis in a cell-free rat liver extract. Calli initiated from leaf, cotyledon, radicle, and hypocotyl and suspension cells initiated from leaf and cotyledon exhibited protein synthesis-inhibiting activity. Ribosome-inhibiting proteins were purified at least 14 times from suspension cells of P. dodecandra. The purified protein fraction contained two proteins as seen by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The relative molecular masses were 30,000 and 31,000 and they showed a pI greater than 9.3. These new RIP's were shown to be different from dodecandrin with respect to molecular mass.

  17. Dynamic protein coronas revealed as a modulator of silver nanoparticle sulphidation in vitro

    Science.gov (United States)

    Miclăuş, Teodora; Beer, Christiane; Chevallier, Jacques; Scavenius, Carsten; Bochenkov, Vladimir E.; Enghild, Jan J.; Sutherland, Duncan S.

    2016-06-01

    Proteins adsorbing at nanoparticles have been proposed as critical toxicity mediators and are included in ongoing efforts to develop predictive tools for safety assessment. Strongly attached proteins can be isolated, identified and correlated to changes in nanoparticle state, cellular association or toxicity. Weakly attached, rapidly exchanging proteins are also present at nanoparticles, but are difficult to isolate and have hardly been examined. Here we study rapidly exchanging proteins and show for the first time that they have a strong modulatory effect on the biotransformation of silver nanoparticles. Released silver ions, known for their role in particle toxicity, are found to be trapped as silver sulphide nanocrystals within the protein corona at silver nanoparticles in serum-containing cell culture media. The strongly attached corona acts as a site for sulphidation, while the weakly attached proteins reduce nanocrystal formation in a serum-concentration-dependent manner. Sulphidation results in decreased toxicity of Ag NPs.

  18. The PEF family proteins sorcin and grancalcin interact in vivo and in vitro

    DEFF Research Database (Denmark)

    Hansen, Christian; Tarabykina, Svetlana; la Cour, Jonas Marstrand

    2003-01-01

    The penta-EF hand (PEF) family of calcium binding proteins includes grancalcin, peflin, sorcin, calpain large and small subunits as well as ALG-2. Systematic testing of the heterodimerization abilities of the PEF proteins using the yeast two-hybrid and glutathione S-transferase pull-down assays...... be a way to regulate and fine tune processes mediated by calcium binding proteins of the penta-EF hand type....

  19. Antiviral effects of milk proteins : Acylation results in polyanionic compounds with potent activity against human immunodeficiency virus types 1 and 2 in vitro

    NARCIS (Netherlands)

    Swart, P J; Kuipers, M E; Smit, C; Pauwels, R; deBéthune, M P; de Clercq, E; Meijer, D K; Huisman, J G

    1996-01-01

    A number of native and modified milk proteins from bovine or human sources were analyzed for their inhibitory effects on human immunodeficiency virus type 1 (HIV-1) and HIV-2 in vitro in an MT4 cell test system, The proteins investigated were lactoferrin, alpha-lactalbumin, beta-lactoglobulin A, and

  20. Alternative Okazaki Fragment Ligation Pathway by DNA Ligase III

    Directory of Open Access Journals (Sweden)

    Hiroshi Arakawa

    2015-06-01

    Full Text Available Higher eukaryotes have three types of DNA ligases: DNA ligase 1 (Lig1, DNA ligase 3 (Lig3 and DNA ligase 4 (Lig4. While Lig1 and Lig4 are present in all eukaryotes from yeast to human, Lig3 appears sporadically in evolution and is uniformly present only in vertebrates. In the classical, textbook view, Lig1 catalyzes Okazaki-fragment ligation at the DNA replication fork and the ligation steps of long-patch base-excision repair (BER, homologous recombination repair (HRR and nucleotide excision repair (NER. Lig4 is responsible for DNA ligation at DNA double strand breaks (DSBs by the classical, DNA-PKcs-dependent pathway of non-homologous end joining (C-NHEJ. Lig3 is implicated in a short-patch base excision repair (BER pathway, in single strand break repair in the nucleus, and in all ligation requirements of the DNA metabolism in mitochondria. In this scenario, Lig1 and Lig4 feature as the major DNA ligases serving the most essential ligation needs of the cell, while Lig3 serves in the cell nucleus only minor repair roles. Notably, recent systematic studies in the chicken B cell line, DT40, involving constitutive and conditional knockouts of all three DNA ligases individually, as well as of combinations thereof, demonstrate that the current view must be revised. Results demonstrate that Lig1 deficient cells proliferate efficiently. Even Lig1/Lig4 double knockout cells show long-term viability and proliferate actively, demonstrating that, at least in DT40, Lig3 can perform all ligation reactions of the cellular DNA metabolism as sole DNA ligase. Indeed, in the absence of Lig1, Lig3 can efficiently support semi-conservative DNA replication via an alternative Okazaki-fragment ligation pathway. In addition, Lig3 can back up NHEJ in the absence of Lig4, and can support NER and HRR in the absence of Lig1. Supporting observations are available in less elaborate genetic models in mouse cells. Collectively, these observations raise Lig3 from a niche

  1. Development of fiber optic spectroscopy for in-vitro and in-planta detection of fluorescent proteins

    Science.gov (United States)

    Liew, Oi Wah; Chen, Jun-Wei; Asundi, Anand K.

    2001-10-01

    The objective of this project is to apply photonics technology to bio-safety management of genetically modified (GM) plants. The conventional method for screening GM plants is through selection using antibiotic resistance markers. There is public concern with such approaches and these are associated with food safety issues, escape of antibiotic resistance genes to pathogenic microorganisms and interference with antibiotic therapy. Thus, the strategy taken in this project is to replace antibiotic resistance markers with fluorescent protein markers that allow for rapid and non-invasive optical screening of genetically modified plants. In this paper, fibre optic spectroscopy was developed to detect and quantify recombinant green (EGFP) and red (DsRED) fluorescent proteins in vitro and in planta. In vitro detection was first carried out to optimize the sensitivity of the optical system. The bacterial expression vectors carrying the coding regions of EGFP and DsRED were introduced into Escherichia coli host cells and fluorescent proteins were produced following induction with IPTG. Soluble EGFP and DsRED proteins were isolated from lysed bacterial cells and serially diluted for quantitative analysis by fibre optic spectroscopy using different light sources, namely, blue LED (475 nm), tungsten halogen (350 - 1000 nm) and double frequency Nd:YAG green laser (532 nm). Fluorescence near the expected emission wavelengths could be detected up to 320X dilution for EGFP and DsRED with blue LED and 532 nm green laser, respectively, as the excitation source. Tungsten halogen was found to be unsuitable for excitation of both EGFP and DsRED. EGFP was successfully purified by size separation under non-denaturing electrophoretic conditions and quantified. The minimum concentration of EGFP detectable with blue LED excitation was 5 mg/ml. To determine the capability of spectroscopy detection in planta, transgenic potato hairy roots and whole modified plant lines expressing the

  2. Anticariogenic and Hemolytic Activity of Selected Seed Protein Extracts In vitro conditions.

    Directory of Open Access Journals (Sweden)

    Kalpesh B Ishnava

    2014-10-01

    Full Text Available This study aimed to assess the anticariogenic and hemolytic activity of crude plant seed protein extracts against tooth decaying bacteria.The proteins from seeds of 12 different plants were extracted and used for antimicrobial assay against six different organisms. The extraction was carried out in 10mM of sodium phosphate buffer (pH 7.0. Protein concentrations were determined as described by Bradford method. Anticariogenic activity was studied by agar well diffusion method and Minimum Inhibitory Concentration (MIC was evaluated by the two-fold serial broth dilution method. Hemolytic activity, treatment of proteinase K and Kinetic study in Mimusops elengi crude seed protein extract.The anticariogenic assay demonstrated the activity of Mimusops elengi against Staphylococcus aureus and Streptococcus pyogenes. A minor activity of Glycine wightii against Streptococcus mutans was also found. The protein content of Mimusops elengi seed protein extract was 5.84mg/ml. The MIC values for Staphylococcus aureus and Streptococcus pyogenes against Mimusops elengi seed protein extract were 364.36μg/ml and 182.19μg/ml, respectively. Kinetic study further elucidated the mode of inhibition in the presence of the Mimusops elengi plant seed protein with respect to time. The concentration of crude extract which gave 50% hemolysis compared to Triton X-100 treatment (HC50 value was 1.58 mg/ml; which is more than five times larger than that of the MIC. Treatment with proteinase K of the Mimusops elengi seed protein resulted in absence of the inhibition zone; which clearly indicates that the activity was only due to protein.Our results showed the prominence of Mimusops elengi plant seed protein extract as an effective herbal medication against tooth decaying bacteria.

  3. A study to evaluate the potential of an in silico approach for predicting dipeptidyl peptidase-IV inhibitory activity in vitro of protein hydrolysates.

    Science.gov (United States)

    Wang, Tzu-Yuan; Hsieh, Cheng-Hong; Hung, Chuan-Chuan; Jao, Chia-Ling; Lin, Pei-Yi; Hsieh, You-Liang; Hsu, Kuo-Chiang

    2017-11-01

    A total of 294 edible protein sequences and 5 commercial proteases listed in the BIOPEP database were analyzed in silico. The frequency (A), a parameter in silico described previously, was examined further to calculating the ratio of truncated peptides with Xaa-proline and/or Xaa-alanine to all peptide fragments in a protein hydrolyzed with a protease, using the BIOPEP database. Then the in vitro DPP-IV inhibitory activity was determined using the same 15 protein and protease combinations to evaluate their relationship. The result shows that A values considering the number of Xaa-proline+Xaa-alanine exhibited a strong correlation with in vitro DPP-IV inhibition rates by Pearson's correlation analysis (r=0.6993; Papproach is effective to predict DPP-IV inhibitory activities in vitro of protein hydrolysates. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. HIV-1 Tat protein alter the tight junction integrity and function of retinal pigment epithelium: an in vitro study

    Directory of Open Access Journals (Sweden)

    Lin Haotian

    2008-06-01

    Full Text Available Abstract Background How HIV-1 enter into the eyes remains obscure. We postulated that HIV-1 Tat protein can alter the expression of specific tight-junction proteins and disturb the blood retinal barrier, and contributes to HIV trafficking into the eyes. This study is to determine the effects of HIV-1 Tat proteins on the barrier function and tight-junction protein expression of retinal pigment epithelial cell (RPE. Methods A human RPE cell line (D407 cultured on microporous filter-supports was used. After treating with HIV-1 Tat protein, transepithelial electrical resistance (TER of confluent RPE cells was measured by epithelial voltmeter. The permeability of the RPE cells to sodium fluorescein was measured. The expressions of the occludin and claudins were determined by real-time polymerase chain reaction, immunofluorescence, and Western blot analysis. Activation of ERK1/2 was detected by Western blot analysis with specific antiphospho protein antibodies. NF-κB DNA binding activity was determined by transcription factor assay. Specific pharmacologic inhibitors directed against the MAPKs were used to analyze the signaling involved in barrier destruction of RPE cells exposed to HIV-1 Tat. Results Treating cultured human retinal pigment epithelial cells with 100 nM Tat for 24 hours increased the permeability and decreased the TER of the epithelial monolayer. HIV-1 Tat also disrupted and downregulated the tight-junction proteins claudin-1, claudin-3, and claudin-4 in these cells, whereas claudin-2 was upregulated, and the expression of occludin was unaffected. HIV-1 Tat protein also induced activation of ERK1/2 and NF-κB. HIV-1 Tat protein induced barrier destruction, changes in expression of TJs, and activation of ERK1/2 and NF-κB were abrogated by inhibitor of ERK1/2 and NF-κB. Conclusion HIV-1 Tat protein causes increases in the paracellular permeability of RPE cells in vitro concomitant with changes in expression of certain transmembrane

  5. Expression of HSP27, HSP72 and MRP proteins in in vitro co-culture ...

    Indian Academy of Sciences (India)

    We studied the expression of inducible heat shock protein (HSP27, HSP72) and multidrug-resistance protein (MRP) in co-cultures of human colon carcinoma cell spheroids obtained from different grades of tumour with normal human colon epithelium, myofibroblast and endothelial cell monolayers. We also measured the ...

  6. Ribosome-inhibiting proteins from in vitro cultures of Phytolacca dodecandra

    DEFF Research Database (Denmark)

    Thomsen, S.; Hansen, Harald S.; Nyman, U.

    1991-01-01

    Phytolacca dodecandra (L'Herit) grown in cell cultures was investigated for content of ribosome-inhibiting proteins, which was evaluated hy measuring inhibition of protein synthesis in a cell-free rat liver extract. Calli initiated from leaf, cotyledon, radicle, and hypocotyl and suspension cells...

  7. Effect of high hydrostatic pressure processing on in vitro digestion of milk proteins and fats

    Science.gov (United States)

    The use of high hydrostatic pressure processing (HPP) is increasing in popularity in the food industry. Its ability to modify milk proteins and fats suggests that it may be useful in creating foods that suppress appetite; however, its effect on the digestibility of proteins and fats is unclear. The...

  8. Protein identification and in vitro digestion of fractions from Tenebrio molitor

    NARCIS (Netherlands)

    Yi, Liya; Boekel, van M.A.J.S.; Boeren, Sjef; Lakemond, Catriona M.M.

    2016-01-01

    The nutritional value of insect protein is evaluated not only in amino acid composition, but also in protein digestibility. The general amino acid composition of Tenebrio molitor has been reported before, but limited knowledge is available on its digestibility. The objective of this study was to

  9. A validated system for ligation-free USER™ -based assembly of expression vectors for mammalian cell engineering

    DEFF Research Database (Denmark)

    Lund, Anne Mathilde; Kildegaard, Helene Faustrup; Hansen, Bjarne Gram

    The development in the field of mammalian cell factories require fast and high-throughput methods, this means a high need for simpler and more efficient cloning techniques. For optimization of protein expression by genetic engineering and for allowing metabolic engineering in mammalian cells, a new...... versatile expression vector system was developed. This vector system applies the ligation-free uracilexcision cloning technique to construct mammalian expression vectors of multiple parts and with maximum flexibility....

  10. Potent in vitro antiviral activity of Cistus incanus extract against HIV and Filoviruses targets viral envelope proteins

    Science.gov (United States)

    Rebensburg, Stephanie; Helfer, Markus; Schneider, Martha; Koppensteiner, Herwig; Eberle, Josef; Schindler, Michael; Gürtler, Lutz; Brack-Werner, Ruth

    2016-01-01

    Novel therapeutic options are urgently needed to improve global treatment of virus infections. Herbal products with confirmed clinical safety features are attractive starting material for the identification of new antiviral activities. Here we demonstrate that Cistus incanus (Ci) herbal products inhibit human immunodeficiency virus (HIV) infections in vitro. Ci extract inhibited clinical HIV-1 and HIV-2 isolates, and, importantly, a virus isolate with multiple drug resistances, confirming broad anti-HIV activity. Antiviral activity was highly selective for virus particles, preventing primary attachment of the virus to the cell surface and viral envelope proteins from binding to heparin. Bioassay-guided fractionation indicated that Ci extract contains numerous antiviral compounds and therefore has favorably low propensity to induce virus resistance. Indeed, no resistant viruses emerged during 24 weeks of continuous propagation of the virus in the presence of Ci extracts. Finally, Ci extracts also inhibited infection by virus particles pseudotyped with Ebola and Marburg virus envelope proteins, indicating that antiviral activity of Ci extract extends to emerging viral pathogens. These results demonstrate that Ci extracts show potent and broad in vitro antiviral activity against viruses that cause life-threatening diseases in humans and are promising sources of agents that target virus particles. PMID:26833261

  11. A low molecular weight ES-20 protein released in vivo and in vitro with diagnostic potential in lymph node tuberculosis

    Directory of Open Access Journals (Sweden)

    Shende N

    2008-01-01

    Full Text Available Purpose: To determine role of antigens released in vivo and in vitro in immunodiagnosis of tuberculosis (TB. Methods: In vivo released circulating tuberculosis antigen (CTA was obtained from TB sera by ammonium sulphate precipitation and in vitro released excretory-secretory (ES antigens from Mycobacterium tuberculosis culture filtrate. CTA and ES antigens were fractionated by SDS-PAGE and electro-eluted gel fractions were analysed for antigen by ELISA. Results: Low molecular weight proteins CTA-9 and ES-9 showed high titre of antigen activity. To explore the diagnostic potential of low molecular weight ES antigen, M. tuberculosis ES antigen was further fractionated by gel filtration chromatography followed by purification on anion exchange column using fast protein liquid chromatography and a highly seroreactive ESG-5D (ES-20 antigen was obtained. Competitive inhibition showed that CTA-9 and ES-9 antigens inhibit the binding of ES-20 antigen to its antibody. Seroanalysis showed sensitivity of 83 and 80% for ES-20 antigen and antibody detection, respectively, in pulmonary TB and 90% in lymph node TB. Conclusions: Seroreactivity studies using M. tuberculosis ES-20 antigen showed usefulness in detection of TB; in particular, lymph node TB.

  12. Early expression of nucleolar SURF-6 protein in mouse spleen lymphocytes activated for proliferation in vitro.

    Science.gov (United States)

    Moraleva, A A; Malysheva, M V; Magoulas, Ch; Polzikov, M A; Zatsepina, O V

    2009-05-01

    Using specific antibodies we studied the content of nucleolar SURF-6 protein, which participates in rRNA processing, in mouser spleen lymphocytes activated for proliferation with concanavalin A and compared it with the content of nucleolar nucleophosmin/B23 protein and DNA replication factor PCNA, well-known markers of proliferating cells. Using immunocytochemistry and immunoblotting methods we demonstrate that the concentration of all these proteins increases simultaneously with increasing the proportion of proliferating cells. Unlike nucleophosmin/B23, SURF-6 protein was not revealed in quiescent lymphocyte nucleoli, while the increase of its level in activated lymphocytes preceded elevation of PCNA level. These observations suggest that nucleolar protein SURF-6 can act as a marker of early T lymphocyte activation for proliferation and that it could participate in cell cycle regulation in mammals.

  13. In vitro anthelmintic effects of Spigelia anthelmia protein fractions against Haemonchus contortus.

    Directory of Open Access Journals (Sweden)

    Sandra Alves Araújo

    Full Text Available Gastrointestinal nematodes are a significant concern for animal health and well-being, and anthelmintic treatment is mainly performed through the use of chemical products. However, bioactive compounds produced by plants have shown promise for development as novel anthelmintics. The aim of this study is to assess the anthelmintic activity of protein fractions from Spigelia anthelmia on the gastrointestinal nematode Haemonchus contortus. Plant parts were separated into leaves, stems and roots, washed with distilled water, freeze-dried and ground into a fine powder. Protein extraction was performed with sodium phosphate buffer (75 mM, pH 7.0. The extract was fractionated using ammonium sulfate (0-90% and extensively dialyzed. The resulting fractions were named LPF (leaf protein fraction, SPF (stem protein fraction and RPF (root protein fraction, and the protein contents and activities of the fractions were analyzed. H. contortus egg hatching (EHA, larval exsheathment inhibition (LEIA and larval migration inhibition (LMIA assays were performed. Proteomic analysis was conducted, and high-performance liquid chromatography (HPLC chromatographic profiles of the fractions were established to identify proteins and possible secondary metabolites. S. anthelmia fractions inhibited H. contortus egg hatching, with LPF having the most potent effects (EC50 0.17 mg mL-1. During LEIA, SPF presented greater efficiency than the other fractions (EC50 0.25 mg mL-1. According to LMIA, the fractions from roots, stems and leaves also reduced the number of larvae, with EC50 values of 0.11, 0.14 and 0.21 mg mL-1, respectively. Protein analysis indicated the presence of plant defense proteins in the S. anthelmia fractions, including protease, protease inhibitor, chitinase and others. Conversely, secondary metabolites were absent in the S. anthemia fractions. These results suggest that S. anthelmia proteins are promising for the control of the gastrointestinal nematode H

  14. In vitro anthelmintic effects of Spigelia anthelmia protein fractions against Haemonchus contortus.

    Science.gov (United States)

    Araújo, Sandra Alves; Soares, Alexandra Martins Dos Santos; Silva, Carolina Rocha; Almeida Júnior, Eduardo Bezerra; Rocha, Cláudia Quintino; Ferreira, André Teixeira da Silva; Perales, Jonas; Costa-Júnior, Livio M

    2017-01-01

    Gastrointestinal nematodes are a significant concern for animal health and well-being, and anthelmintic treatment is mainly performed through the use of chemical products. However, bioactive compounds produced by plants have shown promise for development as novel anthelmintics. The aim of this study is to assess the anthelmintic activity of protein fractions from Spigelia anthelmia on the gastrointestinal nematode Haemonchus contortus. Plant parts were separated into leaves, stems and roots, washed with distilled water, freeze-dried and ground into a fine powder. Protein extraction was performed with sodium phosphate buffer (75 mM, pH 7.0). The extract was fractionated using ammonium sulfate (0-90%) and extensively dialyzed. The resulting fractions were named LPF (leaf protein fraction), SPF (stem protein fraction) and RPF (root protein fraction), and the protein contents and activities of the fractions were analyzed. H. contortus egg hatching (EHA), larval exsheathment inhibition (LEIA) and larval migration inhibition (LMIA) assays were performed. Proteomic analysis was conducted, and high-performance liquid chromatography (HPLC) chromatographic profiles of the fractions were established to identify proteins and possible secondary metabolites. S. anthelmia fractions inhibited H. contortus egg hatching, with LPF having the most potent effects (EC50 0.17 mg mL-1). During LEIA, SPF presented greater efficiency than the other fractions (EC50 0.25 mg mL-1). According to LMIA, the fractions from roots, stems and leaves also reduced the number of larvae, with EC50 values of 0.11, 0.14 and 0.21 mg mL-1, respectively. Protein analysis indicated the presence of plant defense proteins in the S. anthelmia fractions, including protease, protease inhibitor, chitinase and others. Conversely, secondary metabolites were absent in the S. anthemia fractions. These results suggest that S. anthelmia proteins are promising for the control of the gastrointestinal nematode H. contortus.

  15. G-protein ligands inhibit in vitro reactions of vacuole inheritance

    OpenAIRE

    1994-01-01

    During budding in Saccharomyces cerevisiae, maternal vacuole material is delivered into the growing daughter cell via tubular or vesicular structures. One of the late steps in vacuole inheritance is the fusion in the bud of vesicles derived from the maternal vacuole. This process has been reconstituted in vitro and requires isolated vacuoles, a physiological temperature, cytosolic factors, and ATP (Conradt, B., J. Shaw, T. Vida, S. Emr, and W. Wickner. 1992. J. Cell Biol. 119:1469- 1479). We ...

  16. Identification of two human nuclear proteins that recognise the cytosine-rich strand of human telomeres in vitro

    Science.gov (United States)

    Lacroix, Laurent; Liénard, Hélène; Labourier, Emmanuel; Djavaheri-Mergny, Mojgan; Lacoste, Jérôme; Leffers, Henrik; Tazi, Jamal; Hélène, Claude; Mergny, Jean-Louis

    2000-01-01

    Most studies on the structure of DNA in telomeres have been dedicated to the double-stranded region or the guanosine-rich strand and consequently little is known about the factors that may bind to the telomere cytosine-rich (C-rich) strand. This led us to investigate whether proteins exist that can recognise C-rich sequences. We have isolated several nuclear factors from human cell extracts that specifically bind the C-rich strand of vertebrate telomeres [namely a d(CCCTAA)n repeat] with high affinity and bind double-stranded telomeric DNA with a 100× reduced affinity. A biochemical assay allowed us to characterise four proteins of apparent molecular weights 66–64, 45 and 35 kDa, respectively. To identify these polypeptides we screened a λgt11-based cDNA expression library, obtained from human HeLa cells using a radiolabelled telomeric oligonucleotide as a probe. Two clones were purified and sequenced: the first corresponded to the hnRNP K protein and the second to the ASF/SF2 splicing factor. Confirmation of the screening results was obtained with recombinant proteins, both of which bind to the human telomeric C-rich strand in vitro. PMID:10710423

  17. In vitro adhesion and invasion inhibition of Shigella dysenteriae, Shigella flexneri and Shigella sonnei clinical strains by human milk proteins

    Directory of Open Access Journals (Sweden)

    Giugliano Loreny

    2004-04-01

    Full Text Available Abstract Background Shigella is the etiological agent of shigellosis, a disease responsible for more than 500,000 deaths of children per year, in developing countries. These pathogens colonize the intestinal colon, invade, spreading to the other enterocytes. Breastfeeding plays a very important role in protecting infants from intestinal infections. Amongst milk compounds, glycosylated proteins prevent the adhesion of many enteropathogens in vitro. The aim of this work was to determine the effect of human milk proteins on the colonization potential of Shigella dysenteriae, S. flexneri and S. sonnei. To fulfill this purpose, pooled milk samples from five donors, were fractionated by gel filtration and affinity chromatography. Using tissue culture, the milk fractions obtained were tested in Shigella adhesion and invasion assays. Results Our revealed showed that both adhesion and invasion of Shigella species were inhibited by low concentration of secretory immunoglobulin A, lactoferrin and free secretory component. This work also showed that, these proteins bind to superficial and whole-cell Shigella proteins. Conclusions Our findings suggest that human milk may act inhibiting adhesion and, consequently, invasion of Shigella, thereafter preventing shigellosis in infants.

  18. Saccharomyces cerevisiae Cmr1 protein preferentially binds to UV-damaged DNA in vitro.

    Science.gov (United States)

    Choi, Do-Hee; Kwon, Sung-Hun; Kim, Joon-Ho; Bae, Sung-Ho

    2012-02-01

    DNA metabolic processes such as DNA replication, recombination, and repair are fundamentally important for the maintenance of genome integrity and cell viability. Although a large number of proteins involved in these pathways have been extensively studied, many proteins still remain to be identified. In this study, we isolated DNA-binding proteins from Saccharomyces cerevisiae using DNA-cellulose columns. By analyzing the proteins using mass spectrometry, an uncharacterized protein, Cmr1/YDL156W, was identified. Cmr1 showed sequence homology to human Damaged-DNA binding protein 2 in its C-terminal WD40 repeats. Consistent with this finding, the purified recombinant Cmr1 protein was found to be intrinsically associated with DNA-binding activity and exhibited higher affinity to UV-damaged DNA substrates. Chromatin isolation experiments revealed that Cmr1 localized in both the chromatin and supernatant fractions, and the level of Cmr1 in the chromatin fraction increased when yeast cells were irradiated with UV. These results suggest that Cmr1 may be involved in DNA-damage responses in yeast.

  19. In vitro and in vivo studies identify important features of dengue virus pr-E protein interactions.

    Directory of Open Access Journals (Sweden)

    Aihua Zheng

    2010-10-01

    Full Text Available Flaviviruses bud into the endoplasmic reticulum and are transported through the secretory pathway, where the mildly acidic environment triggers particle rearrangement and allows furin processing of the prM protein to pr and M. The peripheral pr peptide remains bound to virus at low pH and inhibits virus-membrane interaction. Upon exocytosis, the release of pr at neutral pH completes virus maturation to an infectious particle. Together this evidence suggests that pr may shield the flavivirus fusion protein E from the low pH environment of the exocytic pathway. Here we developed an in vitro system to reconstitute the interaction of dengue virus (DENV pr with soluble truncated E proteins. At low pH recombinant pr bound to both monomeric and dimeric forms of E and blocked their membrane insertion. Exogenous pr interacted with mature infectious DENV and specifically inhibited virus fusion and infection. Alanine substitution of E H244, a highly conserved histidine residue in the pr-E interface, blocked pr-E interaction and reduced release of DENV virus-like particles. Folding, membrane insertion and trimerization of the H244A mutant E protein were preserved, and particle release could be partially rescued by neutralization of the low pH of the secretory pathway. Thus, pr acts to silence flavivirus fusion activity during virus secretion, and this function can be separated from the chaperone activity of prM. The sequence conservation of key residues involved in the flavivirus pr-E interaction suggests that this protein-protein interface may be a useful target for broad-spectrum inhibitors.

  20. Development and Preliminary Assessment of Hemoperfusion Cartridge with Tannic Acid for Toxic Proteins' Precipitation: An In Vitro Model

    Directory of Open Access Journals (Sweden)

    Valquíria Miwa Hanai Yoshida

    2016-09-01

    Full Text Available Charcoal hemoperfusion (CHP is one of the extracorporeal removal techniques that are used to remove toxins from the body. CHP generally is considered the preferred method for extracorporeal extraction of several toxins—toxins that are adsorbed by activated charcoal. Assessments of the tannic acid's protective effects on ophidian poisoning are associated with the toxic proteins' precipitation by tannic acid. The challenge in treating a snakebite lies in removing the injected poison with minimal damage to blood constituent proteins. An alternative is CHP, and this investigation proposed to develop a column for hemoperfuser cartridge, combining charcoal granules trapped between layers of polymeric material conjugated to tannic acid, using an in vitro model scaled to the Wistar rat, which can be tested in an animal model. The cartridge was evaluated using the 22 full factorial design, in duplicate, as a method to study the effects of granulated-charcoal size and tannic acid concentration on the hematologic profile (platelet and leukocyte counts and biochemical profile (total serum protein and albumin dosages of sheep blood. The results demonstrate that charcoal in hemoperfuser cartridge: (1 decreases the serum in sheep blood volume, as consequence, (2 increases the serum proteins' concentration, and (iii exerts slight influence on albumin. The inclusion of tannic acid in hemoperfuser column precipitates some of serum proteins and albumin, decreasing their concentrations in the plasma serum. In conclusion, based on these effects we can suggest the use of 0.02 g tannic acid concentration and 8–20 mesh granulated charcoal in hemoperfuser cartridge for precipitating toxic proteins from snake venoms.

  1. Design and validation of DNA libraries for multiplexing proximity ligation assays.

    Directory of Open Access Journals (Sweden)

    Nicolas Gobet

    Full Text Available Here, we present an in silico, analytical procedure for designing and testing orthogonal DNA templates for multiplexing of the proximity ligation assay (PLA. PLA is a technology for the detection of protein interactions, post-translational modifications, and protein concentrations. To enable multiplexing of the PLA, the target information of antibodies was encoded within the DNA template of a PLA, where each template comprised four single-stranded DNA molecules. Our DNA design procedure followed the principles of minimizing the free energy of DNA cross-hybridization. To validate the functionality, orthogonality, and efficiency of the constructed template libraries, we developed a high-throughput solid-phase rolling-circle amplification assay and solid-phase PLA on a microfluidic platform. Upon integration on a microfluidic chip, 640 miniaturized pull-down assays for oligonucleotides or antibodies could be performed in parallel together with steps of DNA ligation, isothermal amplification, and detection under controlled microenvironments. From a large computed PLA template library, we randomly selected 10 template sets and tested all DNA combinations for cross-reactivity in the presence and absence of antibodies. By using the microfluidic chip application, we determined rapidly the false-positive rate of the design procedure, which was less than 1%. The combined theoretical and experimental procedure is applicable for high-throughput PLA studies on a microfluidic chip.

  2. Wild-type alternatively spliced p53: binding to DNA and interaction with the major p53 protein in vitro and in cells.

    OpenAIRE

    Wu, Y.; Liu, Y; L. Lee; Miner, Z; Kulesz-Martin, M

    1994-01-01

    A p53 variant protein (p53as) generated from alternatively spliced p53 RNA is expressed in normal and malignant mouse cells and tissues, and p53as antigen activity is preferentially associated with the G2 phase of the cell cycle, suggesting that p53as and p53 protein may have distinct properties. Using p53as and p53 proteins translated in vitro, we now provide evidence that p53as protein has efficient sequence-specific DNA-binding ability. DNA binding by p53 protein is inefficient in comparis...

  3. Adrenal function in preterm infants undergoing patent ductus arteriosus ligation.

    LENUS (Irish Health Repository)

    El-Khuffash, Afif

    2013-01-01

    Targeted milrinone treatment for low left ventricular output (LVO) reduces the incidence of acute cardiorespiratory instability following ligation of patent ductus arteriosus (PDA) in preterm infants. Despite this, some infants continue to experience postoperative deterioration. Adrenal insufficiency related to prematurity has been postulated as a possible mechanism.

  4. Tubal ectopic pregnancy after bilateral tubal ligation: A case report ...

    African Journals Online (AJOL)

    Tubal ectopic pregnancy after bilateral tubal ligation: A case report. N Ameh, NH Madugu, US Bawa, MS Adelaiye, M Akpa. Abstract. No Abstract. Nigerian Journal of Medicine Vol. 15 (4) October-December 2006: 453-454. Full Text: EMAIL FULL TEXT EMAIL FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT.

  5. Experience with rubber band ligation of hemorrhoids in northern ...

    African Journals Online (AJOL)

    Background: Treatment of hemorrhoids in Nigeria is usually done by the traditional open method that requires hospital admission; anesthesia and is associated with high morbidity. Rubber band ligation is a suitable alternative to open hemorrhoidectomy and has the potential to reduce the need for hospital admission.

  6. Esophageal variceal ligation for hemostasis of acute variceal bleeding

    African Journals Online (AJOL)

    Our aim was to evaluate the effectiveness and safety of endoscopic variceal ligation in the management of oesophageal variceal bleeding in cirrhosis in a located population in Morocco. Methods: Via a retrospective study over 118 months (December 2001- October 2011), cirrhotic patients with endoscopically proven ...

  7. The effectiveness of Doppler controlled hemorrhoidal artery ligation ...

    African Journals Online (AJOL)

    In this work, we discuss the preliminary results of the effectiveness of the hemorrhoidal artery ligation under control Doppler as a new technique for the treatment of hemorrhoids. We report the results of patients with hemorrhoids we have followed over a period of one year who were treated with HAL Doppler. The intra-and ...

  8. Synthesis of coumarin or ferrocene labeled nucleosides via Staudinger ligation

    Directory of Open Access Journals (Sweden)

    Kois Pavol

    2006-11-01

    Full Text Available Abstract Background Reaction of azides with triaryl phosphines under mild conditions gives iminophosphoranes which can react with almost any kind of electrophilic reagent, e.g. aldehydes/ketones to form imines or esters to form amides. This so-called Staudinger ligation has been employed in a wide range of applications as a general tool for bioconjugation including specific labeling of nucleic acids. Results A new approach for the preparation of labeled nucleosides via intermolecular Staudinger ligation is described. Reaction of azidonucleosides with triphenylphosphine lead to iminophosphorane intermediates, which react subsequently with derivatives of coumarin or ferrocene to form coumarin or ferrocene labeled nucleosides. Fluorescent properties of coumarin labeled nucleosides are determined. Conclusion New coumarin and ferrocene labeled nucleosides were prepared via intermolecular Staudinger ligation. This reaction joins the fluorescent coumarin and biospecific nucleoside to the new molecule with promising fluorescent and electrochemical properties. The isolated yields of products depend on the structure of azidonucleoside and carboxylic acids. A detailed study of the kinetics of the Staudinger ligation with nucleoside substrates is in progress.

  9. Endoscopic variceal band ligation: a local experience | Jani | East ...

    African Journals Online (AJOL)

    Objective: To evaluate the results of endoscopic variceal band ligation (EVBL) in the local set-up. Design: Retrospective analysis of data of all patients who had EVBL. Setting: Patients having EVBL at the office endoscopy suite. The Nairobi Hospital, the Aga Khan Hospital and M.P Shah Hospital. Methods: The varices were ...

  10. Spontaneous retraction of the ligated hernial sac during herniotomy ...

    African Journals Online (AJOL)

    Inguinal hernias constitute one of the most common surgical conditions for which a child presents to a surgeon 1,2. The treatment for this procedure in children is through an inguinal herniotomy with a high ligation of the hernial sac at the deep inguinal ring or at the level of the preperitoneal fat 3. In boys, it is important to.

  11. Impact of contraception use among women seeking tubal ligation in ...

    African Journals Online (AJOL)

    2007-02-21

    Feb 21, 2007 ... To describe user characteristics and analyse the impact of reversible contraception use among women who underwent tubal ligation ... suggesting a high prevalence of inconsistent or incorrect use of contraception. pg15-18.indd 15. 2/21/07 .... use is associated with smaller family units (Table II). Discussion.

  12. Assessment and treatment of post patent ductus arteriosus ligation syndrome.

    LENUS (Irish Health Repository)

    El-Khuffash, Afif F

    2014-07-01

    To compare differences in tissue Doppler imaging, global longitudinal strain (GLS), and cardiac troponin T (cTnT) between infants with low (<200 mL\\/kg\\/min) and high (>200 mL\\/kg\\/min) left ventricular (LV) output 1 hour after duct ligation and assess the impact of milrinone treatment on cardiac output and myocardial performance.

  13. Oesophageal variceal band ligation using a Saeed Six-Shooter ...

    African Journals Online (AJOL)

    He had a total of three sessions after which he was maintained on propranolol. Result: The last recheck endoscopy demonstrated obliterated varices after which he was maintained on propranolol. Conclusion: We present a case of successful variceal band ligation of a cirrhotic with extensive oesophageal varices presenting ...

  14. To evaluate the results of endoscopic variceal band ligation (EVBL)

    African Journals Online (AJOL)

    hi-tech

    2004-04-04

    Apr 4, 2004 ... Lay, C.S. Tsai, Y.T. Teg, C.Y. et al. Endoscopic variceal ligation in prophylaxis of first variceal bleeding in cirrhotic patients with high-risk esophageal varices. Hepatology. 1997;. 25:346-350. 24. Stiegmann, G.V. Goff, J.S. Michaletz-Onody, P.A. et al. Endoscopic sclerotherapy as compared with endoscopic.

  15. Impact of contraception use among women seeking tubal ligation in ...

    African Journals Online (AJOL)

    To describe user characteristics and analyse the impact of reversible contraception use among women who underwent tubal ligation in a rural health district of the Democratic Republic of Congo over a 4-year period. Methods. A retrospective analysis of family planning programme registers for 4 years (1990 - 1994). During ...

  16. Thermoresponsive Injectable Hydrogels Cross-Linked by Native Chemical Ligation

    NARCIS (Netherlands)

    Boere, Kristel W M; Soliman, Bram G.; Rijkers, Dirk T S; Hennink, Wim E.; Vermonden, Tina

    2014-01-01

    Temperature-induced physical gelation was combined with native chemical ligation (NCL) as a chemical cross-linking mechanism to yield rapid network formation and mechanically strong hydrogels. To this end, a novel monomer N-(2-hydroxypropyl)methacrylamide-cysteine (HPMA-Cys) was synthesized that

  17. Ectopic pregnancy after two times tubal ligation: a case report

    Directory of Open Access Journals (Sweden)

    Farideh Keypour

    2013-06-01

    Full Text Available Background: Tubal sterilization is the permanent and effective contraception method. This can be performed at any time, but at least half are performed in conjunction with cesarean or vaginal delivery and are termed puerperal. The most complication after tubal ligation is ectopic pregnancy. Ectopic pregnancy is the leading cause of maternal death in first trimester.Case presentation: We present a 33 years old woman gravida5, para4, all normal vaginal delivery, presented with complaints of delayed menstrual period, pelvic pain and spotting. She underwent tubal ligation for two times. For the first time she had puerperal Pomeroy tubal sterilization after third child delivery. Intra uterine pregnancy occurred three years later. One day after vaginal delivery of fourth child, she underwent post partum tubal ligation with the Parkland method. Tubal pregnancy occurred nine months later. Physical examination identified acute abdomen. Pelvic ultrasound showed no gestational sac in uterine cavity. The sac with fetal pole was in right adnexa. Beta-HCG was 2840mIU/ml. She underwent laparotomy. Surgical management included salpingectomy with cornual resection in both sides. The surgery identified Ectopic pregnancy.Conclusion: Any symptoms of pregnancy in a woman after tubal ligation must be investigated; an ectopic pregnancy should be excluded. Ectopic pregnancy must be considered, in any woman with lower abdominal pain, missed period and vaginal bleed-ing. Conception after tubal sterilization can be explained by fistula formation and re-canalization of fallopian tube.

  18. Female sterilization by tubal ligation at caesarean section in Makurdi ...

    African Journals Online (AJOL)

    Background: Female sterilization is an important tool in reducing unplanned pregnancy and maternal mortality in our environment. The aim of this study was to determine the incidence, sociodemographic characteristics, technique, effectiveness and complications associated with female sterilization by bilateral tubal ligation ...

  19. Neovascularisation of the ovary post ligation of ovarian vessels in ...

    African Journals Online (AJOL)

    The viable ovarian tissues noticed on histology were evidence of revascularization suggestive of newer invasion of blood vessels from the ovarian attachment from the retroperitoneal tissues and likely from the subcutaneous supplies. Consequently, it is not advisable to neuter a bitch by ligation of the ovarian vessels ...

  20. Kinetics and equilibria for the axial ligation of bromomethyl (aqua ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Chemical Sciences; Volume 121; Issue 6. Kinetics and equilibria for the axial ligation of bromomethyl (aqua)cobaloxime with pyridines - Isolation characterization and DNA binding. Kotha Laxma Reddy K Ashwini Kumar N Ravi Kumar Reddy Penumaka Nagababu A Panasa Reddy S ...

  1. To evaluate the results of endoscopic variceal band ligation (EVBL)

    African Journals Online (AJOL)

    hi-tech

    2004-04-04

    Apr 4, 2004 ... Villanueva, C. Ortiz, J. Minana, J. et al. Somatostatin treatment and risk stratification by continuous portal pressure monitoring during acute variceal bleeding. Gastroenterology. 2001; 121:ll0-117. 5. Nevens, F. and Rutgeerts, P. Variceal band ligation in the management of bleeding oesophageal varices: an ...

  2. Inducing Acute Traumatic Coagulopathy In Vitro: The Effects of Activated Protein C on Healthy Human Whole Blood.

    Directory of Open Access Journals (Sweden)

    Benjamin M Howard

    Full Text Available Acute traumatic coagulopathy has been associated with shock and tissue injury, and may be mediated via activation of the protein C pathway. Patients with acute traumatic coagulopathy have prolonged PT and PTT, and decreased activity of factors V and VIII; they are also hypocoagulable by thromboelastometry (ROTEM and other viscoelastic assays. To test the etiology of this phenomenon, we hypothesized that such coagulopathy could be induced in vitro in healthy human blood with the addition of activated protein C (aPC.Whole blood was collected from 20 healthy human subjects, and was "spiked" with increasing concentrations of purified human aPC (control, 75, 300, 2000 ng/mL. PT/PTT, factor activity assays, and ROTEM were performed on each sample. Mixed effect regression modeling was performed to assess the association of aPC concentration with PT/PTT, factor activity, and ROTEM parameters.In all subjects, increasing concentrations of aPC produced ROTEM tracings consistent with traumatic coagulopathy. ROTEM EXTEM parameters differed significantly by aPC concentration, with stepwise prolongation of clotting time (CT and clot formation time (CFT, decreased alpha angle (α, impaired early clot formation (a10 and a20, and reduced maximum clot firmness (MCF. PT and PTT were significantly prolonged at higher aPC concentrations, with corresponding significant decreases in factor V and VIII activity.A phenotype of acute traumatic coagulopathy can be induced in healthy blood by the in vitro addition of aPC alone, as evidenced by viscoelastic measures and confirmed by conventional coagulation assays and factor activity. This may lend further mechanistic insight to the etiology of coagulation abnormalities in trauma, supporting the central role of the protein C pathway. Our findings also represent a model for future investigations in the diagnosis and treatment of acute traumatic coagulopathy.

  3. Sea buckthorn (Hippophae rhamnoides) proanthocyanidins inhibit in vitro enzymatic hydrolysis of protein.

    Science.gov (United States)

    Arimboor, Ranjith; Arumughan, C

    2011-08-01

    Interactions of phenolics with other food constituents and digestive enzymes are likely to have interference with the digestion and bioavailability of food and phenolics. In this study the effect of sea buckthorn proanthocyanidins on in vitro digestion of protein was evaluated. Optimization of the extraction conditions showed that maximum recovery of sea buckthorn proanthocyanidins was obtained with acidified acetone; water mixture (60% to 70%, v/v). Crude proanthocyanidin extracts thus prepared were purified using sephadex gel column chromatography and their average degree of polymerization and the effects on enzymatic hydrolysis of bovine serum albumin as influenced by their protein precipitation capacities were studied. Average degree of polymerization of proanthocyanidins in berry pulp, kernel, seed coat, and leaves was 7.4, 5.6, 8.2, and 10.6, respectively. The EC50 values for the protein precipitation by the PA of berry pulp, kernel seed coat, and leaves were 44.2, 44.1, 65.8, and 39.8 μg, respectively. Relative enzymatic hydrolysis of the protein-proanthocyanidin complexes was 44.1% to 60.3% for pepsin and 57.5% to 67.7% for trypsin. Interactions of sea buckthorn proanthocyanidins with food proteins and digestive enzymes might alter the protein digestibility and phenolic bioavailabilty. © 2011 Institute of Food Technologists®

  4. Understanding renal nuclear protein accumulation: an in vitro approach to explain an in vivo phenomenon.

    Science.gov (United States)

    Luks, Lisanne; Maier, Marcia Y; Sacchi, Silvia; Pollegioni, Loredano; Dietrich, Daniel R

    2017-11-01

    Proper subcellular trafficking is essential to prevent protein mislocalization and aggregation. Transport of the peroxisomal enzyme D-amino acid oxidase (DAAO) appears dysregulated by specific pharmaceuticals, e.g., the anti-overactive bladder drug propiverine or a norepinephrine/serotonin reuptake inhibitor (NSRI), resulting in massive cytosolic and nuclear accumulations in rat kidney. To assess the underlying molecular mechanism of the latter, we aimed to characterize the nature of peroxisomal and cyto-nuclear shuttling of human and rat DAAO overexpressed in three cell lines using confocal microscopy. Indeed, interference with peroxisomal transport via deletion of the PTS1 signal or PEX5 knockdown resulted in induced nuclear DAAO localization. Having demonstrated the absence of active nuclear import and employing variably sized mCherry- and/or EYFP-fusion proteins of DAAO and catalase, we showed that peroxisomal proteins ≤134 kDa can passively diffuse into mammalian cell nuclei-thereby contradicting the often-cited 40 kDa diffusion limit. Moreover, their inherent nuclear presence and nuclear accumulation subsequent to proteasome inhibition or abrogated peroxisomal transport suggests that nuclear localization is a characteristic in the lifecycle of peroxisomal proteins. Based on this molecular trafficking analysis, we suggest that pharmaceuticals like propiverine or an NSRI may interfere with peroxisomal protein targeting and import, consequently resulting in massive nuclear protein accumulation in vivo.

  5. Evaluation of antisecretory, gastroprotective and in-vitro antacid capacity of Fumaria indica in rats.

    Science.gov (United States)

    Chandra, Phool; Kishore, Kamal; Ghosh, Ashoke Kumar

    2015-09-01

    Fumaria indica is used for its anthelmintic, antidyspeptic, cholagogue, diaphoretic, diuretic, laxative, stomachic, tonic properties and claimed to possess various properties for the ailments of blood, skin, gastrointestinal systems and central nervous system. The present study was undertaken to evaluate antisecretory, gastroprotective and in-vitro antacid capacity of ethanol extract from F. indica in rats. Evaluation of F. indica extract as antisecretory was carried out by pyloric ligation induced ulcer model. The gastroprotective effect was carried out by absolute ethanol induced ulcer model. Integrity of gastric mucosa was evaluated by estimation of GSH and gastric mucus level. The in-vitro antacid capacity was evaluated by titration method. Ethanol extract of F. indica at 200 mg kg(-1), orally showed inhibition of secretion in pyloric ligation model. GSH level (1.67 μg mg(-1) protein), gastricwall mucus (240.76 μg g(-1) wet glandular tissue) and percentage protection (77.59%) of ulcer were significantly (P indica was compared with the standard. Conclusively, it appears that F. indica possess antisecretory (inhibition of acid secretion), gastroprotective (potentiation of defensive factors) and in-vitro antacid activity.

  6. Traceless chemical ligation from S-, O-, and N-acyl isopeptides.

    Science.gov (United States)

    Panda, Siva S; Hall, C Dennis; Oliferenko, Alexander A; Katritzky, Alan R

    2014-04-15

    Peptides are ubiquitous in nature where they play crucial roles as catalysts (enzymes), cell membrane ion transporters, and structural elements (proteins) within biological systems. In addition, both linear and cyclic peptides have found use as pharmaceuticals and components of various conjugate molecular systems. Small wonder then that chemists throughout the ages have sought to mimic nature by synthesis of the amide polymers known as peptides and proteins. The fundamental reaction in the formation of a peptide bond is condensation of an amine of one amino acid with the activated carbonyl group of another. This "fragment condensation" has been achieved in many ways both in solution and by solid-phase peptide synthesis (SPSS) on resin. The most successful method for in-solution coupling is known as native chemical ligation (NCL), and the technique dates back to the pioneering work of Wieland (1953) and subsequently Kent (1994) among many others. This Account builds on the established principles of NCL as applied specifically to S-, O-, and N-isopeptides, molecules that are generally more soluble and less prone to aggregation than native peptides. This Account also covers NCL of isopeptides containing terminal and nonterminal S-acylated cysteine units, reactions that enable the synthesis of native peptides from S-acyl peptides without the use of auxiliaries. With C-terminal S-acyl isopeptides, NCL was carried out under microwave irradiation in phosphate buffer (pH 7.3) at 50 °C. Intramolecular acyl migration was observed through 5-19-membered transition states with relative rates, as assessed by product analysis, in the order, 5 > 10 > 11 > 14, 16, or 17 > 12 > 13, 15, or 19 > 18 ≫ 9 > 8. The rate/pH profile for the 15-membered TS showed a maximum for ligated product versus transacylation at pH 7.0-7.3 presumably associated with the pKa of the N-nucleophile in the hydrogen-bonded TS. Cysteine occurs at low abundance (1.7%) in natural peptides and is rarely

  7. Effects of Autoclaving Soy-Free and Soy-Containing Diets for Laboratory Rats on Protein and Energy Values Determined In Vitro and In Vivo

    Science.gov (United States)

    Taciak, Marcin; Tuśnio, Anna; Święch, Ewa; Barszcz, Marcin; Staśkiewicz, Łukasz; Skomiał, Jacek; Paradziej-Łukowicz, Jolanta; Pastuszewska, Barbara

    2015-01-01

    Autoclaving diminishes the nutritional value of rat diets, depending on the duration and temperature of the process and the type of dietary protein. We evaluated in vivo and in vitro the effects of autoclaving on the protein and energy values of soy-free and soy-containing rat diets. The true digestibility and biological value of the dietary protein were determined in a 10-d experiment involving 28-d-old Wistar Crl:WI(Han) male rats fed casein- or soy-containing diet that was autoclaved for 20 min at 121 °C (T1), 10 min at 134 °C (T2), or not autoclaved (T0). The apparent protein digestibility and metabolizable energy concentration of experimental diets were assayed during an 18-d trial involving 6-wk-old Wistar-Crl:WI(Han) male rats and compared with a commercial diet. The neutral detergent fiber (NDF) content, amount of protein bound to NDF, protein solubility, and in vitro ileal protein digestibility were determined. Autoclaving decreased protein solubility, with the T2 condition having a greater effect than that of T1, and decreased the protein parameters determined in vivo, except for the apparent digestibility of the standard rat diet. Autoclaving decreased metabolizable energy slightly. The Atwater formula yielded higher values than those determined in rats, in vitro, and calculated according to the pig equation. We conclude that autoclaving diets according to the T1 program was less detrimental to dietary protein than was T2 and that the NDF content and protein solubility may be helpful in assessing the effect of autoclaving. The pig formula and in vitro method appear to be valid for estimating the metabolizable energy of rat diets. PMID:26424248

  8. Interaction of Agouti protein with the melanocortin 1 receptor in vitro and in vivo

    Science.gov (United States)

    Ollmann, Michael M.; Lamoreux, M. Lynn; Wilson, Brent D.; Barsh, Gregory S.

    1998-01-01

    Agouti protein and Agouti-related protein (Agrp) are paracrine-signaling molecules that normally regulate pigmentation and body weight, respectively. These proteins antagonize the effects of α-melanocyte-stimulating hormone (α-MSH) and other melanocortins, and several alternatives have been proposed to explain their biochemical mechanisms of action. We have used a sensitive bioassay based on Xenopus melanophores to characterize pharmacologic properties of recombinant Agouti protein, and have directly measured its cell-surface binding to mammalian cells by use of an epitope-tagged form (HA–Agouti) that retains biologic activity. In melanophores, Agouti protein has no effect in the absence of α-MSH, but its action cannot be explained solely by inhibition of α-MSH binding. In 293T cells, expression of the Mc1r confers a specific, high-affinity binding site for HA-Agouti. Binding is inhibited by α-MSH, or by Agrp, which indicates that α-MSH and Agouti protein bind in a mutually exclusive way to the Mc1r, and that the similarity between Agouti protein and Agrp includes their binding sites. The effects of Agouti and the Mc1r in vivo have been examined in a sensitized background provided by the chinchilla (Tyrc-ch) mutation, which uncovers a phenotypic difference between overexpression of Agouti in lethal yellow (Ay/a) mice and loss of Mc1r function in recessive yellow (Mc1re/Mc1re) mice. Double and triple mutant studies indicate that a functional Mc1r is required for the pigmentary effects of Agouti, and suggest that Agouti protein can act as an agonist of the Mc1r in a way that differs from α-MSH stimulation. These results resolve questions regarding the biochemical mechanism of Agouti protein action, and provide evidence of a novel signaling mechanism whereby α-MSH and Agouti protein or Agrp function as independent ligands that inhibit each other’s binding and transduce opposite signals through a single receptor. PMID:9450927

  9. Kinetic Controlled Tag-Catcher Interactions for Directed Covalent Protein Assembly.

    Science.gov (United States)

    Tan, Lee Ling; Hoon, Shawn S; Wong, Fong T

    2016-01-01

    Over the last few years, a number of different protein assembly strategies have been developed, greatly expanding the toolbox for controlling macromolecular assembly. One of the most promising developments is a rapid protein ligation approach using a short polypeptide SpyTag and its partner, SpyCatcher derived from Streptococcus pyogenes fibronectin-binding protein, FbaB. To extend this technology, we have engineered and characterized a new Tag-Catcher pair from a related fibronectin-binding protein in Streptococcus dysgalactiae. The polypeptide Tag, named SdyTag, was constructed based on the native Cna protein B-type (CnaB) domain and was found to be highly unreactive to SpyCatcher. SpyCatcher has 320-fold specificity for its native SpyTag compared to SdyTag. Similarly, SdyTag has a 75-fold specificity for its optimized Catcher, named SdyCatcherDANG short, compared to SpyCatcher. These Tag-Catcher pairs were used in combination to demonstrate specific sequential assembly of tagged proteins in vitro. We also demonstrated that the in vivo generation of circularized proteins in a Tag-Catcher specific manner where specific Tags can be left unreacted for use in subsequent ligation reactions. From the success of these experiments, we foresee the application of SdyTags and SpyTags, not only, for multiplexed control of protein assembly but also for the construction of novel protein architectures.

  10. Kinetic Controlled Tag-Catcher Interactions for Directed Covalent Protein Assembly.

    Directory of Open Access Journals (Sweden)

    Lee Ling Tan

    Full Text Available Over the last few years, a number of different protein assembly strategies have been developed, greatly expanding the toolbox for controlling macromolecular assembly. One of the most promising developments is a rapid protein ligation approach using a short polypeptide SpyTag and its partner, SpyCatcher derived from Streptococcus pyogenes fibronectin-binding protein, FbaB. To extend this technology, we have engineered and characterized a new Tag-Catcher pair from a related fibronectin-binding protein in Streptococcus dysgalactiae. The polypeptide Tag, named SdyTag, was constructed based on the native Cna protein B-type (CnaB domain and was found to be highly unreactive to SpyCatcher. SpyCatcher has 320-fold specificity for its native SpyTag compared to SdyTag. Similarly, SdyTag has a 75-fold specificity for its optimized Catcher, named SdyCatcherDANG short, compared to SpyCatcher. These Tag-Catcher pairs were used in combination to demonstrate specific sequential assembly of tagged proteins in vitro. We also demonstrated that the in vivo generation of circularized proteins in a Tag-Catcher specific manner where specific Tags can be left unreacted for use in subsequent ligation reactions. From the success of these experiments, we foresee the application of SdyTags and SpyTags, not only, for multiplexed control of protein assembly but also for the construction of novel protein architectures.

  11. Ligating perforators in abdominoplasty reduces the risk of seroma.

    Science.gov (United States)

    Skillman, J M; Venus, M R; Nightingale, P; Titley, O G; Park, A

    2014-04-01

    Seroma formation, a common complication of abdominoplasty, can cause patient discomfort and inconvenience. This study aimed to compare seroma rates after ligation and diathermy of large abdominal perforating vessels during abdominoplasty. Consecutive patients undergoing abdominoplasty with epigastric undermining between 2004 and 2011 were studied. Body mass index (BMI), age at operation, smoking history, preoperative weight loss, operative details, perioperative fluid infiltration, concomitant abdominal liposuction, ligation of perforators by clips, suture or diathermy, use of quilting sutures, weight of tissue removed, postoperative drainage, inpatient stay, and seroma rates were recorded. Statistical analysis was undertaken using the unpaired t test, Fisher's exact test, the Mann-Whitney U test, and Kendall's tau-b test. The study included 90 patients. The incidence of seroma was significantly lower among the patients who had perforators ligated (4/60, 6.7%) than among those who had diathermy (10/30, 33%) (p=0.002, Fisher's exact test). Seroma formation was significantly associated with a higher BMI, (27.45 vs. 25.16 kg/m2; p=0.025, t test) but not with preoperative weight loss. Postoperative fluid drainage did not differ significantly between ligated and diathermied perforators (p=0.716 Mann-Whitney U test). Use of ligation by clip or suture rather than by diathermy to ablate large abdominal perforators significantly reduced the incidence of seroma among abdominoplasty patients. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

  12. Infrared coagulation versus rubber band ligation in early stage hemorrhoids

    Directory of Open Access Journals (Sweden)

    P.J. Gupta

    2003-10-01

    Full Text Available The ideal therapy for early stages of hemorrhoids is always debated. Some are more effective but are more painful, others are less painful but their efficacy is also lower. Thus, comfort or efficacy is a major concern. In the present randomized study, a comparison is made between infrared coagulation and rubber band ligation in terms of effectiveness and discomfort. One hundred patients with second degree bleeding piles were randomized prospectively to either rubber band ligation (N = 54 or infrared coagulation (N = 46. Parameters measured included postoperative discomfort and pain, time to return to work, relief in incidence of bleeding, and recurrence rate. The mean age was 38 years (range 19-68 years. The mean duration of disease was 17.5 months (range 12 to 34 months. The number of male patients was double that of females. Postoperative pain during the first week was more intense in the band ligation group (2-5 vs 0-3 on a visual analogue scale. Post-defecation pain was more intense with band ligation and so was rectal tenesmus (P = 0.0059. The patients in the infrared coagulation group resumed their duties earlier (2 vs 4 days, P = 0.03, but also had a higher recurrence or failure rate (P = 0.03. Thus, we conclude that band ligation, although more effective in controlling symptoms and obliterating hemorrhoids, is associated with more pain and discomfort to the patient. As infrared coagulation can be conveniently repeated in case of recurrence, it could be considered to be a suitable alternative office procedure for the treatment of early stage hemorrhoids.

  13. Corrosion behavior of self-ligating and conventional metal brackets

    Directory of Open Access Journals (Sweden)

    Lúcio Henrique Esmeraldo Gurgel Maia

    2014-04-01

    Full Text Available Objective: To test the null hypothesis that the aging process in self-ligating brackets is not higher than in conventional brackets. Methods: Twenty-five conventional (GN-3M/Unitek; GE-GAC; VE-Aditek and 25 self-ligating (SCs-3M/Unitek; INs-GAC; ECs-Aditek metal brackets from three manufacturers (n = 150 were submitted to aging process in 0.9% NaCl solution at a constant temperature of 37 ± 1ºC for 21 days. The content of nickel, chromium and iron ions in the solution collected at intervals of 7, 14 and 21 days was quantified by atomic absorption spectrophotometry. After the aging process, the brackets were analyzed by scanning electron microscopy (SEM under 22X and 1,000X magnifications. Results: Comparison of metal release in self-ligating and conventional brackets from the same manufacturer proved that the SCs group released more nickel (p < 0.05 than the GN group after 7 and 14 days, but less chromium (p < 0.05 after 14 days and less iron (p < 0.05 at the three experimental time intervals. The INs group released less iron (p < 0.05 than the GE group after 7 days and less nickel, chromium and iron (p < 0.05 after 14 and 21 days. The ECs group released more nickel, chromium and iron (p < 0.05 than the VE group after 14 days, but released less nickel and chromium (p < 0.05 after 7 days and less chromium and iron (p < 0.05 after 21 days. The SEM analysis revealed alterations on surface topography of conventional and self-ligating brackets. Conclusions: The aging process in self-ligating brackets was not greater than in conventional brackets from the same manufacturer. The null hypothesis was accepted.

  14. The core protein of classical Swine Fever virus is dispensable for virus propagation in vitro.

    Directory of Open Access Journals (Sweden)

    Christiane Riedel

    Full Text Available Core protein of Flaviviridae is regarded as essential factor for nucleocapsid formation. Yet, core protein is not encoded by all isolates (GBV- A and GBV- C. Pestiviruses are a genus within the family Flaviviridae that affect cloven-hoofed animals, causing economically important diseases like classical swine fever (CSF and bovine viral diarrhea (BVD. Recent findings describe the ability of NS3 of classical swine fever virus (CSFV to compensate for disabling size increase of core protein (Riedel et al., 2010. NS3 is a nonstructural protein possessing protease, helicase and NTPase activity and a key player in virus replication. A role of NS3 in particle morphogenesis has also been described for other members of the Flaviviridae (Patkar et al., 2008; Ma et al., 2008. These findings raise questions about the necessity and function of core protein and the role of NS3 in particle assembly. A reverse genetic system for CSFV was employed to generate poorly growing CSFVs by modification of the core gene. After passaging, rescued viruses had acquired single amino acid substitutions (SAAS within NS3 helicase subdomain 3. Upon introduction of these SAAS in a nonviable CSFV with deletion of almost the entire core gene (Vp447(Δc, virus could be rescued. Further characterization of this virus with regard to its physical properties, morphology and behavior in cell culture did not reveal major differences between wildtype (Vp447 and Vp447(Δc. Upon infection of the natural host, Vp447(Δc was attenuated. Hence we conclude that core protein is not essential for particle assembly of a core-encoding member of the Flaviviridae, but important for its virulence. This raises questions about capsid structure and necessity, the role of NS3 in particle assembly and the function of core protein in general.

  15. Binding of human serum proteins to titanium dioxide particles in vitro.

    Science.gov (United States)

    Zaqout, Mazen S K; Sumizawa, Tomoyuki; Igisu, Hideki; Higashi, Toshiaki; Myojo, Toshihiko

    2011-01-01

    To determine the capacity of human serum proteins to bind to titanium dioxide (TiO(2)) particles of different polymorphs and sizes. TiO(2) particles were mixed with diluted human serum, purified human serum albumin (HSA) or purified human serum gamma-globulin (HGG) solutions. After incubation at 37°C for 1 h, the particles were sedimented by centrifugation, and proteins in the supernatant, as well as those bound to the particles, were analyzed. The total protein concentration in the supernatant was lowered by TiO(2), whereas the albumin/globulin ratio was elevated by the particles. Incubation with TiO(2) also lowered the immunoglobulin, pre-albumin, beta2-microglobulin, ceruloplasmin and retinol-binding protein levels, but not ferritin levels, in the supernatant. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins in the supernatant, especially HGG, were observed to decrease, while those released from the particles (after adding 1% SDS and heating) increased, depending on the dose of TiO(2). Purified HGG and HSA were also bound to TiO(2), although the former appeared to have a higher affinity. All the proteins tested showed the highest binding potency to the amorphous particles (<50 nm) and the lowest to the rutile particles (<5,000 nm), while binding to anatase particles was intermediate. The affinity to the larger anatase was higher than that to smaller anatase particles in most cases. Human serum proteins, including the two major components, HSA and HGG, are bound by TiO(2) particles. The polymorph of the particles seems to be important for determining the binding capacity of the particles and it may affect distribution of the particles in the body.

  16. Cloning, Overexpression and in vitro Antifungal Activity of Zea Mays PR10 Protein.

    Science.gov (United States)

    Zandvakili, Niloofar; Zamani, Mohammadreza; Motallebi, Mostafa; Moghaddassi Jahromi, Zahra

    2017-03-01

    Plants have various defense mechanisms such as production of antimicrobial peptides, particularly pathogenesis related proteins (PR proteins). PR10 family is an essential member of this group, with antifungal, antibacterial and antiviral activities. The goal of this study is to assess the antifungal activity of maize PR10 against some of fungal phytopathogens. Zea mays PR10 gene (TN-05-147) was cloned from genomic DNA and cDNA and overexpressed in Escherichia coli. The existence of a 77- bp intron and two exons in PR10 was confi rmed by comparing the genomic and cDNA sequences. The PR10 cDNA was cloned in pET26b (+) expression vector and transformed into E. coli strain Rosetta DE3 in order to express PR10 recombinant protein. Expression of the recombinant protein was checked by western analysis. Recombinant PR10 appeared as insoluble inclusion bodies and thus solubilized and refolded. PR10 was isolated using Ni- NTA column. The activity of the refolded protein was confi rmed by DNA degradation test. The antifungal activity of PR10 was assessed using radial diff usion, disc diff usion and spore germination. The hemolytic assay was performed to investigate the biosafety of recombinant PR10. Recombinant maize PR10 exerted broad spectrum antifungal activity against Botrytis cinerea, Sclerotinia sclerotiorum, Fusarium oxysporum, Verticillium dahlia and Alternaria solani. Hemolysis biosafety test indicated that the protein is not poisonous to mammalian cells. Maize PR10 has the potential to be used as the antifungal agent against diff erent fungal phytopathogens. Therefore, this protein can be used in order to produce antifungal agents and fungi resistance transgenic plants.

  17. [Handling G-protein-coupled receptors: expression, purification and in vitro stabilization].

    Science.gov (United States)

    Banères, Jean-Louis; Mouillac, Bernard

    2012-10-01

    Among the different classes of integral membrane proteins, G protein-coupled receptors (GPCR) constitute the largest family. They are involved in most essential physiological functions and particularly play a key role in cell-to-cell communication and sensory signal transduction. They represent targets for approximately 30% of currently marketed drugs. In order to better understand their functioning, define their tridimensional structure and develop novel selective and efficient therapeutic compounds, it is crucial to purify these proteins for a full characterization. However, this biochemical step is not trivial since GPCR are present in membranes at very low levels and they require detergents to be extracted from their natural lipid environment and be handled as functional proteins. No universal strategy for GPCR production, purification and stabilization is currently available; each single GPCR possesses a unique set of physicochemical characteristics, preference for some detergents upon solubilization and specific conditions for purification. During the last decade, major breakthroughs regarding overexpression, purification and above all GPCR stabilization, thanks to amphipols and nanodiscs, opened very exciting perspectives for structural and dynamic investigations of these membrane proteins. The aim of this chapter is to provide an overview of the different aspects of GPCR handling. © 2012 médecine/sciences – Inserm / SRMS.

  18. Sensitive detection of Aβ protofibrils by proximity ligation - relevance for Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Gustafsdottir Sigrun

    2010-10-01

    Full Text Available Abstract Background Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of Aβ peptides are cornerstones in the pathology of Alzheimer's disease. Soluble protein aggregates are therefore potential diagnostic and prognostic biomarkers for their cognate disorders. Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins. Here we have established a proximity ligation assay (PLA for specific and sensitive detection of Aβ protofibrils via simultaneous recognition of three identical determinants present in the aggregates. PLA is a versatile technology in which the requirement for multiple target recognitions is combined with the ability to translate signals from detected target molecules to amplifiable DNA strands, providing very high specificity and sensitivity. Results For specific detection of Aβ protofibrils we have used a monoclonal antibody, mAb158, selective for Aβ protofibrils in a modified PLA, where the same monoclonal antibody was used for the three classes of affinity reagents required in the assay. These reagents were used for detection of soluble Aβ aggregates in solid-phase reactions, allowing detection of just 0.1 pg/ml Aβ protofibrils, and with a dynamic range greater than six orders of magnitude. Compared to a sandwich ELISA setup of the same antibody the PLA increases the sensitivity of the Aβ protofibril detection by up to 25-fold. The assay was used to measure soluble Aβ aggregates in brain homogenates from mice transgenic for a human allele predisposing to Aβ aggregation. Conclusions The proximity ligation assay is a versatile analytical technology for proteins, which can provide highly sensitive and specific detection of Aβ aggregates - and by implication other protein aggregates of relevance in Alzheimer's disease and other neurodegenerative disorders.

  19. Characterization of early and terminal complement proteins associated with polymorphonuclear leukocytes in vitro and in vivo after spinal cord injury

    Directory of Open Access Journals (Sweden)

    Galvan Manuel D

    2008-06-01

    Full Text Available Abstract Background The complement system has been suggested to affect injury or disease of the central nervous system (CNS by regulating numerous physiological events and pathways. The activation of complement following traumatic CNS injury can also result in the formation and deposition of C5b-9 membrane attack complex (C5b-9/MAC, causing cell lysis or sublytic effects on vital CNS cells. Although complement proteins derived from serum/blood-brain barrier breakdown can contribute to injury or disease, infiltrating immune cells may represent an important local source of complement after injury. As the first immune cells to infiltrate the CNS within hours post-injury, polymorphonuclear leukocytes (PMNs may affect injury through mechanisms associated with complement-mediated events. However, the expression/association of both early and terminal complement proteins by PMNs has not been fully characterized in vitro, and has not observed previously in vivo after traumatic spinal cord injury (SCI. Method We investigated the expression of complement mRNAs using rt-PCR and the presence of complement proteins associated with PMNs using immunofluroescence and quantitative flow cytometry. Results Stimulated or unstimulated PMNs expressed mRNAs encoding for C1q, C3, and C4, but not C5, C6, C7 or C9 in culture. Complement protein C1q or C3 was also detected in less than 30% of cultured PMNs. In contrast, over 70% of PMNs that infiltrated the injured spinal cord were associated with C1q, C3, C7 and C5b-9/MAC 3 days post-SCI. The localization/association of C7 or C5b-9/MAC with infiltrating PMNs in the injured spinal cord suggests the incorporation or internalization of C7 or C5b-9/MAC bound cellular debris by infiltrating PMNs because C7 and C5b-9/MAC were mostly localized to granular vesicles within PMNs at the spinal cord epicenter region. Furthermore, PMN presence in the injured spinal cord was observed for many weeks post-SCI, suggesting that this

  20. The effect of tranilast on fibroblast activation protein α (FAP-α expression in normal and keloid fibroblasts in vitro

    Directory of Open Access Journals (Sweden)

    Paweł P. Antończak

    2017-07-01

    Full Text Available Introduction . Tranilast (N-(3’,4’-demethoxycinnamoyl-anthranilic acid is an anti-allergic drug. Its mechanism of action is based on the inhibition of antigen-induced release of chemical mediators from mast cells and basophils. It also reveals antifibroproliferative activities. These properties of tranilast are used in the treatment of hypertrophic scars and keloids. Keloids are characterized by incorrect extracellular matrix components turnover. Fibroblasts derived from keloids reveal overproduction of collagen type I and decreased degradation of extracellular matrix in comparison with normal fibroblasts. Fibroblast activation protein α (FAP-α may play an important role in remodeling of extracellular matrix and the invasive properties of keloids. Objective . In the present study, the effect of tranilast on expression of FAP-α gene and its protein was evaluated in normal human dermal fibroblasts and fibroblasts derived from keloids cultured in vitro . Materials and methods. In the first stage of the study, the influence of tranilast on cell viability was estimated. The second stage of the study included the quantitative evaluation of FAP-α mRNA expression in normal and keloid fibroblasts treated with tranilast. The third stage of the study comprised fibroblast activation protein α expression analysis in the examined cells treated with tranilast. Results and conclusions . The expression of FAP-α gene and fibroblast activation protein α is higher in keloid fibroblasts. Tranilast at concentrations of 3 μM and 30 μM up-regulated mRNA FAP-α expression in normal fibroblasts but did not influence keloid fibroblasts. The drug, at concentrations of 30 μM and 300 μM up-regulated fibroblast activation protein α expression in normal fibroblasts and did not influence keloid fibroblasts. Tranilast antiproliferative effect is not associated with FAP-α expression in keloid fibroblasts.

  1. In vitro expression and analysis of the 826 human G protein-coupled receptors

    Directory of Open Access Journals (Sweden)

    Xuechen Lv

    2016-04-01

    Full Text Available ABSTRACT G protein-coupled receptors (GPCRs are involved in all human physiological systems where they are responsible for transducing extracellular signals into cells. GPCRs signal in response to a diverse array of stimuli including light, hormones, and lipids, where these signals affect downstream cascades to impact both health and disease states. Yet, despite their importance as therapeutic targets, detailed molecular structures of only 30 GPCRs have been determined to date. A key challenge to their structure determination is adequate protein expression. Here we report the quantification of protein expression in an insect cell expression system for all 826 human GPCRs using two different fusion constructs. Expression characteristics are analyzed in aggregate and among each of the five distinct subfamilies. These data can be used to identify trends related to GPCR expression between different fusion constructs and between different GPCR families, and to prioritize lead candidates for future structure determination feasibility.

  2. Inhibitory effect of corcin on aggregation of 1N/4R human tau protein in vitro

    Directory of Open Access Journals (Sweden)

    Ali Mohammadi Karakani

    2015-05-01

    Full Text Available Objective(s:Alzheimer's disease (AD is the most common age-related neurodegenerative disorder. One of the hallmarks of AD is an abnormal accumulation of fibril forms of tau protein which is known as a microtubule associated protein. In this regard, inhibition of tau aggregation has been documented to be a potent therapeutic approach in AD and tauopathies. Unfortunately, the available synthetic drugs have modest beneficial efficacy with several side effects. Therefore, pipeline drugs from natural sources with anti-aggregation properties can be useful in the prevention and treatment of AD. Among medicinal plants, saffron (Crocus sativus, L., as a traditional herbal medicine has different pharmacological properties and can be used as treatment for several nervous system impairment including depression and dementia. Crocin as a major constituent of saffron is the glycosylated form of crocetin. Materials and Methods:  In this study, we investigated the inhibitory effect of crocin on aggregation of recombinant human tau protein 1N/4R isoform using biochemical methods and cell culture. Results:  Results revealed that tau protein under the fibrillation condition and in the presence of crocin had enough stability with low tendency for aggregation. Crocin inhibited tau aggregation with IC50 of 100 µg/ml.  Furthermore, transmission electron microscopy images confirmed that crocin could suppress the formation of tau protein filaments. Conclusion: Inhibitory effect of crocin could be related to its interference with nucleation phase that led to increases in monomer species of tau protein. Based on our results, crocin is recommended as a proper candidate to be used in AD treatment.

  3. LegC3, an Effector Protein from Legionella pneumophila, Inhibits Homotypic Yeast Vacuole Fusion In Vivo and In Vitro

    Science.gov (United States)

    Bennett, Terry L.; Kraft, Shannon M.; Reaves, Barbara J.; Mima, Joji; O’Brien, Kevin M.; Starai, Vincent J.

    2013-01-01

    During infection, the intracellular pathogenic bacterium Legionella pneumophila causes an extensive remodeling of host membrane trafficking pathways, both in the construction of a replication-competent vacuole comprised of ER-derived vesicles and plasma membrane components, and in the inhibition of normal phagosome:endosome/lysosome fusion pathways. Here, we identify the LegC3 secreted effector protein from L. pneumophila as able to inhibit a SNARE- and Rab GTPase-dependent membrane fusion pathway in vitro, the homotypic fusion of yeast vacuoles (lysosomes). This vacuole fusion inhibition appeared to be specific, as similar secreted coiled-coiled domain containing proteins from L. pneumophila, LegC7/YlfA and LegC2/YlfB, did not inhibit vacuole fusion. The LegC3-mediated fusion inhibition was reversible by a yeast cytosolic extract, as well as by a purified soluble SNARE, Vam7p. LegC3 blocked the formation of trans-SNARE complexes during vacuole fusion, although we did not detect a direct interaction of LegC3 with the vacuolar SNARE protein complexes required for fusion. Additionally, LegC3 was incapable of inhibiting a defined synthetic model of vacuolar SNARE-driven membrane fusion, further suggesting that LegC3 does not directly inhibit the activity of vacuolar SNAREs, HOPS complex, or Sec17p/18p during membrane fusion. LegC3 is likely utilized by Legionella to modulate eukaryotic membrane fusion events during pathogenesis. PMID:23437241

  4. In vitro digestion of Pickering emulsions stabilized by soft whey protein microgel particles: influence of thermal treatment.

    Science.gov (United States)

    Sarkar, Anwesha; Murray, Brent; Holmes, Melvin; Ettelaie, Rammile; Abdalla, Azad; Yang, Xinyi

    2016-04-21

    Emulsions stabilized by soft whey protein microgel particles have gained research interest due to their combined advantages of biocompatibility and a high degree of resistance to coalescence. We designed Pickering oil-in-water emulsions using whey protein microgels by a facile route of heat-set gel formation followed by mechanical shear and studied the influence of heat treatment on emulsions stabilized by these particles. The aim of this study was to compare the barrier properties of the microgel particles and heat-treated fused microgel particles at the oil-water interface in delaying the digestion of the emulsified lipids using an in vitro digestion model. A combination of transmission electron microscopy and surface coverage measurements revealed an increased coverage of heat-treated microgel particles at the interface. The heat-induced microgel particle aggregation and, therefore, a fused network at the oil-water interface were more beneficial to delay the rate of digestion in the presence of pure lipase and bile salts compared to intact whey protein microgel particles, as shown by the measurements of zeta potential and free fatty acid release, plus theoretical calculations. However, simulated gastric digestion with pepsin impacted significantly on such barrier effects, due to the proteolysis of the particle network at the interface irrespective of the heat treatment, as visualized using sodium dodecyl sulfate polyacryl amide gel electrophoresis measurements.

  5. Effects of milk proteins on release properties and particle morphology of β-carotene emulsions during in vitro digestion.

    Science.gov (United States)

    Liu, Yuwei; Lei, Fei; Yuan, Fang; Gao, Yanxiang

    2014-11-01

    In the present study, β-lactoglobulin, sodium caseinate, lactalbumin and lactoferrin were used to prepare β-carotene emulsions. The milk protein-stabilized emulsions were explored using an in vitro release model to elucidate the effects of different milk proteins on β-carotene release properties in the stomach, duodenum and small intestine, respectively. Notable changes in the droplet size and size distribution were observed among these four oil-in-water (O/W) milk protein emulsions. In the gastric environment, the highest β-carotene release rate (2.9%) was achieved in β-lactoglobulin emulsion with a remarkable change in the particle size. In the simulated intestine, the best β-carotene micellarization potency (92%) was observed in β-lactoglobulin emulsion and its droplet diameter moderately increased from 215 nm to 471 nm. Moreover, substantial release of β-carotene was found in the small intestine for the four types of emulsions. It was concluded that β-carotene release in different digestive stages was characterized by the emulsion interfacial composition.

  6. Activities of the bimodal fluorescent protein produced by Photobacterium phosphoreum strain bmFP in the luciferase reaction in vitro.

    Science.gov (United States)

    Karatani, H; Konaka, T

    2000-02-01

    The activity of the bimodal fluorescent protein (bmFP) (lambda max, 488 and 517 nm) in the in vitro luciferase reaction has been studied. The bmFP that is produced by Photobacterium phosphoreum strain bmFP is a dimer of two homologous subunits binding four riboflavin 5'-phosphate (FMN)-myristate chromophores. The addition of bmFP to the luciferase reaction in the presence of the lumazine protein prevented the lumazine protein-induced blue shift in the emission band. The bmFP reduced electrochemically serves as a substrate in the luciferase reaction in the absence of added FMN, resulting in light emission with a single maximum at about 487 nm. The bmFP was also active in lieu of FMN in the NADH/FMN oxidoreductase (flavin reductase)-luciferase coupled bioluminescence reaction in the absence of added FMN. In the coupled reaction, bioluminescence with the isolated bmFP chromophore was weaker than that with the holo-bmFP. After bmFP was used in luciferase reactions initiated either chemically or electrochemically, it was still capable of emitting bimodal fluorescence.

  7. Effect of the presence of protein on lipolysis and lipid oxidation occurring during in vitro digestion of highly unsaturated oils.

    Science.gov (United States)

    Nieva-Echevarría, Bárbara; Goicoechea, Encarnación; Guillén, María D

    2017-11-15

    The effect of the presence of ovalbumin and soy protein isolate on lipolysis and oxidation taking place during in vitro gastrointestinal digestion of slightly oxidized sunflower and flaxseed oils was addressed. The extent of lipolysis, the molar proportions of acyl groups/fatty acids after digestion, and the oxidation products formed were studied by Proton Nuclear Magnetic Resonance. The presence of proteins provoked a higher hydrolysis in triglycerides, a lower decrease of polyunsaturated chains, and a lower generation of oxidation compounds (conjugated dienes in chains having also hydroperoxy/hydroxy groups, epoxides and aldehydes); the formation of hydroxides was clearly favoured over that of hydroperoxides. Study of headspace composition by Solid Phase Microextraction-Gas Chromatography/Mass Spectrometry confirmed that oxidation advanced to a lesser extent in the presence of protein. Thus, amino acids/peptides released during digestion may show antioxidant properties, affecting not only the extent of lipid oxidation, but also reactions pathways. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Effect of protein corona magnetite nanoparticles derived from bread in vitro digestion on Caco-2 cells morphology and uptake.

    Science.gov (United States)

    Di Silvio, Desirè; Rigby, Neil; Bajka, Balazs; Mackie, Alan; Baldelli Bombelli, Francesca

    2016-06-01

    Nanoparticles (NPs) in biological fluids immediately interact with proteins forming a biomolecular corona (PC) that imparts their biological identity. While several studies on the formation of the PC in human plasma have been reported, the PC of orally administrated NPs has been less investigated, mostly in the presence of a food matrix. In fact, food matrixes when digested are subject of several dynamic changes that will certainly affect the PC formed on the NPs. The lack of studies on this topic is clearly related to the difficulty in isolating representative PC NPs from such a complex environment. In this work magnetite NPs were added to in vitro simulated digestion simultaneously with bread and PC NPs were isolated after gastric and duodenal phases by sucrose gradient ultracentrifugation (UC). The PC NPs were characterized in terms of size and protein composition. Translocation studies were then performed on Caco-2 monolayers in a serum free environment and cell morphology was characterized by confocal microscopy. PC NPs isolated from gastric and duodenal phases were different in size, surface charge and protein corona composition. NP cellular uptake was enhanced by the digestive PC inducing morphology changes in the cell monolayer. Overall, in this work we were able to isolate PC NPs from digested fluids in the presence of a food matrix and study their biological response on Caco-2 cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Effect of heat treatment and subsequent urea supplementation of sunflower meal on the in vitro ruminal degradability of crude protein content and its postruminal digestibility.

    Science.gov (United States)

    Veresegyházy, T; Kutas, F; Fekete, S; Csapó, J

    1989-01-01

    A three-phase laboratory procedure was used for predicting the degradability of the protein of extracted sunflower meal before (SFM) and after heat treatment (HSFM). The rumen fluid degraded 69.1% and 67.1% of the SFM and HSFM protein, respectively. The digestibility values of rumen undegraded protein (UDP) were 57.3% (SFM) and 57.0% (HSFM) with pepsin and 17.6% (SFM) and 15.9% (HSFM) with pancreatin. Urea supplementation practically did not alter the rumen degradability of HSFM protein, while the pepsin digestibility of UDP decreased to 47.2%. Four fractions (NH3, dissolved amino acids, oligopeptides and proteins) of rumen degradable crude protein (RDP) were also determined in vitro: 81 to 92% of the degraded crude protein was found in the fractions tested. Heat treatment reduced free NH3 content but did not alter the other three fractions. Urea supplementation decreased the quantity of NH3, peptides and proteins as well.

  10. Predicting omasal flow of nonammonia N and milk protein yield from in vitro-determined utilizable crude protein at the duodenum.

    Science.gov (United States)

    Gidlund, H; Vaga, M; Ahvenjärvi, S; Rinne, M; Ramin, M; Huhtanen, P

    2018-02-01

    This study evaluated the relationship between utilizable crude protein (uCP) at the duodenum estimated in vitro and omasal flow of crude protein (CP; omasal flow of nonammonia N × 6.25) measured in lactating dairy cows. In vivo data were obtained from previous studies estimating omasal digesta flow using a triple-marker method and 15N as microbial marker. A total of 34 different diets based on grass and red clover silages were incubated with buffered rumen fluid previously preincubated with carbohydrates for 3 h. The buffer solution was modified to contain 38 g of NaHCO3 and 1 g of (NH4)HCO3 in 1,000 mL of distilled water. Continuous sampling of the liquid phase for determination of ammonia-N was performed at 0.5, 4, 8, 12, 24, and 30 h after the start of incubation. The ammonia N concentrations after incubation were used to calculate uCP. The natural logarithm of uCP [g/kg of dry matter (DM)] at time points 0.5, 4, 8, 12, 24, and 30 h of incubation was plotted against time to estimate the concentration of uCP (g/kg of DM) at time points 16, 20, and 24 h using an exponential function. Fixed model regression analysis and mixed model regression analysis with random study effect were used to evaluate the relationships between predicted uCP (supply and concentration) and observed omasal CP flow and milk protein yield. Residual analysis was also conducted to evaluate whether any dietary factors influenced the relationships. The in vitro uCP method ranked the diets accurately in terms of total omasal CP flow (kg/d) or omasal CP flow per kilogram of DM intake. We also noted a close relationship between estimated uCP supply and adjusted omasal CP flow, as demonstrated by a coefficient of determination of 0.87, although the slope of 0.77 indicated that estimated uCP supply (kg/d) was greater than the value determined in vivo. The linear bias with mixed model analysis indicated that uCP supply overestimated the difference in omasal CP flow between the diets within a study

  11. Adherens junction proteins are expressed in collagen corneal equivalents produced in vitro with human cells.

    Science.gov (United States)

    Giasson, Claude J; Deschambeault, Alexandre; Carrier, Patrick; Germain, Lucie

    2014-01-01

    To test whether adherens junction proteins are present in the epithelium and the endothelium of corneal equivalents. Corneal cell types were harvested from human eyes and grown separately. Stromal equivalents were constructed by seeding fibroblasts into a collagen gel on which epithelial and endothelial cells were added on each side. Alternatively, bovine endothelial cells were used. At maturity, sections of stromal equivalents were processed for Masson's trichrome or indirect immunofluorescence using antibodies against pan-, N-, or E-cadherins or α- or β-catenins. Alternatively, stromal equivalents were dissected, to separate the proteins from the epithelium, endothelium, and stroma with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blots of the transferred proteins exposed to these primary antibodies were detected with chemiluminescence. Native corneas were processed similarly. Three or four layers of epithelial cells reminiscent of the native cornea (basal cuboidal and superficial flatter cells) lay over a stromal construct containing fibroblastic cells under which an endothelium is present. Western blots and indirect immunofluorescence revealed that, similarly to the native cornea, the epithelium reacted positively to antibodies against catenins (α and β) and E-cadherin. The endothelium of corneal constructs, whether of human or bovine origin, reacted mildly to catenins and N-cadherin. This collagen-based corneal equivalent simulated the native cornea. Cells from the epithelial and endothelial layers expressed adherens junction proteins, indicating the presence of cell-cell contacts and the existence of polarized morphology of these layers over corneal equivalents.

  12. Anabolic Properties of High Mobility Group Box Protein-1 in Human Periodontal Ligament Cells In Vitro

    Directory of Open Access Journals (Sweden)

    Michael Wolf

    2014-01-01

    Full Text Available High mobility group box protein-1 (HMGB1 is mainly recognized as a chemoattractant for macrophages in the initial phase of host response to pathogenic stimuli. However, recent findings provide evidence for anabolic properties in terms of enhanced proliferation, migration, and support of wound healing capacity of mesenchymal cells suggesting a dual role of the cytokine in the regulation of immune response and subsequent regenerative processes. Here, we examined potential anabolic effects of HMGB1 on human periodontal ligament (PDL cells in the regulation of periodontal remodelling, for example, during orthodontic tooth movement. Preconfluent human PDL cells (hPDL were exposed to HMGB1 protein and the influence on proliferation, migration, osteogenic differentiation, and biomineralization was determined by MTS assay, real time PCR, immunofluorescence cytochemistry, ELISA, and von Kossa staining. HMGB1 protein increased hPDL cell proliferation, migration, osteoblastic marker gene expression, and protein production as well as mineralized nodule formation significantly. The present findings support the dual character of HMGB1 with anabolic therapeutic potential that might support the reestablishment of the structural and functional integrity of the periodontium following periodontal trauma such as orthodontic tooth movement.

  13. LOCALIZATION OF THE SPERM PROTEIN SP22 AND INHIBITION OF FERTILITY IN VIVO AND IN VITRO

    Science.gov (United States)

    We previously established that the levels sperm membrane protein SP22 are highly correlated with the fertility of sperm from the cauda epididymidis of rats exposed to both epididymal and testicular toxicants, and that a testis-specific SP22 transcript is expressed in post-meiotic...

  14. Inhibition of protein synthesis in vitro by a lectin from Momordica charantia and by other haemagglutinins.

    Science.gov (United States)

    Barbieri, L; Lorenzoni, E; Stirpe, F

    1979-08-15

    Protein synthesis by a rabbit reticulocyte lysate is inhibited by the haemagglutinating lectins from Momordica charantia and Crotalaria juncea seeds and from the roe of Rutilus rutilus, and by a commercial preparation of the mitogenic lectin from Phytolacca americana. The haemagglutinins from the seeds of Ricinus communis and of Vicia cracca acquired inhibitory activity after their reduction with 2-mercaptoethanol.

  15. Biological value (in vitro and in sacco of chemically treated feather as rumen by pass protein source

    Directory of Open Access Journals (Sweden)

    W Puastuti

    2004-06-01

    Full Text Available A series of experiments has been conducted to study chemical processing method of feather meal using hydrocloric acid (HCl and to evaluate the biological values by in vitro and is sacco methods of the hydrolitic feather meal (HBA. Feather meal was hydrolyzed using four levels of HCl concentration (i.e.0, 6, 12 and 24% in three incubation times (i.e. 2, 4, and 6 days. The hydrolysis reaction was carried out in closed container in the ratio of feather meal and HCl of 2:1 (w/v. In vitro evaluation was conducted to measure dry matter (DM and organic matter (OM digestibility, DM solubility, ammonia (NH3 and volatile fatty acid (VFA content. In sacco to observe the degradation of HBA crude protein. Results of in sacco evaluation in rumen showed that soluble and degraded crude proteins (CP were significantly only affected by HCl concentration (P0.05. In vitro DM and OM digestibilities of HBA increased as the concentration of HCl was increased. The increase of DM digestibility followed the equation Y = -0.0231x3 + 0.7323x2 – 1.5716x + 12.383 (r = 0.994; and the OM digestibility followed the equation Y = -0.0229x3 + 0.7194x2 – 1.0606x + 15.951 (r = 0.993. Time of incubation, on the other hand, did not affect OM and DM digestibilities (P>0.05. DM solubility of HBA was significantly affected by HCl concentration and the length of incubation time (P<0.01. The increase of DM solubility was followed by the increase of NH3 content (P<0.01. The relation between DM solubility and NH3 content followed the equation Y = 0.4365x + 5.4047 (r = 0.966. The increase of DM solubility followed the equation Y = -0.027x3 + 0.9596x2 – 4.8142x + 5.3878 (r = 0.973 and the increase of NH3 content followed the equation Y = -0.0085x3 + 0.3175x2 – 1.4139x + 7.0889 (r = 0.992. Result of in sacco evaluation showed that fraction of crude protein (CP disolved and fraction of CP degraded in rumen was significantly affected by HCl concentration (P<0.01, while the rate of CP

  16. New Lanthanide Tag for the Generation of Pseudocontact Shifts in DNA by Site-Specific Ligation to a Phosphorothioate Group.

    Science.gov (United States)

    Wu, Zuyan; Lee, Michael D; Carruthers, Thomas J; Szabo, Monika; Dennis, Matthew L; Swarbrick, James D; Graham, Bim; Otting, Gottfried

    2017-06-21

    Pseudocontact shifts (PCS) generated by paramagnetic lanthanides provide a rich source of long-range structural restraints that can readily be measured by nuclear magnetic resonance (NMR) spectroscopy. Many different lanthanide-binding tags have been designed for site-specific tagging of proteins, but established routes for tagging DNA with a single metal ion rely on difficult chemical synthesis. Here we present a simple and practical strategy for site-specific tagging of inexpensive phosphorothioate (PT) oligonucleotides. Commercially available PT oligonucleotides are diastereomers with S and R stereoconfiguration at the backbone PT site. The respective SP and RP diastereomers can readily be separated by HPLC. A new alkylating lanthanide-binding tag, C10, was synthesized that delivered quantitative tagging yields with both diastereomers. PCSs were observed following ligation with the complementary DNA strand to form double-stranded DNA duplexes. The PCSs were larger for the SP than the RP oligonucleotide and good correlation between back-calculated and experimental PCSs was observed. The C10 tag can also be attached to cysteine residues in proteins, where it generates a stable thioether bond. Ligated to the A28C mutant of ubiquitin, the tag produced excellent fits of magnetic susceptibility anisotropy (Δχ) tensors, with larger tensors than for the tagged PT oligonucleotides, indicating that the tag is not completely immobilized after ligation with a PT group.

  17. In vitro, in silico and integrated strategies for the estimation of plasma protein binding. A review.

    Science.gov (United States)

    Lambrinidis, George; Vallianatou, Theodosia; Tsantili-Kakoulidou, Anna

    2015-06-23

    Plasma protein binding (PPB) strongly affects drug distribution and pharmacokinetic behavior with consequences in overall pharmacological action. Extended plasma protein binding may be associated with drug safety issues and several adverse effects, like low clearance, low brain penetration, drug-drug interactions, loss of efficacy, while influencing the fate of enantiomers and diastereoisomers by stereoselective binding within the body. Therefore in holistic drug design approaches, where ADME(T) properties are considered in parallel with target affinity, considerable efforts are focused in early estimation of PPB mainly in regard to human serum albumin (HSA), which is the most abundant and most important plasma protein. The second critical serum protein α1-acid glycoprotein (AGP), although often underscored, plays also an important and complicated role in clinical therapy and thus the last years it has been studied thoroughly too. In the present review, after an overview of the principles of HSA and AGP binding as well as the structure topology of the proteins, the current trends and perspectives in the field of PPB predictions are presented and discussed considering both HSA and AGP binding. Since however for the latter protein systematic studies have started only the last years, the review focuses mainly to HSA. One part of the review highlights the challenge to develop rapid techniques for HSA and AGP binding simulation and their performance in assessment of PPB. The second part focuses on in silico approaches to predict HSA and AGP binding, analyzing and evaluating structure-based and ligand-based methods, as well as combination of both methods in the aim to exploit the different information and overcome the limitations of each individual approach. Ligand-based methods use the Quantitative Structure-Activity Relationships (QSAR) methodology to establish quantitate models for the prediction of binding constants from molecular descriptors, while they provide

  18. Formation of a covalent complex between the 80,000-dalton adenovirus terminal protein and 5'-dCMP in vitro.

    OpenAIRE

    Lichy, J H; Horwitz, M. S.; Hurwitz, J

    1981-01-01

    An in vitro adenovirus DNA replication system catalyzed the formation of a covalent complex between an 80,000-dalton protein and 5'-dCMP in the presence of [alpha-32P-dCTP, MgCl2, ATP, and adenovirus (Ad) DNA with a protein covalently bound to the 5' end of each strand (Ad DNA-prot). The requirement for Ad DNA-prot in this reaction was similar to that for in vitro DNA replication. When dATP, dTTP, and the 2',3'-dideoxynucleoside triphosphate (ddNTP) ddGTP were included in the reaction mixture...

  19. In vitro studies of platelet adhesion, activation, and protein adsorption on curcumin-eluting biodegradable stent materials.

    Science.gov (United States)

    Pan, C J; Shao, Z Y; Tang, J J; Wang, J; Huang, N

    2007-09-01

    A major complication of coronary stenting is in-stent restenosis (ISR) due to thrombus formation. We hypothesized that locally released curcumin from coronary stent surface would inhibit ISR due to thrombus formation because of antithrombosis of curcumin. In the present work, curcumin-eluting polylactic acid-co-glycolic acid (PLGA) films were fabricated and their properties in vitro were investigated. The in vitro platelet adhesion and activation, as well as protein adsorption on curcumin-loading PLGA films were investigated to evaluate the blood compatibility of curcumin-eluting films. The structure of curcumin-eluting PLGA film and control was examined by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy indicating that the peaks of curcumin did not shift in curcumin-eluting films. The results of contact angle and surface free energy indicated that loading curcumin in PLGA would make PLGA become more hydrophilic, which contributed to the increase of polar fraction of surface free energy. With the increase of curcumin in films, platelets adhering to the curcumin-eluting films decreased significantly. The number of activation platelets decreased after incorporating curcumin in PLGA films. Loading curcumin in PLGA film can markedly reduce the fibrinogen adsorption. All results indicated that incorporating curcumin in PLGA film can improve the blood compatibility of PLGA films. It can be used to fabricate drug-eluting stent to prevent thrombosis formation.

  20. De novo protein sequencing, humanization and in vitro effects of an antihuman CD34 mouse monoclonal antibody.

    Science.gov (United States)

    Fan, Chia-Yu; Huang, Sheng-Yu; Chou, Min-Yuan; Lyu, Ping-Chiang

    2017-03-01

    QBEND/10 is a mouse immunoglobulin lambda-chain monoclonal antibody with strict specificity against human hematopoietic progenitor cell antigen CD34. Our in vitro study showed that QBEND/10 impairs the tube formation of human umbilical vein endothelial cells (HUVECs), suggesting that the antibody may be of potential benefit in blocking tumor angiogenesis. We provided a de novo protein sequencing method through tandem mass spectrometry to identify the amino acid sequences in the variable heavy and light chains of QBEND/10. To reduce immunogenicity for clinical applications, QBEND/10 was further humanized using the resurfacing approach. We demonstrate that the de novo sequenced and humanized QBEND/10 retains the biological functions of the parental mouse counterpart, including the binding kinetics to CD34 and blockage of the tube formation of the HUVECs.

  1. Effect of probiotic fermentation on antinutrients and in vitro protein and starch digestibilities of indigenously developed RWGT food mixture.

    Science.gov (United States)

    Sindhu, Sangeeta C; Khetarpaul, Neelam

    2002-01-01

    Indigenously developed RWGT food mixture which contained rice flour, whey, sprouted green gram paste and tomato pulp (2:1:1:1 w/w) was autoclaved 1.5 kg/cm2, 15 min, 121 degrees C), cooled and fermented with 2% liquid culture (containing 10(6) cells/ml broth). Two types of fermentations were carried out i.e. single culture fermentation [L. casei, L. plantarum (37 degrees C, 24 hr.)] and sequential culture fermentation [S. boulardii (25 degrees C, 24 hr.) + L. casei (37 degrees C, 24 hr.)]; S. boulardii [(25 degrees C, 24 hr.) + L. plantarum (37 degrees C, 24 hr.)]. All the fermentations drastically reduced the contents of phytic acid, polyphenols and trypsin inhibitor activity while significantly improving the in vitro digestibilities of starch and protein. Sequential culture fermentations brought about higher changes as compared to single culture fermentations.

  2. 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.

  3. Thermostable factor(s) in soya producing a net excess of secretion in the ligated gut test in pigs.

    Science.gov (United States)

    Nabuurs, M J

    1986-09-01

    The ligated gut test (LGT) is the standard method for the examination of Escherichia coli strains for enterotoxin production in pigs. As solid pig feed has been associated with diarrhea, soya products (the main protein source for piglets) were investigated with the same test as E. coli strains. After injection of different soya products into ligated segments of the small intestine fluid accumulation was observed, indicating a net excess of secretion. The factor in soya products responsible for this effect was found to be thermostable, as its effect was unaltered after heating at 120 degrees C during an hour. No indications of a possible allergic phenomenon accounting for the fluid accumulation were found. From the results of this study it is concluded that soyabean products can produce results in the LGT similar to those produced by enterotoxigenic E. coli strains.

  4. Identification of new cancer biomarkers based on aberrant mucin glycoforms by in situ proximity ligation

    DEFF Research Database (Denmark)

    Pinto, Rita; Carvalho, Ana S; Conze, Tim

    2012-01-01

    Mucin glycoproteins are major secreted or membrane-bound molecules that, in cancer, show modifications in both the mucin proteins expression and in the O-glycosylation profile, generating some of the most relevant tumour markers in clinical use for decades. Thus far, the identification...... of these biomarkers has been based on the detection of either the protein or the O-glycan modifications. We therefore aimed to identify the combined mucin and O-glycan features, that is, specific glycoforms, in an attempt to increase specificity of these cancer biomarkers. Using in situ proximity ligation assays (PLA......) based on existing monoclonal antibodies directed to MUC1, MUC2, MUC5AC and MUC6 mucins and to cancer-associated carbohydrate antigens Tn, Sialyl-Tn (STn), T, Sialyl-Le(a) (SLe(a) ) and Sialyl-Le(x) (SLe(x) ) we screened a series of 28 mucinous adenocarcinomas from different locations (stomach, ampulla...

  5. Proximity ligation in situ assay for monitoring the global DNA methylation in cells

    Directory of Open Access Journals (Sweden)

    Vallette François M

    2011-04-01

    Full Text Available Abstract Background DNA methylation has a central role in the epigenetic control of mammalian gene expression, and is required for X inactivation, genomics imprinting and silencing of retrotransposons and repetitive sequences. Thus, several technologies have been developed to measure the degree of DNA methylation. Results We here present the development of the detection of protein-protein interactions via the adaptation of the proximity ligation in situ technology to evaluate the DNA methylation status in cells since the quantification of Dnmt1/PCNA interaction in cells reflects the degree of DNA methylation. Conclusion This method being directly realizable on cells, it appears that it could suggest a wide range of applications in basic research and drug development. More particularly, this method is specially adapted for the investigations realized from a weak quantity of biologic materiel such as stem cells or primary cultured tumor cells for examples.

  6. Proximity ligation in situ assay for monitoring the global DNA methylation in cells.

    Science.gov (United States)

    Hervouet, Eric; Hulin, Philippe; Vallette, François M; Cartron, Pierre-François

    2011-04-06

    DNA methylation has a central role in the epigenetic control of mammalian gene expression, and is required for X inactivation, genomics imprinting and silencing of retrotransposons and repetitive sequences. Thus, several technologies have been developed to measure the degree of DNA methylation. We here present the development of the detection of protein-protein interactions via the adaptation of the proximity ligation in situ technology to evaluate the DNA methylation status in cells since the quantification of Dnmt1/PCNA interaction in cells reflects the degree of DNA methylation. This method being directly realizable on cells, it appears that it could suggest a wide range of applications in basic research and drug development. More particularly, this method is specially adapted for the investigations realized from a weak quantity of biologic materiel such as stem cells or primary cultured tumor cells for examples.

  7. Ligations of Gold Atoms with Iron Porphyrin

    DEFF Research Database (Denmark)

    Zhang, Ling; Kepp, Kasper Planeta; Ulstrup, Jens

    electrochemistry and electrochemical scanning tunneling microscopy (in situ STM) are explained with this theory. Iron porphyrin is a well-known active redox center of cytochrome c and hemoglobin/myoglobin assisting membrane-crossing electron transfer or blood oxygentransport. The electronic states...... and configurations of iron porphyrin affect the electrochemical properties of the metalloproteins, where the artificial constructed proteins are designed by the mutations of amino residues or the structural optimizations of iron porphyrins. Iron porphyrin adsorption on graphite and graphene surfaces by п-п electron...... stacking has been widely studied and the catalytic activity found to be enhanced warranting the notion of enzyme mimics. Weak physisorption was, however, recently observed by in situ STM, but the electronic properties of iron porphyrin adsorbed on gold has not been addressed before. This issue is, however...

  8. Application of Fluorescent Protein Expressing Strains to Evaluation of Anti-Tuberculosis Therapeutic Efficacy In Vitro and In Vivo.

    Directory of Open Access Journals (Sweden)

    Ying Kong

    Full Text Available The slow growth of Mycobacterium tuberculosis (Mtb, the causative agent of tuberculosis (TB, hinders development of new diagnostics, therapeutics and vaccines. Using non-invasive real-time imaging technologies to monitor the disease process in live animals would facilitate TB research in all areas. We developed fluorescent protein (FP expressing Mycobacterium bovis BCG strains for in vivo imaging, which can be used to track bacterial location, and to quantify bacterial load in live animals. We selected an optimal FP for in vivo imaging, by first cloning six FPs: tdTomato, mCherry, mPlum, mKate, Katushka and mKeima, into mycobacteria under either a mycobacterial Hsp60 or L5 promoter, and compared their fluorescent signals in vitro and in vivo. Fluorescence from each FP-expressing strain was measured with a multimode reader using the optimal excitation and emission wavelengths for the FP. After normalizing bacterial numbers with optical density, the strain expressing L5-tdTomato displayed the highest fluorescence. We used the tdTomato-labeled M. bovis BCG to obtain real-time images of pulmonary infections in living mice and rapidly determined the number of bacteria present. Further comparison between L5-tdTomato and Hsp60-tdTomato revealed that L5-tdTomato carried four-fold more tdTomato gene copies than Hsp60-tdTomato, which eventually led to higher protein expression of tdTomato. Evaluating anti-TB efficacy of rifampicin and isoniazid therapy in vitro and in vivo using the L5-tdTomato strain demonstrated that this strain can be used to identify anti-TB therapeutic efficacy as quickly as 24 h post-treatment. These M. bovis BCG reporter strains represent a valuable new tool for evaluation of therapeutics, vaccines and virulence.

  9. Development of a Synthetic Surfactant Using a Surfactant Protein-C Peptide Analog: In Vitro Studies of Surface Physical Properties.

    Science.gov (United States)

    Bae, Chong Woo; Chung, Sung Hoon; Choi, Yong Sung

    2016-01-01

    Pulmonary surfactant (PS) replacement has been the gold standard therapy for neonatal respiratory distress syndrome; however, almost all commercial PSs contain animal proteins. We prepared a synthetic PS by using a human surfactant protein (SP) analog and evaluated its in vitro properties. A peptide sequence (CPVHLKRLLLLLLLLLLLLLLLL) of human SP-C was chosen to develop the peptide analog (SPa-C). The new synthetic SP-C PS (sSP-C PS) was synthesized from SPa-C, dipalmitoyl phosphatidylcholine, phosphatidyl glycerol, and palmitic acid. Physical properties of the sSP-C PS were evaluated by measuring the maximum and minimum surface tensions (STs), surfactant spreading, and adsorption rate. In addition, we recorded an ST-area diagram. The data obtained on sSP-C PS were subsequently compared with those of purified natural bovine surfactant (PNBS), and the commercial product, Surfacten®. The sSP-C PS and Surfacten® were found to have maximum ST values of 32-33 mN/m, whereas that of PNBS was much lower at 19 mN/m. The minimum ST values of all three products were less than 10 mN/m. The values that were measured for the equilibrium ST of rapidly spreading sSP-C PS, Surfacten®, and PNBS were 27, 27, and 24 mN/m, respectively. The surface adsorptions were found to be the same for all three PSs (20 mN/m). ST-area diagrams of sSP-C PS and Surfacten® revealed similar properties. In an in vitro experiment, the physical properties exhibited by sSP-C PS were similar to those of Surfacten®. Further study is required to evaluate the in vivo efficacy.

  10. Composition of incubation solution impacts in vitro protein uptake to silicone hydrogel contact lenses

    Science.gov (United States)

    Heynen, Miriam; Luensmann, Doerte; Jones, Lyndon

    2012-01-01

    Purpose To determine the impact of incubation solution composition on protein deposition to silicone hydrogel (SH) contact lenses using a simplistic and a complex model of the tear film. Methods Three SH materials – senofilcon A (SA), lotrafilcon B (LB), and balafilcon A (BA) – were incubated in two different solutions; Solution A was a simplistic augmented buffered saline solution containing a single protein, whereas Solution B was a complex artificial tear solution (ATS), containing the augmented buffered saline solution in addition to proteins, lipids, and mucins (pH=7.4). The proteins of interest (lysozyme, lactoferrin, albumin) were radiolabeled with Iodine-125 (2% protein of interest) and the accumulation of the conjugated protein to the lens materials was determined after 1, 7, 14, and 28 days of incubation. Protein deposition was measured using a gamma counter and the raw data were translated into absolute amounts (µg/lens) via extrapolation from standards. Results After 28 days, lysozyme uptake was significantly lower on BA lenses when incubated in Solution A (33.7 μg) compared to Solution B (56.2 μg), plenses deposited similar amounts of lysozyme when incubated in either Solution A (2.6 μg) or Solution B (4.1 μg), p>0.05. LB lenses also deposited similar amounts of lysozyme for both solutions (Solution A: 5.0 μg, Solution B: 4.7 μg, p>0.05). After 28 days, BA lenses accumulated approximately twice the amount of lactoferrin than the other lens materials, with 30.3 μg depositing when exposed to Solution A and 22.0 μg with Solution B. The difference between the two solutions was statistically significant (plenses regardless of incubation solution composition (Solution A: 8.2 μg, Solution B: 11.2 μg, p>0.05). After 28 days, albumin deposition onto BA lenses was significantly greater when lenses were incubated in Solution B (1.7 μg) compared to Solution A (0.9 μg), plenses when incubated in either solution (0.6 μg versus 0.7 μg, p>0.05). LB

  11. Enzymes involved in DNA ligation and end-healing in the radioresistant bacterium Deinococcus radiodurans

    Science.gov (United States)

    Blasius, Melanie; Buob, Rebecca; Shevelev, Igor V; Hubscher, Ulrich

    2007-01-01

    Background Enzymes involved in DNA metabolic events of the highly radioresistant bacterium Deinococcus radiodurans are currently examined to understand the mechanisms that protect and repair the Deinococcus radiodurans genome after extremely high doses of γ-irradiation. Although several Deinococcus radiodurans DNA repair enzymes have been characterised, no biochemical data is available for DNA ligation and DNA endhealing enzymes of Deinococcus radiodurans so far. DNA ligases are necessary to seal broken DNA backbones during replication, repair and recombination. In addition, ionizing radiation frequently leaves DNA strand-breaks that are not feasible for ligation and thus require end-healing by a 5'-polynucleotide kinase or a 3'-phosphatase. We expect that DNA ligases and end-processing enzymes play an important role in Deinococcus radiodurans DNA strand-break repair. Results In this report, we describe the cloning and expression of a Deinococcus radiodurans DNA ligase in Escherichia coli. This enzyme efficiently catalyses DNA ligation in the presence of Mn(II) and NAD+ as cofactors and lysine 128 was found to be essential for its activity. We have also analysed a predicted second DNA ligase from Deinococcus radiodurans that is part of a putative DNA repair operon and shows sequence similarity to known ATP-dependent DNA ligases. We show that this enzyme possesses an adenylyltransferase activity using ATP, but is not functional as a DNA ligase by itself. Furthermore, we identified a 5'-polynucleotide kinase similar to human polynucleotide kinase that probably prepares DNA termini for subsequent ligation. Conclusion Deinococcus radiodurans contains a standard bacterial DNA ligase that uses NAD+ as a cofactor. Its enzymatic properties are similar to E. coli DNA ligase except for its preference for Mn(II) as a metal cofactor. The function of a putative second DNA ligase remains unclear, but its adenylyltransferase activity classifies it as a member of the

  12. Enzymes involved in DNA ligation and end-healing in the radioresistant bacterium Deinococcus radiodurans

    Directory of Open Access Journals (Sweden)

    Shevelev Igor V

    2007-08-01

    Full Text Available Abstract Background Enzymes involved in DNA metabolic events of the highly radioresistant bacterium Deinococcus radiodurans are currently examined to understand the mechanisms that protect and repair the Deinococcus radiodurans genome after extremely high doses of γ-irradiation. Although several Deinococcus radiodurans DNA repair enzymes have been characterised, no biochemical data is available for DNA ligation and DNA endhealing enzymes of Deinococcus radiodurans so far. DNA ligases are necessary to seal broken DNA backbones during replication, repair and recombination. In addition, ionizing radiation frequently leaves DNA strand-breaks that are not feasible for ligation and thus require end-healing by a 5'-polynucleotide kinase or a 3'-phosphatase. We expect that DNA ligases and end-processing enzymes play an important role in Deinococcus radiodurans DNA strand-break repair. Results In this report, we describe the cloning and expression of a Deinococcus radiodurans DNA ligase in Escherichia coli. This enzyme efficiently catalyses DNA ligation in the presence of Mn(II and NAD+ as cofactors and lysine 128 was found to be essential for its activity. We have also analysed a predicted second DNA ligase from Deinococcus radiodurans that is part of a putative DNA repair operon and shows sequence similarity to known ATP-dependent DNA ligases. We show that this enzyme possesses an adenylyltransferase activity using ATP, but is not functional as a DNA ligase by itself. Furthermore, we identified a 5'-polynucleotide kinase similar to human polynucleotide kinase that probably prepares DNA termini for subsequent ligation. Conclusion Deinococcus radiodurans contains a standard bacterial DNA ligase that uses NAD+ as a cofactor. Its enzymatic properties are similar to E. coli DNA ligase except for its preference for Mn(II as a metal cofactor. The function of a putative second DNA ligase remains unclear, but its adenylyltransferase activity classifies it as a

  13. Modifications of nano-titania surface for in vitro evaluations of hemolysis, cytotoxicity, and nonspecific protein binding

    Energy Technology Data Exchange (ETDEWEB)

    Datta, Aparna, E-mail: adatta.research@gmail.com [Jadavpur University, School of Materials Science and Nanotechnology (India); Dasgupta, Sayantan [NRS Medical College and Hospital, Department of Biochemistry (India); Mukherjee, Siddhartha [Jadavpur University, Department of Metallurgical and Material Engineering (India)

    2017-04-15

    In the past decade, a variety of drug carriers based on mesoporous silica nanoparticles has been extensively reported. However, their biocompatibility still remains debatable, which motivated us to explore the porous nanostructures of other metal oxides, for example titanium dioxide (TiO{sub 2}), as potential drug delivery vehicles. Herein, we report the in vitro hemolysis, cytotoxicity, and protein binding of TiO{sub 2} nanoparticles, synthesized by a sol–gel method. The surface of the TiO{sub 2} nanoparticles was modified with hydroxyl, amine, or thiol containing moieties to examine the influence of surface functional groups on the toxicity and protein binding aspects of the nanoparticles. Our study revealed the superior hemocompatibility of pristine, as well as functionalized TiO{sub 2} nanoparticles, compared to that of mesoporous silica, the present gold standard. Among the functional groups studied, aminosilane moieties on the TiO{sub 2} surface substantially reduced the degree of hemolysis (down to 5%). Further, cytotoxicity studies by MTT assay suggested that surface functional moieties play a crucial role in determining the biocompatibility of the nanoparticles. The presence of NH{sub 2}– functional groups on the TiO{sub 2} nanoparticle surface enhanced the cell viability by almost 28% as compared to its native counterpart (at 100 μg/ml), which was in agreement with the hemolysis assay. Finally, nonspecific protein adsorption on functionalized TiO{sub 2} surfaces was examined using human serum albumin and it was found that negatively charged surface moieties, like –OH and –SH, could mitigate protein adsorption to a significant extent.

  14. MicroRNA and protein profiles in invasive versus non-invasive oral tongue squamous cell carcinoma cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Korvala, Johanna, E-mail: johanna.korvala@oulu.fi [Cancer and Translational Medicine Research Unit, University of Oulu, The Medical Research Center Oulu, Oulu University Hospital, Aapistie 5A, 90014 Oulu (Finland); Jee, Kowan [Department of Pathology, University of Turku, Turku University Hospital, Turku (Finland); Department of Pathology, Haartman Institute, University of Helsinki, Helsinki (Finland); Porkola, Emmi [Cancer and Translational Medicine Research Unit, University of Oulu, The Medical Research Center Oulu, Oulu University Hospital, Aapistie 5A, 90014 Oulu (Finland); Almangush, Alhadi [Department of Pathology, Haartman Institute, University of Helsinki, Helsinki (Finland); Mosakhani, Neda [Department of Pathology, HUSLAB, Helsinki (Finland); Bitu, Carolina [Cancer and Translational Medicine Research Unit, University of Oulu, The Medical Research Center Oulu, Oulu University Hospital, Aapistie 5A, 90014 Oulu (Finland); Cervigne, Nilva K. [Department of Oral Diagnosis, School of Dentistry, University of Campinas (UNICAMP), Av. Limeira, 901 – Bairro Areião, CEP: 13414-903 Piracicaba, São Paulo (Brazil); Department of Clinical and Pathology, Faculty of Medicine of Jundiai - FMJ, Jundiai, SP (Brazil); Zandonadi, Flávia S.; Meirelles, Gabriela V.; Leme, Adriana Franco Paes [Laboratório Nacional de Biociências, LNBio, CNPEM, Rua Giuseppe Máximo Scolfaro, 10.000, Polo II de Alta Tecnologia de Campinas, Campinas/SP, P.O.Box 6192, CEP 13083-970 Campinas, São Paulo (Brazil); Coletta, Ricardo D. [Department of Oral Diagnosis, School of Dentistry, University of Campinas (UNICAMP), Av. Limeira, 901 – Bairro Areião, CEP: 13414-903 Piracicaba, São Paulo (Brazil); and others

    2017-01-01

    Complex molecular pathways regulate cancer invasion. This study overviewed proteins and microRNAs (miRNAs) involved in oral tongue squamous cell carcinoma (OTSCC) invasion. The human highly aggressive OTSCC cell line HSC-3 was examined in a 3D organotypic human leiomyoma model. Non-invasive and invasive cells were laser-captured and protein expression was analyzed using mass spectrometry-based proteomics and miRNA expression by microarray. In functional studies the 3D invasion assay was replicated after silencing candidate miRNAs, miR-498 and miR-940, in invasive OTSCC cell lines (HSC-3 and SCC-15). Cell migration, proliferation and viability were also studied in the silenced cells. In HSC-3 cells, 67 proteins and 53 miRNAs showed significant fold-changes between non-invasive vs. invasive cells. Pathway enrichment analyses allocated “Focal adhesion” and “ECM-receptor interaction” as most important for invasion. Significantly, in HSC-3 cells, miR-498 silencing decreased the invasion area and miR-940 silencing reduced invasion area and depth. Viability, proliferation and migration weren’t significantly affected. In SCC-15 cells, down-regulation of miR-498 significantly reduced invasion and migration. This study shows HSC-3 specific miRNA and protein expression in invasion, and suggests that miR-498 and miR-940 affect invasion in vitro, the process being more influenced by mir-940 silencing in aggressive HSC-3 cells than in the less invasive SCC-15.

  15. Formaldehyde at low concentration induces protein tau into globular amyloid-like aggregates in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Chun Lai Nie

    Full Text Available Recent studies have shown that neurodegeneration is closely related to misfolding and aggregation of neuronal tau. Our previous results show that neuronal tau aggregates in formaldehyde solution and that aggregated tau induces apoptosis of SH-SY5Y and hippocampal cells. In the present study, based on atomic force microscopy (AFM observation, we have found that formaldehyde at low concentrations induces tau polymerization whilst acetaldehyde does not. Neuronal tau misfolds and aggregates into globular-like polymers in 0.01-0.1% formaldehyde solutions. Apart from globular-like aggregation, no fibril-like polymerization was observed when the protein was incubated with formaldehyde for 15 days. SDS-PAGE results also exhibit tau polymerizing in the presence of formaldehyde. Under the same experimental conditions, polymerization of bovine serum albumin (BSA or alpha-synuclein was not markedly detected. Kinetic study shows that tau significantly misfolds and polymerizes in 60 minutes in 0.1% formaldehyde solution. However, presence of 10% methanol prevents protein tau from polymerization. This suggests that formaldehyde polymerization is involved in tau aggregation. Such aggregation process is probably linked to the tau's special "worm-like" structure, which leaves the epsilon-amino groups of Lys and thiol groups of Cys exposed to the exterior. Such a structure can easily bond to formaldehyde molecules in vitro and in vivo. Polymerizing of formaldehyde itself results in aggregation of protein tau. Immunocytochemistry and thioflavin S staining of both endogenous and exogenous tau in the presence of formaldehyde at low concentrations in the cell culture have shown that formaldehyde can induce tau into amyloid-like aggregates in vivo during apoptosis. The significant protein tau aggregation induced by formaldehyde and the severe toxicity of the aggregated tau to neural cells may suggest that toxicity of methanol and formaldehyde ingestion is related to

  16. MicroRNA and protein profiles in invasive versus non-invasive oral tongue squamous cell carcinoma cells in vitro.

    Science.gov (United States)

    Korvala, Johanna; Jee, Kowan; Porkola, Emmi; Almangush, Alhadi; Mosakhani, Neda; Bitu, Carolina; Cervigne, Nilva K; Zandonadi, Flávia S; Meirelles, Gabriela V; Leme, Adriana Franco Paes; Coletta, Ricardo D; Leivo, Ilmo; Salo, Tuula

    2017-01-01

    Complex molecular pathways regulate cancer invasion. This study overviewed proteins and microRNAs (miRNAs) involved in oral tongue squamous cell carcinoma (OTSCC) invasion. The human highly aggressive OTSCC cell line HSC-3 was examined in a 3D organotypic human leiomyoma model. Non-invasive and invasive cells were laser-captured and protein expression was analyzed using mass spectrometry-based proteomics and miRNA expression by microarray. In functional studies the 3D invasion assay was replicated after silencing candidate miRNAs, miR-498 and miR-940, in invasive OTSCC cell lines (HSC-3 and SCC-15). Cell migration, proliferation and viability were also studied in the silenced cells. In HSC-3 cells, 67 proteins and 53 miRNAs showed significant fold-changes between non-invasive vs. invasive cells. Pathway enrichment analyses allocated "Focal adhesion" and "ECM-receptor interaction" as most important for invasion. Significantly, in HSC-3 cells, miR-498 silencing decreased the invasion area and miR-940 silencing reduced invasion area and depth. Viability, proliferation and migration weren't significantly affected. In SCC-15 cells, down-regulation of miR-498 significantly reduced invasion and migration. This study shows HSC-3 specific miRNA and protein expression in invasion, and suggests that miR-498 and miR-940 affect invasion in vitro, the process being more influenced by mir-940 silencing in aggressive HSC-3 cells than in the less invasive SCC-15. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Modifications of nano-titania surface for in vitro evaluations of hemolysis, cytotoxicity, and nonspecific protein binding

    Science.gov (United States)

    Datta, Aparna; Dasgupta, Sayantan; Mukherjee, Siddhartha

    2017-04-01

    In the past decade, a variety of drug carriers based on mesoporous silica nanoparticles has been extensively reported. However, their biocompatibility still remains debatable, which motivated us to explore the porous nanostructures of other metal oxides, for example titanium dioxide (TiO2), as potential drug delivery vehicles. Herein, we report the in vitro hemolysis, cytotoxicity, and protein binding of TiO2 nanoparticles, synthesized by a sol-gel method. The surface of the TiO2 nanoparticles was modified with hydroxyl, amine, or thiol containing moieties to examine the influence of surface functional groups on the toxicity and protein binding aspects of the nanoparticles. Our study revealed the superior hemocompatibility of pristine, as well as functionalized TiO2 nanoparticles, compared to that of mesoporous silica, the present gold standard. Among the functional groups studied, aminosilane moieties on the TiO2 surface substantially reduced the degree of hemolysis (down to 5%). Further, cytotoxicity studies by MTT assay suggested that surface functional moieties play a crucial role in determining the biocompatibility of the nanoparticles. The presence of NH2- functional groups on the TiO2 nanoparticle surface enhanced the cell viability by almost 28% as compared to its native counterpart (at 100 μg/ml), which was in agreement with the hemolysis assay. Finally, nonspecific protein adsorption on functionalized TiO2 surfaces was examined using human serum albumin and it was found that negatively charged surface moieties, like -OH and -SH, could mitigate protein adsorption to a significant extent.

  18. Acute pancreatitis after thoracic duct ligation for iatrogenic chylothorax. A case report.

    Science.gov (United States)

    Bédat, Benoît; Scarpa, Cosimo Riccardo; Sadowski, Samira Mercedes; Triponez, Frédéric; Karenovics, Wolfram

    2017-01-23

    To report the association between thoracic duct ligation and acute pancreatitis. The association between sudden stop of lymphatic flow and pancreatitis has been established in experimental models. A 57-year-old woman operated for thymoma presented a iatrogenic chylothorax. After thoracic duct ligation, she presented an acute pancreatitis which resolved after conservative treatment. The chylothorax disappeared within 4 days of thoracic duct ligation. This is the first report of acute pancreatitis following thoracic duct ligation. The pancreas and digestive tract should be assessed in symptomatic patients after thoracic duct ligation.

  19. Certain aspects of uncoupling due to mitochondrial uncoupling proteins in vitro and in vivo

    Czech Academy of Sciences Publication Activity Database

    Dlasková, Andrea; Špaček, Tomáš; Škobisová, Eva; Šantorová, Jitka; Ježek, Petr

    2006-01-01

    Roč. 1757, č. 5-6 (2006), s. 467-473 ISSN 0005-2728 R&D Projects: GA MŠk(CZ) 1P05ME794; GA ČR(CZ) GA521/02/0183; GA AV ČR(CZ) IAA5011106 Institutional research plan: CEZ:AV0Z50110509 Keywords : fatty acid-induced uncoupling * uncoupling protein -1 * carnitine cycle Subject RIV: CE - Biochemistry Impact factor: 4.237, year: 2006

  20. Adherens junction proteins are expressed in collagen corneal equivalents produced in vitro with human cells

    OpenAIRE

    Giasson, Claude J.; Deschambeault, Alexandre; Carrier, Patrick; Germain, Lucie

    2014-01-01

    Purpose To test whether adherens junction proteins are present in the epithelium and the endothelium of corneal equivalents. Methods Corneal cell types were harvested from human eyes and grown separately. Stromal equivalents were constructed by seeding fibroblasts into a collagen gel on which epithelial and endothelial cells were added on each side. Alternatively, bovine endothelial cells were used. At maturity, sections of stromal equivalents were processed for Masson's trichrome or indirect...

  1. Lcp1 Is a Phosphotransferase Responsible for Ligating Arabinogalactan to Peptidoglycan in Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    James Harrison

    2016-08-01

    Full Text Available Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB, has a unique cell envelope which accounts for its unusual low permeability and contributes to resistance against common antibiotics. The main structural elements of the cell wall consist of a cross-linked network of peptidoglycan (PG in which some of the muramic acid residues are covalently attached to a complex polysaccharide, arabinogalactan (AG, via a unique α-l-rhamnopyranose–(1→3-α-d-GlcNAc-(1→P linker unit. While the molecular genetics associated with PG and AG biosynthetic pathways have been largely delineated, the mechanism by which these two major pathways converge has remained elusive. In Gram-positive organisms, the LytR-CpsA-Psr (LCP family of proteins are responsible for ligating cell wall teichoic acids to peptidoglycan, through a linker unit that bears a striking resemblance to that found in mycobacterial arabinogalactan. In this study, we have identified Rv3267 as a mycobacterial LCP homolog gene that encodes a phosphotransferase which we have named Lcp1. We demonstrate that lcp1 is an essential gene required for cell viability and show that recombinant Lcp1 is capable of ligating AG to PG in a cell-free radiolabeling assay.

  2. Ligation of MHC class I molecules on peripheral blood T lymphocytes induces new phenotypes and functions

    DEFF Research Database (Denmark)

    Bregenholt, S; Röpke, M; Skov, S

    1996-01-01

    Microgram concentrations of immobilized anti-MHC class I (MHC-I) Ab induced proliferation of resting CD3+ T cells from peripheral blood. In contrast, soluble Ab did not activate T cells. Exposure of T cells to immobilized anti-MHC-I Ab for only 24 h was followed by proliferation and development...... of T cell-mediated cytotoxicity. Immediately following MHC-I ligation, the T cells responded with increased protein tyrosine phosphorylation, with new bands appearing in the SDS-PAGE. Exposure of T cells to immobilized anti-MHC-I Ab for 24 h induced an increased surface expression of the TCR/CD3 and CD......28 molecules. MHC-I-induced proliferation of purified T cells was dependent on cellular interactions with non-T cells. Under certain conditions, in which MHC-I was ligated by picogram concentrations of immobilized anti-MHC-I Ab, anti-TCR/CD3 Ab-induced proliferation of T cells was strongly inhibited...

  3. Influence of two hop (Humulus lupulus L.) varieties on in vitro dry matter and crude protein degradability and digestibility in ruminants.

    Science.gov (United States)

    Lavrenčič, Andrej; Levart, Alenka; Košir, Iztok Jože; Cerenak, Andreja

    2014-04-01

    Hop cones contain several antimicrobial substances. The aim of the study was to determine the effects of two hop varieties, Aurora and Dana, on substrate (diet for a dairy cow, producing 30 kg milk daily) in vitro dry matter (DM) and crude protein (CP) degradability and digestibility. In the in vitro trial freshly ground hops were added to the buffered rumen fluid in concentrations simulating the cow's daily intake of 50, 100 and 200 g of hops. Increasing the concentration of hops decreased (P digestibility. The difference between CP digestibility and degradability represents an estimate of the amount of rumen 'bypass' protein which increased with increasing concentration of hops from 172 to 454 g kg(-1). Decreased DM and CP degradability and increased amount of rumen 'bypass' protein could lower the amounts of protein required by high-producing ruminant animals. However, this supposition needs a validation with in vivo trials. © 2013 Society of Chemical Industry.

  4. Mutagenic analysis of potato virus X movement protein (TGBp1) and the coat protein (CP): in vitro TGBp1-CP binding and viral RNA translation activation.

    Science.gov (United States)

    Zayakina, Olga; Arkhipenko, Marina; Kozlovsky, Stanislav; Nikitin, Nikolai; Smirnov, Alexander; Susi, Petri; Rodionova, Nina; Karpova, Olga; Atabekov, Joseph

    2008-01-01

    Previously, we have shown that encapsidated Potato virus X (PVX) RNA was non-translatable in vitro, but could be converted into a translatable form by binding of the PVX movement protein TGBp1 to one end of the virion or by coat protein (CP) phosphorylation. Here, a mutagenic analysis of PVX CP and TGBp1 was used to identify the regions involved in TGBp1-CP binding and translational activation of PVX RNA by TGBp1. It was found that the C-terminal (C-ter) 10/18 amino acids region was not essential for virus-like particle (VP) assembly from CP and RNA. However, the VPs assembled from the CP lacking C-ter 10/18 amino acids were incapable of TGBp1 binding and being translationally activated. It was suggested that the 10-amino-acid C-ter regions of protein subunits located at one end of a polar helical PVX particle contain a domain accessible to TGBp1 binding and PVX remodelling. The non-translatable particles assembled from the C-ter mutant CP could be converted into a translatable form by CP phosphorylation. The TGBp1-CP binding activity was preserved unless a conservative motif IV was removed from TGBp1. By contrast, TGBp1-dependent activation of PVX RNA translation was abolished by deletions of various NTPase/helicase conservative motifs and their combinations. The motif IV might be essential for TGBp1-CP binding, but insufficient for PVX RNA translation activation. The evidence to discriminate between these two events, i.e. TGBp1 binding to the CP-helix and TGBp1-dependent RNA translation activation, is discussed.

  5. Formation of a covalent complex between the 80,000-dalton adenovirus terminal protein and 5'-dCMP in vitro.

    Science.gov (United States)

    Lichy, J H; Horwitz, M S; Hurwitz, J

    1981-05-01

    An in vitro adenovirus DNA replication system catalyzed the formation of a covalent complex between an 80,000-dalton protein and 5'-dCMP in the presence of [alpha-32P-dCTP, MgCl2, ATP, and adenovirus (Ad) DNA with a protein covalently bound to the 5' end of each strand (Ad DNA-prot). The requirement for Ad DNA-prot in this reaction was similar to that for in vitro DNA replication. When dATP, dTTP, and the 2',3'-dideoxynucleoside triphosphate (ddNTP) ddGTP were included in the reaction mixture, an elongated complex was detected, which consisted of an 80,000-dalton protein bound to a 26-base oligonucleotide. Formation of the elongated product, but not of the protein-dCMP complex, was inhibited by ddATP, ddCTP, or ddTTP. The requirements for formation of the protein-dCMP complex, the nature of the linkage between protein and dCMP, the size of the protein, and the existence of elongated forms indicated that the protein associated with the complex was identical to the 80,000-dalton Ad terminal protein found on replicating DNA molecules as described by Challberg et al. [Challberg, M. D., Desiderio, S. V. & Kelly, T. J., Jr. (1980) Proc. Natl. Acad. Sci. USA 77, 5105-5109].

  6. Feasibility of laparoscopic portal vein ligation prior to major hepatectomy.

    Science.gov (United States)

    Are, C; Iacovitti, S; Prete, F; Crafa, F M

    2008-01-01

    Patients noted to have an inadequate future liver remnant on pre operative volumetric assessment are considered to be candidates for portal vein embolization (PVE). A subset of patients undergo laparoscopic intervention prior to PVE for staging purposes or to address the primary in Stage IV colon cancer. These patients usually undergo PVE as a subsequent additional procedure by the transhepatic route. The aim of this study was to assess the feasibility of portal vein ligation by the laparoscopic approach in suitable patients. A retrospective review of a prospectively maintained database was performed to identify patients that underwent laparoscopic portal vein ligation (LPVL). The demographic, clinical, radiographic, operative and volumetric details were collected to determine the feasibility of portal vein ligation. A total of nine patients underwent LPVL as part of a two stage procedure in preparation for subsequent major hepatectomy. With a median age of 67 yrs, the diagnoses included: colorectal metastasis (five patients), cholangiocarcinoma (three patients) and hepatocellular carcinoma (one patient). The ligation involved the right portal vein in all and was performed with silk ligature (seven patients) and clips (two patients). Volumetric data was available in six patients which showed a mean increase from 209.1 cc+/-97.76 to 495.83 cc+/-310.91 (increase by 181.5%) In two patients, inadequate hypertrophy mandated later embolization by percutaneous technique. Five patients underwent subsequent major hepatic resection as planned. The remaining four patients were noted to have progression of disease that precluded the planned procedure. There were no complications associated with LPVL. LPVL is feasible and can be safely performed. In a select group of patients, it may be considered as an alternative to subsequent embolization and thereby potentially absolve the need for an additional procedure with its attendant complications.

  7. The human metapneumovirus matrix protein stimulates the inflammatory immune response in vitro.

    Directory of Open Access Journals (Sweden)

    Audrey Bagnaud-Baule

    Full Text Available Each year, during winter months, human Metapneumovirus (hMPV is associated with epidemics of bronchiolitis resulting in the hospitalization of many infants. Bronchiolitis is an acute illness of the lower respiratory tract with a consequent inflammation of the bronchioles. The rapid onset of inflammation suggests the innate immune response may have a role to play in the pathogenesis of this hMPV infection. Since, the matrix protein is one of the most abundant proteins in the Paramyxoviridae family virion, we hypothesized that the inflammatory modulation observed in hMPV infected patients may be partly associated with the matrix protein (M-hMPV response. By western blot analysis, we detected a soluble form of M-hMPV released from hMPV infected cell as well as from M-hMPV transfected HEK 293T cells suggesting that M-hMPV may be directly in contact with antigen presenting cells (APCs during the course of infection. Moreover, flow cytometry and confocal microscopy allowed determining that M-hMPV was taken up by dendritic cells (moDCs and macrophages inducing their activation. Furthermore, these moDCs enter into a maturation process inducing the secretion of a broad range of inflammatory cytokines when exposed to M-hMPV. Additionally, M-hMPV activated DCs were shown to stimulate IL-2 and IFN-γ production by allogeneic T lymphocytes. This M-hMPV-mediated activation and antigen presentation of APCs may in part explain the marked inflammatory immune response observed in pathology induced by hMPV in patients.

  8. In vitro antibacterial activity of venom protein isolated from sea snake Enhydrina schistosa against drugresistant human pathogenic bacterial strains

    Directory of Open Access Journals (Sweden)

    Palani Damotharan

    2015-06-01

    Full Text Available Objective: To evaluate the antibacterial activity of sea snake (Enhydrina schistosa venom protein against drug-resistant human pathogenic bacterial strains. Methods: The venom was collected by milking process from the live specimens of sea snake are using capillary tubes or glass plates. Venom was purified by ion exchange chromatography and it was tested for in-vitro antibacterial activity against 10 drug-resistant human pathogenic bacterial strains using the standard disc diffusion method. Results: The notable antibacterial activity was observed at 150 µg/mL concentration of purified venom and gave its minimum inhibitory concentrations values exhibited between 200-100 µg/mL against all the tested bacterial strains. The maximum zone of inhibition was observed at 16.4 mm against Salmonella boydii and the minimum activity was observed at 7.5 mm against Pseudomonas aeruginosa. After the sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis there were a clear single band was detected in the gel that corresponding to purified venom protein molecular weight of 44 kDa. Conclusions: These results suggested that the sea snake venom might be a feasible source for searching potential antibiotics agents against human pathogenic diseases.

  9. Evaluation of the in vitro antioxidant properties of a cod (Gadus morhua) protein hydrolysate and peptide fractions.

    Science.gov (United States)

    Girgih, Abraham T; He, Rong; Hasan, Fida M; Udenigwe, Chibuike C; Gill, Tom A; Aluko, Rotimi E

    2015-04-15

    Mechanically-deboned cod muscle proteins were sequentially hydrolysed using pepsin and a trypsin+chymotrypsin combination, which was followed by passing the digest through a 1 kDa equipped tangential flow filtration system; the permeate (<1 kDa peptides) was collected as the cod protein hydrolysate (CPH). Reversed-phase high performance liquid chromatography (RP-HPLC) was used to separate the CPH into four peptide fractions (CF1-CF4) and their in vitro antioxidant properties investigated. Results showed that most of the peptide fractions (CF2-CF4) displayed significantly higher (p<0.05) oxygen radical absorbance capacity values (698-942 μM Trolox equivalents, TE/g) and 2,2-diphenyl-1-picrylhydrazyl scavenging activities (17-32%) than those of CPH (613 μM TE/g and 19%, respectively). However, the unfractionated CPH displayed improved capability to scavenge superoxide and hydroxyl radicals as well as significantly higher (p<0.05) ferric iron reduction and chelation of iron than the RP-HPLC peptides. The CPH and peptide fractions displayed a dose-dependent inhibition of linoleic acid oxidation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Diarylethenes Display In Vitro Anti-TB Activity and Are Efficient Hits Targeting the Mycobacterium tuberculosis HU Protein

    Directory of Open Access Journals (Sweden)

    María Angélica Suarez

    2017-07-01

    Full Text Available Tuberculosis continues to be a great source of concern in global health because of the large reservoir of humans infected with the bacilli and the appearance of clinical isolates resistant to a wide array of anti-tuberculosis drugs. New drugs with novel mechanisms of action on new targets are urgently required to reduce global tuberculosis burden. Mycobacterium tuberculosis nucleoid associated protein (NAP HU has been shown to be druggable and essential for the organism’s survival. In this study, four diarylethenes were synthesized using a one-pot decarboxylated Heck-coupling of coumaric acids with iodoanisoles. The prepared compounds 1–4 were tested for their in vitro growth inhibition of M. tuberculosis H37Rv using the spot culture growth inhibition assay, displaying minimum inhibitory concentrations between 9 and 22 µM. Their cytotoxicity against BHK-21 cell line showed half inhibition at concentrations between 98 and 729 µM. The most selective hit (SI = 81, demonstrated inhibition of M. tuberculosis HU protein involved in maintaining bacterial genome architecture.

  11. The Matrix protein M1 from influenza C virus induces tubular membrane invaginations in an in vitro cell membrane model.

    Science.gov (United States)

    Saletti, David; Radzimanowski, Jens; Effantin, Gregory; Midtvedt, Daniel; Mangenot, Stéphanie; Weissenhorn, Winfried; Bassereau, Patricia; Bally, Marta

    2017-01-25

    Matrix proteins from enveloped viruses play an important role in budding and stabilizing virus particles. In order to assess the role of the matrix protein M1 from influenza C virus (M1-C) in plasma membrane deformation, we have combined structural and in vitro reconstitution experiments with model membranes. We present the crystal structure of the N-terminal domain of M1-C and show by Small Angle X-Ray Scattering analysis that full-length M1-C folds into an elongated structure that associates laterally into ring-like or filamentous polymers. Using negatively charged giant unilamellar vesicles (GUVs), we demonstrate that M1-C full-length binds to and induces inward budding of membrane tubules with diameters that resemble the diameter of viruses. Membrane tubule formation requires the C-terminal domain of M1-C, corroborating its essential role for M1-C polymerization. Our results indicate that M1-C assembly on membranes constitutes the driving force for budding and suggest that M1-C plays a key role in facilitating viral egress.

  12. Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins.

    OpenAIRE

    Wickham, T.J.; E.; Tzeng; Shears, L. L.; Roelvink, P. W.; Li, Y; Lee, G. M.; Brough, D E; Lizonova, A; Kovesdi, I

    1997-01-01

    Alteration of the natural tropism of adenovirus (Ad) will permit gene transfer into specific cell types and thereby greatly broaden the scope of target diseases that can be treated by using Ad. We have constructed two Ad vectors which contain modifications to the Ad fiber coat protein that redirect virus binding to either alpha(v) integrin [AdZ.F(RGD)] or heparan sulfate [AdZ.F(pK7)] cellular receptors. These vectors were constructed by a novel method involving E4 rescue of an E4-deficient Ad...

  13. In vitro characterization of the Bacillus subtilis protein tyrosine phosphatase YwqE

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Musumeci, Lucia; Tautz, Lutz

    2005-01-01

    Both gram-negative and gram-positive bacteria possess protein tyrosine phosphatases (PTPs) with a catalytic Cys residue. In addition, many gram-positive bacteria have acquired a new family of PTPs, whose first characterized member was CpsB from Streptococcus pneumoniae. Bacillus subtilis contains......, we characterize YwqE, compare the activities of the three B. subtilis PTPs (YwqE, YwlE, and YfkJ), and demonstrate that the two B. subtilis class II PTPs do not dephosphorylate the physiological substrates of YwqE....

  14. Comparison of Hi-C results using in-solution versus in-nucleus ligation.

    Science.gov (United States)

    Nagano, Takashi; Várnai, Csilla; Schoenfelder, Stefan; Javierre, Biola-Maria; Wingett, Steven W; Fraser, Peter

    2015-08-26

    Chromosome conformation capture and various derivative methods such as 4C, 5C and Hi-C have emerged as standard tools to analyze the three-dimensional organization of the genome in the nucleus. These methods employ ligation of diluted cross-linked chromatin complexes, intended to favor proximity-dependent, intra-complex ligation. During development of single-cell Hi-C, we devised an alternative Hi-C protocol with ligation in preserved nuclei rather than in solution. Here we directly compare Hi-C methods employing in-nucleus ligation with the standard in-solution ligation. We show in-nucleus ligation results in consistently lower levels of inter-chromosomal contacts. Through chromatin mixing experiments we show that a significantly large fraction of inter-chromosomal contacts are the result of spurious ligation events formed during in-solution ligation. In-nucleus ligation significantly reduces this source of experimental noise, and results in improved reproducibility between replicates. We also find that in-nucleus ligation eliminates restriction fragment length bias found with in-solution ligation. These improvements result in greater reproducibility of long-range intra-chromosomal and inter-chromosomal contacts, as well as enhanced detection of structural features such as topologically associated domain boundaries. We conclude that in-nucleus ligation captures chromatin interactions more consistently over a wider range of distances, and significantly reduces both experimental noise and bias. In-nucleus ligation creates higher quality Hi-C libraries while simplifying the experimental procedure. We suggest that the entire range of 3C applications are likely to show similar benefits from in-nucleus ligation.

  15. Effect of domestic processing on the cooking time, nutrients, antinutrients and in vitro protein digestibility of the African yambean (Sphenostylis stenocarpa).

    Science.gov (United States)

    Ene-obong, H N; Obizoba, I C

    1996-01-01

    The effects of processing (soaking, dehulling, fermentation and heat treatment) on the cooking time, protein, mineral, tannin, phytate and in vitro protein digestibility (IVPD) of the African yambean (AYB) were examined. The cooking time ranged from 90-155 minutes. Soaking reduced cooking time by about 50 percent. Soaking for 12 hours was the most appropriate to reduce cooking time, tannin and phytate levels. It improved in vitro protein digestibility (IVPD). Prolonged soaking (24 hours) decreased calcium (Ca) and iron (Fe) values by 19 percent and 35 percent, respectively. Dehulling showed that Ca, Fe, magnesium (Mg) and zinc (Zn) were concentrated in the seed coat of the AYB. The seeds soaked and dehulled retained Mg and Zn. Dehulling reduced tannin but had no significant effect on phytate and the IVPD of the AYB except for seeds soaked for 12 hours before dehulling. Soaking for 24 hours before dehulling significantly increased crude protein content by 16 percent (p < 0.05). Blanching and roasting increased the IVPD by 8-11 percent. Fermentation had no effect on the crude protein, Ca, Fe, Mg and Zn but significantly reduced phytate content of the AYB. Fermentation had no advantage over heat treatment with respect to improving the in vitro protein digestibility of the AYB.

  16. [Protein kinase inhibitor flavopiridol inhibits the replication of influenza virus in vitro].

    Science.gov (United States)

    Wang, Shixiong; Zhang, Junjie; Ye, Xin

    2012-09-04

    To investigate the antiviral effect of the flavonoid compound flavopiridol on influenza A virus and explore its antiviral mechanism. The A549 or Madin-Darby canine kidney (MDCK) cells were infected with influenza A virus A/WSN/33 and treated with flavopiridol. The viral proteins were determined by immunolotting and immunofluorescence. The virus titer was measured by plaque assay. To verify whether the activity of host RNA polymerase II was affected by flavopiridol, the phosphorylation status of RNA polymerase II CTD domain was analyzed by immunoblotting with phosphor-specific antibody. The amount of viral mRNA, vRNA and cRNA was measured by reverse transcription and PCR. The amount of viral proteins was significantly decreased and the titer of virus was greatly reduced in cells treated with flavopiridol. Further analysis showed that the phosphorylation of Ser-2 in the heptad repeat of the CTD domain in RNA polymerase II was decreased in falvopiridol treated cell. This result indicated that the transcription elongation activity of RNA pol II was impaired upon treatment with flavopiridol. Then we found that the amount of viral vRNA was significantly decreased in flavopiridol treated cells while only moderate decrease of mRNA was observed and almost no reduction of cRNA was detected. Flavopiridol can greatly suppress the replication of influenza virus. We propose that the inhibition of the transcription elongation activity of host RNA polymerase II would cause the decrease of viral mRNA transcription.

  17. Distribution of Major Basic Protein on Human Airway following In Vitro Eosinophil Incubation

    Directory of Open Access Journals (Sweden)

    Ailing Xue

    2010-01-01

    Full Text Available Major basic protein (MBP released from activated eosinophils may influence airway hyperresponsiveness (AHR by either direct effects on airway myocytes or by an indirect effect. In this study, human bronchi, freshly isolated human eosinophils, or MBP purified from human eosinophil granules were incubated for studying eosinophil infiltration and MBP localization. Eosinophils immediately adhered to intact human airway as well as to cultured human airway myocytes and epithelium. Following incubation 18–24 h, eosinophils migrated into the airway media, including the smooth muscle layer, but had no specific recruitment to airway neurons. Eosinophils released significant amounts of MBP within the airway media, including areas comprising the smooth muscle layer. Most deposits of MBP were focally discrete and restricted by immunologic detection to a maximum volume of ∼300 μm3 about the eosinophil. Native MBP applied exogenously was immediately deposited on the surface of the airway, but required at least 1 h to become detected within the media of the airway wall. Tissue MBP infiltration and deposition increased in a time- and concentration-dependent manner. Taken together, these findings suggest that eosinophil-derived cationic proteins may alter airway hyperresponsiveness (AHR in vivo by an effect that is not limited to the bronchial epithelium.

  18. DNA-protein, DNA interstrand cross-links induced by camphoramine chloroacetic platinum in vitro.

    Science.gov (United States)

    Wang, L G; Zeng, H

    1989-05-01

    Effects of camphoramine chloroacetic platinum (CCP) on DNA migration and transcription, DNA-protein and DNA interstrand cross-links induced by CCP were investigated by using agarose gel electrophoresis, alkaline elution and enzymatic techniques, respectively. Chromosome break down and migration alteration of DNa modified by CCP were observed. Plasmid pAR 436 DNA transcription was also blocked when the DNA was treated with CCP. The cross-links took place 8 h after HeLa cells were exposed to CCP 10 mumol/L and higher number of cross-links were obtained after treatment with the agent 20 mumol/L. The number of cross-links was also found to be decreased when the cell lysis was digested with proteinase K. These results suggest that CCP can also induce DNA-protein cross links. Enzymatic studies indicated that CCP preferentially attacks guanine in DNA and restriction enzymes are unable to cleave G-platinated at interval of one base to the restriction sequence.

  19. Multiplikasi, Induksi Planlet dan Seleksi Tembakau Hasil Transformasi Gen Coat Protein SMV Secara Kultur in Vitro

    Directory of Open Access Journals (Sweden)

    N.A. FITRIYAH

    2004-11-01

    Full Text Available A study has been conducted to screen and multiply calluses and buds oftobacco transformed with the Soybean Mosaic Virus (SMV coat protein gene and to induce them to plantlet formation by using MS medium with phytohormon treatments. The multiplication of calluses and buds was carried out by sub-culturing on MS medium using 0.3 mg/L NAA+1 mg/L BAP, followed by induction of plantlet formation on MS medium supplemented with NAA and BAP at varied concentration. The results showed that tobacco calluses transformed with SMV coat protein gene was multipliable on MS medium supplemented with NAA at a concentration of 0.3 mg/mL and BAP at 1 mg/L concentration. In this experiment kanamycin was used at a concentration of 100 μg/mL which resulted in the viability level of 84%. On MS medium supplemented with 0.3 mg/L NAA, 0.1 mg/L BAP, and 100 μg/mL kanamycin, induction of calluses to plantlet formation reached 20% level.

  20. Inhibition of nucleoside diphosphate kinase activity by in vitro phosphorylation by protein kinase CK2. Differential phosphorylation of NDP kinases in HeLa cells in culture

    DEFF Research Database (Denmark)

    Biondi, R M; Engel, M; Sauane, M

    1996-01-01

    that in vitro protein kinase CK2 catalyzed phosphorylation of human NDPK A inhibits its enzymatic activity by inhibiting the first step of its ping-pong mechanism of catalysis: its autophosphorylation. Upon in vivo 32P labeling of HeLa cells, we observed that both human NDPKs, A and B, were autophosphorylated...

  1. Effects of exposure of epididymal boar spermatozoa to seminal plasma on the binding of zona pellucida proteins during in vitro capacitation

    NARCIS (Netherlands)

    Harkema, W.; Colenbrander, B.; Engel, B.; Woelders, H.

    2004-01-01

    The purpose of the investigation was to determine whether seminal plasma plays a role in the increase during in vitro capacitation of the number of boar spermatozoa with enhanced binding of zona pellucida proteins. Ejaculated spermatozoa and spermatozoa collected from the caudae epididymides of

  2. Chrysophanic Acid Suppresses Adipogenesis and Induces Thermogenesis by Activating AMP-activated Protein Kinase Alpha in vivo and in vitro

    Directory of Open Access Journals (Sweden)

    Hara Lim

    2016-12-01

    Full Text Available Chrysophanic acid (CA is a member of the anthraquinone family abundant in rhubarb, a widely used herb for obesity treatment in Traditional Korean Medicine. Though several studies have indicated numerous features of CA, no study has yet reported the effect of CA on obesity. In this study, we tried to identify the anti-obesity effects of CA. By using 3T3-L1 adipocytes and primary cultured brown adipocytes as in vitro models, high-fat diet (HFD-induced obese mice, and zebrafish as in vivo models, we determined the anti-obesity effects of CA. CA reduced weight gain in HFD-induced obese mice. They also decreased lipid accumulation and the expressions of adipogenesis factors including peroxisome proliferator-activated receptor gamma (PPARγ and CCAAT/enhancer-binding protein alpha (C/EBPα in 3T3-L1 adipocytes. In addition, uncoupling protein 1 (UCP1 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α, the brown fat specific thermogenic genes, were up-regulated in brown adipocytes by CA treatment. Furthermore, when co-treated with Compound C, the AMP-activated protein kinase (AMPK inhibitor, CA was able to restore the activation of AMPKα in both types of adipocytes, indicating the multi-controlling effect of CA was partially via the AMPKα pathway. Given all together, these results indicate that CA can ameliorate obesity by controlling the adipogenic and thermogenic pathway at the same time. On these bases we suggest the new potential of CA as an anti-obese pharmacotherapy.

  3. In vitro evaluation of cytotoxicity, possible alteration of apoptotic regulatory proteins, and antibacterial activity of synthesized copper oxide nanoparticles.

    Science.gov (United States)

    Khan, Shahanavaj; Ansari, Anees A; Khan, Azmat Ali; Abdulla, Maha; Al-Obaid, Omar; Ahmad, Rehan

    2017-05-01

    Copper oxide nanoparticles (CuO-NPs) were synthesized using a urea-based thermal decomposition technique, and characterized using different techniques. X-ray diffraction (XRD) and Raman spectroscopy confirmed the phase purity and crystalline structure of CuO-NPs. The size of CuO-NPs was investigated using XRD and was confirmed via dynamic light scattering analysis. CuO-NPs showed an average diameter of ∼20nm. The possible cytotoxicity of CuO-NPs was evaluated in HT-29 and SW620 cancer cell lines. The median inhibitory concentration of CuO-NPs in HT-29 and SW-620 cells was 4.99 and 3.75μg/mL, respectively. The underlying mechanism responsible for apoptosis in colon cancer cells after CuO-NP exposure has not been well understood. In this study, we investigated the possible mechanisms of induction of apoptosis via analysis of the expression of Bcl-2 and Bcl-xL proteins in HT-29 human colon cancer cells after CuO-NP exposure. Western blot assay showed downregulation of Bcl-2 and Bcl-xL protein expression after CuO-NP exposure. Our findings may aid in the understanding of the potential mechanisms responsible for induction of apoptosis owing to inhibition of Bcl-2 and Bcl-xL protein expression. Furthermore, the antibacterial activity assay showed that the synthesized CuO-NPs did not exert significant inhibitory effects against different gram-positive and gram-negative bacteria in vitro. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. An evolved ribosome-inactivating protein targets and kills human melanoma cells in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Green David E

    2010-02-01

    Full Text Available Abstract Background Few treatment options exist for patients with metastatic melanoma, resulting in poor prognosis. One standard treatment, dacarbazine (DTIC, shows low response rates ranging from 15 to 25 percent with an 8-month median survival time. The development of targeted therapeutics with novel mechanisms of action may improve patient outcome. Ribosome-inactivating proteins (RIPs such as Shiga-like Toxin 1 (SLT-1 represent powerful scaffolds for developing selective anticancer agents. Here we report the discovery and properties of a single chain ribosome-inactivating protein (scRIP derived from the cytotoxic A subunit of SLT-1 (SLT-1A, harboring the 7-amino acid peptide insertion IYSNKLM (termed SLT-1AIYSNKLM allowing the toxin variant to selectively target and kill human melanoma cells. Results SLT-1AIYSNKLM was able to kill 7 of 8 human melanoma cell lines. This scRIP binds to 518-A2 human melanoma cells with a dissociation constant of 18 nM, resulting in the blockage of protein synthesis and apoptosis in such cells. Biodistribution and imaging studies of radiolabeled SLT-1AIYSNKLM administered intravenously into SCID mice bearing a human melanoma xenograft indicate that SLT-1AIYSNKLM readily accumulates at the tumor site as opposed to non-target tissues. Furthermore, the co-administration of SLT-1AIYSNKLM with DTIC resulted in tumor regression and greatly increased survival in this mouse xenograft model in comparison to DTIC or SLT-1AIYSNKLM treatment alone (115 day median survival versus 46 and 47 days respectively; P values IYSNKLM is stable in serum and its intravenous administration resulted in modest immune responses following repeated injections in CD1 mice. Conclusions These results demonstrate that the evolution of a scRIP template can lead to the discovery of novel cancer cell-targeted compounds and in the case of SLT-1AIYSNKLM can specifically kill human melanoma cells in vitro and in vivo.

  5. Production of Fibronectin Binding Protein A at the surface of Lactococcus lactis increases plasmid transfer in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Daniela Pontes

    Full Text Available Lactococci are noninvasive lactic acid bacteria frequently used as protein delivery vectors and, more recently, as DNA delivery vehicles. We previously showed that Lactococcus lactis (LL expressing the Fibronectin-Binding Protein A of Staphylococcus aureus (LL-FnBPA+ showed higher internalization rates in vitro in Caco-2 cells than the native (wt lactococci and were able to deliver a eukaryotic Green Fluorescent Protein (GFP expression plasmid in 1% of human Caco-2 cells. Here, using the bovine beta-lactoglobulin (BLG, one of the major cow's milk allergen, and GFP we characterized the potential of LL-FnBPA+ as an in vivo DNA vaccine delivery vehicle. We first showed that the invasive strain LL-FnBPA+ carrying the plasmid pValac:BLG (LL-FnBPA+ BLG was more invasive than LL-BLG and showed the same invasivity as LL-FnBPA+. Then we demonstrated that the Caco-2 cells, co-incubated with LL-FnBPA+ BLG produced up to 30 times more BLG than the Caco-2 cells co-incubated with the non invasive LL-BLG. Using two different gene reporters, BLG and GFP, and two different methods of detection, EIA and fluorescence microscopy, we showed in vivo that: i in order to be effective, LL-FnBPA+ required a pre-coating with Fetal Calf Serum before oral administration; ii plasmid transfer occurred in enterocytes without regard to the strains used (invasive or not; iii the use of LL-FnBPA+ increased the number of mice producing BLG, but not the level of BLG produced. We thus confirmed the good potential of invasive recombinant lactic acid bacteria as DNA delivery vector in vivo.

  6. Immune responses elicited against rotavirus middle layer protein VP6 inhibit viral replication in vitro and in vivo

    Science.gov (United States)

    Lappalainen, Suvi; Pastor, Ana Ruth; Tamminen, Kirsi; López-Guerrero, Vanessa; Esquivel-Guadarrama, Fernando; Palomares, Laura A; Vesikari, Timo; Blazevic, Vesna

    2014-01-01

    Rotavirus (RV) is a common cause of severe gastroenteritis (GE) in children worldwide. Live oral RV vaccines protect against severe RVGE, but the immune correlates of protection are not yet clearly defined. Inner capsid VP6 protein is a highly conserved, abundant, and immunogenic RV protein, and VP6-specific mucosal antibodies, especially IgA, have been implicated to protect against viral challenge in mice. In the present study systemic and mucosal IgG and IgA responses were induced by immunizing BALB/c mice intranasally with a combination of recombinant RV VP6 protein (subgroup II [SGII]) and norovirus (NoV) virus-like particles (VLPs) used in a candidate vaccine. Following immunization mice were challenged orally with murine RV strain EDIMwt (SG non-I-non-II, G3P10[16]). In order to determine neutralizing activity of fecal samples, sera, and vaginal washes (VW) against human Wa RV (SGII, G1P1A[8]) and rhesus RV (SGI, G3P5B[3]), the RV antigen production was measured with an ELISA-based antigen reduction neutralization assay. Only VWs of immunized mice inhibited replication of both RVs, indicating heterotypic protection of induced antibodies. IgA antibody depletion and blocking experiments using recombinant VP6 confirmed that neutralization was mediated by anti-VP6 IgA antibodies. Most importantly, after the RV challenge significant reduction in viral shedding was observed in feces of immunized mice. These results suggest a significant role for mucosal RV VP6-specific IgA for the inhibition of RV replication in vitro and in vivo. In addition, these results underline the importance of non-serotype-specific immunity induced by the conserved subgroup-specific RV antigen VP6 in clearance of RV infection. PMID:25424814

  7. Protein kinase D2 regulates migration and invasion of U87MG glioblastoma cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Bernhart, Eva; Damm, Sabine; Wintersperger, Andrea [Institute of Molecular Biology and Biochemistry, Medical University of Graz, Graz (Austria); DeVaney, Trevor [Institute of Biophysics, Medical University of Graz (Austria); Zimmer, Andreas [Institute of Pharmaceutical Sciences, Department of Pharmaceutical Technology, Karl-Franzens University, Graz (Austria); Raynham, Tony; Ireson, Christopher [Cancer Research Technology Ltd, London (United Kingdom); Sattler, Wolfgang, E-mail: wolfgang.sattler@medunigraz.at [Institute of Molecular Biology and Biochemistry, Medical University of Graz, Graz (Austria)

    2013-08-01

    Glioblastoma multiforme (GBM) is the most common malignant brain tumor, which, despite combined modality treatment, reoccurs and is invariably fatal for affected patients. Recently, a member of the serine/threonine protein kinase D (PRKD) family, PRKD2, was shown to be a potent mediator of glioblastoma growth. Here we studied the role of PRKD2 in U87MG glioblastoma cell migration and invasion in response to sphingosine-1-phosphate (S1P), an activator of PRKD2 and a GBM mitogen. Time-lapse microscopy demonstrated that random cell migration was significantly diminished in response to PRKD2 silencing. The pharmacological PRKD family inhibitor CRT0066101 decreased chemotactic migration and invasion across uncoated or matrigel-coated Transwell inserts. Silencing of PRKD2 attenuated migration and invasion of U87MG cells even more effectively. In terms of downstream signaling, CRT0066101 prevented PRKD2 autophosphorylation and inhibited p44/42 MAPK and to a smaller extent p54/46 JNK and p38 MAPK activation. PRKD2 silencing impaired activation of p44/42 MAPK and p54/46 JNK, downregulated nuclear c-Jun protein levels and decreased c-Jun{sup S73} phosphorylation without affecting the NFκB pathway. Finally, qPCR array analyses revealed that silencing of PRKD2 downregulates mRNA levels of integrin alpha-2 and -4 (ITGA2 and -4), plasminogen activator urokinase (PLAU), plasminogen activator urokinase receptor (PLAUR), and matrix metallopeptidase 1 (MMP1). Findings of the present study identify PRKD2 as a potential target to interfere with glioblastoma cell migration and invasion, two major determinants contributing to recurrence of glioblastoma after multimodality treatment. Highlights: • Sphingosine-1-phosphate induces glioma cell migration and invasion. • Part of the effects is mediated by protein kinase D2 (PRKD2) activation. • Inactivation of PRKD2 attenuates glioblastoma cell migration and invasion. • Both, RNAi and pharmacological inhibition of PRKD2 inhibits MAPK

  8. Influence of lipid extraction from different protein sources on in vitro digestibility Influência da extração de lipídio de diferentes fontes protéicas na digestibilidade in vitro

    Directory of Open Access Journals (Sweden)

    Rita de Cássia Oliveira Sant'Ana

    2011-08-01

    Full Text Available Proteins are the most abundant macromolecules in living cells and their primary role in the diet is to supply the body with essential amino acids in adequate quantities for the synthesis and maintenance of body tissues. The determination of protein digestibility of foods is an important factor to estimate their quality and the in vitro methodology is a fast and easy way to perform it. This study aimed to determine the influence of lipids on the in vitro digestibility of animal and vegetable proteins. The following protein sources: oat, beef, chicken, fish and pork meats, red beans, milk powder, textured soy protein (TSP, quinoa and five soybean varieties were evaluated. Animal proteins presented higher in vitro values than vegetable proteins, except for the textured soy protein, which presented higher digestibility based on the thermal treatment. In this study, there was no statistic difference between lipid content and protein digestibility. Therefore, there is no need that samples be defatted prior the analysis of the in vitro digestibility, using an enzymatic system containing the enzymes trypsin and pancreatin, which facilitates even more the use of these methods for foods with high lipid levels in food industries.As proteínas são as macromoléculas mais abundantes nas células vivas, tendo como principal função na dieta suprir o organismo de aminoácidos indispensáveis em quantidades adequadas para síntese e manutenção dos tecidos corporais. Desse modo, a determinação da digestibilidade proteica de um alimento é um fator importante para estimar a sua qualidade, sendo o método in vitro uma alternativa rápida e fácil. Neste trabalho, objetivou-se determinar a influência dos lipídios na digestibilidade in vitro de proteínas de origem animal e vegetal. Foram utilizadas as seguintes fontes de proteína: aveia, carnes: bovina, de frango, de peixe e suína, feijão vermelho, leite em pó, proteína texturizada de soja (PTS, quinoa

  9. The use of antifreeze protein type III for vitrification of in vitro matured bovine oocytes.

    Science.gov (United States)

    Chaves, Dowglish F; Campelo, Iana S; Silva, Mirelly M A S; Bhat, Maajid H; Teixeira, Darcio I A; Melo, Luciana M; Souza-Fabjan, Joanna M G; Mermillod, Pascal; Freitas, Vicente J F

    2016-12-01

    The aim of this study was to evaluate the use of antifreeze protein type III (AFP III) into vitrification medium on meiotic spindle morphology of in vitro matured bovine oocytes as well as the fertilization and blastocyst rates. Mature cumulus-oocyte complexes (COC) were distributed in four groups: control (untreated), vitrified without supplementation (AFP0) or supplemented with 500 (AFP500) or 1000 ng/mL (AFP1000) into vitrification solutions. Samples from each group were used to analyze the organization of meiotic spindle by confocal microscopy and the remaining COC were submitted to in vitro fertilization and culture for eight days. Control group exhibited only 15% of abnormal spindle. However, the spindle morphology was affected in all vitrified groups regardless to AFP concentration: 75.8%, 76.1% and 69.2% (P > 0.05) for AFP0, AFP500 and AFP1000, respectively. Similar cleavage rate was obtained among the vitrified groups (AFP0 = 17.9%, AFP500 = 16.9% and AFP1000 = 17.8%), but lower (P < 0.05) compared with control group (68.7%). At Day 5 of culture, embryo production rate of AFP500 (30.8%) and AFP1000 (25.0%) were similar to control group (49.4%). However, at Day 8 of culture, AFP0, AFP500 and AFP1000 groups exhibited lower (P < 0.05) blastocyst rates (10.0%, 3.8% and 9.4%, respectively) when compared to control (41.1%). In conclusion, AFP III did not preserve meiotic spindle organization against the cryoinjuries. However, the use of AFP III improved embryo development at Day 5 of culture, although this effect was not maintained up to the blastocyst formation. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. A Histone-Like Protein Induces Plasmid DNA to Form Liquid Crystals in Vitro and Gene Compaction in Vivo

    Directory of Open Access Journals (Sweden)

    Shiyong Sun

    2013-12-01

    Full Text Available The liquid crystalline state is a universal phenomenon involving the formation of an ordered structure via a self-assembly process that has attracted attention from numerous scientists. In this study, the dinoflagellate histone-like protein HCcp3 is shown to induce super-coiled pUC18 plasmid DNA to enter a liquid crystalline state in vitro, and the role of HCcp3 in gene condensation in vivo is also presented. The plasmid DNA (pDNA-HCcp3 complex formed birefringent spherical particles with a semi-crystalline selected area electronic diffraction (SAED pattern. Circular dichroism (CD titrations of pDNA and HCcp3 were performed. Without HCcp3, pUC18 showed the characteristic B conformation. As the HCcp3 concentration increased, the 273 nm band sharply shifted to 282 nm. When the HCcp3 concentration became high, the base pair (bp/dimer ratio fell below 42/1, and the CD spectra of the pDNA-HCcp3 complexes became similar to that of dehydrated A-form DNA. Microscopy results showed that HCcp3 compacted the super-coiled gene into a condensed state and that inclusion bodies were formed. Our results indicated that HCcp3 has significant roles in gene condensation both in vitro and in histone-less eukaryotes in vivo. The present study indicates that HCcp3 has great potential for applications in non-viral gene delivery systems, where HCcp3 may compact genetic material to form liquid crystals.

  11. Molecular Architecture of the Blood Brain Barrier Tight Junction Proteins--A Synergistic Computational and In Vitro Approach.

    Science.gov (United States)

    Irudayanathan, Flaviyan Jerome; Trasatti, John P; Karande, Pankaj; Nangia, Shikha

    2016-01-14

    The blood-brain barrier (BBB) constituted by claudin-5 tight junctions is critical in maintaining the homeostasis of the central nervous system, but this highly selective molecular interface is an impediment for therapeutic interventions in neurodegenerative and neurological diseases. Therapeutic strategies that can exploit the paracellular transport remain elusive due to lack of molecular insights of the tight junction assembly. This study focuses on analyzing the membrane driven cis interactions of claudin-5 proteins in the formation of the BBB tight junctions. We have adopted a synergistic approach employing in silico multiscale dynamics and in vitro cross-linking experiments to study the claudin-5 interactions. Long time scale simulations of claudin-5 monomers, in seven different lipid compositions, show formation of cis dimers that subsequently aggregate into strands. In vitro formaldehyde cross-linking studies also conclusively show that cis-interacting claudin-5 dimers cross-link with short methylene spacers. Using this synergistic approach, we have identified five unique dimer interfaces in our simulations that correlate with the cross-linking experiments, four of which are mediated by transmembrane (TM) helices and the other mediated by extracellular loops (ECL). Potential of mean force calculations of these five dimers revealed that the TM mediated interfaces, which can have distinctive leucine zipper interactions in some cases, are more stable than the ECL mediated interface. Additionally, simulations show that claudin-5 dimerization is significantly influenced by the lipid microenvironment. This study captures the fundamental interactions responsible for the BBB tight junction assembly and offers a framework for extending this work to other tight junctions found in the body.

  12. Impact of breast cancer resistance protein expression on the in vitro efficacy of anticancer drugs in pancreatic cancer cell lines.

    Science.gov (United States)

    Washio, Ikumi; Nakanishi, Takeo; Ishiguro, Naoki; Yamamura, Norio; Tamai, Ikumi

    2017-12-15

    Breast cancer resistance protein (BCRP) overexpression confers multidrug resistance to cancer cells, and the efficacy of anticancer drugs has been reported to be significantly affected by BCRP in cell lines transfected with BCRP or selected with drugs. It is unclear whether the in vitro efficacy of anticancer drugs is affected by endogenous BCRP, although cancer cell line panels consisting of defined tumor cell lines with endogenous BCRP have been used to screen for anticancer drugs in the pharmaceutical industry. We assessed the impact of BCRP expression on efficacy of anticancer drugs using pancreatic cancer cell lines expressing varying levels of endogenous BCRP. Pancreatic cancer cell lines were selected from the Cancer Cell Line Encyclopedia (CCLE). EC50 of SN-38, topotecan, and mitoxantrone decreased in the presence of a BCRP inhibitor in PANC-1 and AsPC-1 cells, which exhibit high BCRP expression. However, no significant alterations in EC50 were observed in HPAF-II, SW 1990, and MIA PaCa-2, which show moderate or low BCRP expression. The shift of EC50 of anticancer drugs with and without a BCRP inhibitor increased with an increase of BCRP mRNA expression levels; however, the shift was obvious only in cells highly expressing BCRP. Thus, the in vitro efficacy of anticancer drugs on cell proliferation may be minimally affected by BCRP in most pancreatic cancer cell lines, considering that 72% pancreatic cancer cell lines in CCLE show moderate or low BCRP expression. The effect of BCRP should be carefully evaluated in pancreatic cell lines that highly express BCRP. The American Society for Pharmacology and Experimental Therapeutics.

  13. E-cadherin homophilic ligation inhibits cell growth and epidermal growth factor receptor signaling independently of other cell interactions.

    Science.gov (United States)

    Perrais, Michaël; Chen, Xiao; Perez-Moreno, Mirna; Gumbiner, Barry M

    2007-06-01

    E-cadherin function leads to the density-dependent contact inhibition of cell growth. Because cadherins control the overall state of cell contact, cytoskeletal organization, and the establishment of many other kinds of cell interactions, it remains unknown whether E-cadherin directly transduces growth inhibitory signals. To address this question, we have selectively formed E-cadherin homophilic bonds at the cell surface of isolated epithelial cells by using functionally active recombinant E-cadherin protein attached to microspheres. We find that E-cadherin ligation alone reduces the frequency of cells entering the S phase, demonstrating that E-cadherin ligation directly transduces growth inhibitory signals. E-cadherin binding to beta-catenin is required for cell growth inhibition, but beta-catenin/T-cell factor transcriptional activity is not involved in growth inhibition resulting from homophilic binding. Neither E-cadherin binding to p120-catenin nor beta-catenin binding to alpha-catenin, and thereby the actin cytoskeleton, is required for growth inhibition. E-cadherin ligation also inhibits epidermal growth factor (EGF) receptor-mediated growth signaling by a beta-catenin-dependent mechanism. It does not affect EGF receptor autophosphorylation or activation of ERK, but it inhibits transphosphorylation of Tyr845 and activation of signal transducers and activators of transcription 5. Thus, E-cadherin homophilic binding independent of other cell contacts directly transduces growth inhibition by a beta-catenin-dependent mechanism that inhibits selective signaling functions of growth factor receptors.

  14. Preparation of Low-Input and Ligation-Free ChIP-seq Libraries Using Template-Switching Technology.

    Science.gov (United States)

    Bolduc, Nathalie; Lehman, Alisa P; Farmer, Andrew

    2016-10-10

    Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) has become the gold standard for mapping of transcription factors and histone modifications throughout the genome. However, for ChIP experiments involving few cells or targeting low-abundance transcription factors, the small amount of DNA recovered makes ligation of adapters very challenging. In this unit, we describe a ChIP-seq workflow that can be applied to small cell numbers, including a robust single-tube and ligation-free method for preparation of sequencing libraries from sub-nanogram amounts of ChIP DNA. An example ChIP protocol is first presented, resulting in selective enrichment of DNA-binding proteins and cross-linked DNA fragments immobilized on beads via an antibody bridge. This is followed by a protocol for fast and easy cross-linking reversal and DNA recovery. Finally, we describe a fast, ligation-free library preparation protocol, featuring DNA SMART technology, resulting in samples ready for Illumina sequencing. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  15. Effects of different industrial heating processes of milk on site-specific protein modifications and their relationship to in vitro and in vivo digestibility.

    Science.gov (United States)

    Wada, Yasuaki; Lönnerdal, Bo

    2014-05-07

    Heating processes are applied to milk and dairy products to ensure their microbiological safety and shelf lives. However, how differences in "industrial" thermal treatments affect protein digestibility is still equivocal. In this study, raw milk was subjected to pasteurization, three kinds of ultra-high-temperature (UHT) treatment, and in-can sterilization and was investigated by in vitro and in vivo digestion and proteomic methods. In-can sterilized milk, followed by UHT milk samples, showed a rapid decrease in protein bands during the course of digestion. However, protein digestibility determined by a Kjeldahl procedure showed insignificant differences. Proteomic analysis revealed that lactulosyllysine, which reflects a decrease in protein digestibility, in α-lactalbumin, β-lactoglobulin, and caseins was higher in in-can sterilized milk, followed by UHT milk samples. Thus, industrial heating may improve the digestibility of milk proteins by denaturation, but the improvement is likely to be offset by heat-derived modifications involved in decreased protein digestibility.

  16. Influence of biflorin on the labelling of red blood cells, plasma protein, cell protein, and lymphocytes with technetium-99m: in vitro study

    Directory of Open Access Journals (Sweden)

    Thiago M. Aquino

    Full Text Available In this paper we report the results of an in vitro study involving the influence of biflorin (an o-quinone isolated from Capraria biflora L. that has potent antimicrobial activity on the Tc-99m labeling of red blood cells, plasma protein, cells protein, and lymphocytes. Blood was withdrawn from Wistar rats and incubated with various concentrations of biflorin, and solutions of stannous chloride and Tc-99m were added. Plasma (P and red blood cells (RBC were isolated, precipitated, and centrifuged, and soluble (SF and insoluble (IF fractions were isolated. The results show that the highest concentration (100% of biflorin is able to reduce the uptake of Tc-99m (%ATI on RBC and the fixation on IF-P. To study the influence of biflorin on 99mTc lymphocyte labeling, human blood was submitted to a technique with Ficoll-Hypac and centrifuged, and white cells were isolated. Lymphocytes (2.5 mL; 1.0 x 10(6 cells/mL were obtained and a 0.2 mL solution was incubated with biflorin (0.1 mL. Solutions of stannous chloride and 99mTc were added. Lymphocytes were separated and the %ATI bound in these cells was evaluated. A reduction in %ATI (from 97.85 ± 0.99 to 88.86 ± 5 was observed for RBC and for IF-P (73.24 ± 5.51 to 20.72 ± 6.95. In this case the results showed no decrease in %ATI for the lymphocytes with biflorin.

  17. Timing of surgical ligation and morbidities in very low birth weight infants.

    Science.gov (United States)

    Youn, YoungAh; Moon, Cheong-Jun; Lee, Jae-Young; Lee, Cheul; Sung, In Kyung

    2017-04-01

    The present study examined whether early patent ductus arteriosus (PDA) surgical ligation at ≤2 weeks of life was associated with increased morbidities and mortality in very low birth weight infants (VLBWIs) who were diagnosed with hemodynamically significant (hs) PDA. Between December 2013 and December 2015, a total of 407 VLBWIs were admitted, of whom 145 (35.6%) infants were diagnosed with an hs PDA. The clinical data for these infants were retrospectively collected for analysis. Among the 145 VLBWIs with an hs PDA, 58 (40%) infants had surgical ligation for PDA; of these, 29 (50%) infants had early ligation (EL; ligation at ≤2 weeks of life) and 29 (50%) infants had late ligation (LL; ligation at ≥2 weeks of life). The mean gestational age and birth weight were significantly lower in the PDA-ligated group compared with the nonligated group. In addition, pulmonary hypertension at ≤1 week of life and neonatal seizures were significantly more prevalent in the ligated group (P hypertension at ≤1 week of life was significantly associated with LL (P = 0.019), which was consistently a risk factor for hs PDA ligation in our multivariable logistic regression analysis. EL was not significantly associated with increased hospital morbidities and mortality in VLBWIs with hs PDA. Pulmonary hypertension at ≤1 week of life can be a risk factor for the need for surgical ligation of hs PDA.

  18. Spectroscopic investigation on the sonodynamic damage to proteins in the presence of berberine in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Wang Xin [School of Pharmaceutical Sciences, Liaoning University, Shenyang 110036 (China); He Lingling, E-mail: helingling76@163.co [College of Applied Chemistry, Shenyang University of Chemical Technology, Shenyang 110142 (China); Liu Bin [School of Pharmaceutical Sciences, Liaoning University, Shenyang 110036 (China); Wang Jun [Department of Chemistry, Liaoning University, Shenyang 110036 (China)

    2011-07-15

    In this paper, bovine serum albumin (BSA) was selected as a target molecule, and the sonodynamic damage to proteins in the presence of berberine (BER) and its mechanism were studied by means of absorption and fluorescence spectra. The results of hyperchromic effect of absorption spectra, and quenching of intrinsic fluorescence spectra indicated that the ultrasound-induced BSA molecules damage was enhanced by BER. The damage degree of BSA molecules increased with the increase in ultrasonic irradiation time and BER concentration. The results of synchronous fluorescence and three-dimensional fluorescence spectra confirmed that the synergistic effects of ultrasound and BER induced the damage of BSA molecules. The results of oxidation-extraction photometry with several reactive oxygen species (ROS) scavengers indicated that the damage of BSA molecules could be mainly due to the generation of ROS, and {sup 1}O{sub 2} was the major mediator of the ultrasound-induced BSA molecules damage in the presence of BER. - Highlights: {yields} Sonodynamic activity of berberine and its mechanism is first reported. {yields} Ultrasound-induced BSA molecules damage is enhanced by berberine. {yields} Damage of BSA molecules is mainly due to the generation of ROS.

  19. Modulation of enamel matrix proteins on the formation and nano-assembly of hydroxyapatite in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Li Hong, E-mail: tlihong@jnu.edu.cn [Department of Materials Science and Engineering, Jinan University, Guangzhou, Guangdong 510630 (China); Department of Bioengineering, Clemson University, Charleston, SC 29425 (United States); Huang Weiya [Department of Chemistry, Jinan University, Guangzhou, Guangdong 510630 (China); Department of Materials Science and Engineering, Taizhou, Taizhou University, Zhejiang 317000 (China); Zhang Yuanming [Department of Chemistry, Jinan University, Guangzhou, Guangdong 510630 (China); Xue Bo [Department of Materials Science and Engineering, Jinan University, Guangzhou, Guangdong 510630 (China); Wen Xuejun [Department of Bioengineering, Clemson University, Charleston, SC 29425 (United States)

    2012-05-01

    Natural enamel has a hierarchically nanoassembled architecture that is regulated by enamel matrix proteins (EMPs) during the formation of enamel crystals. To understand the role of EMPs on enamel mineralization, calcium phosphate (CaP) growth experiments in both the presence and absence of native rat EMPs in a single diffusion system were conducted. The morphology and organization of formed CaP crystals were examined by X-Ray Diffraction (XRD), High-Resolution Transmission Microscopy (HRTEM) and Selected Area Electron Diffraction (SAED). In the system containing the EMPs, hydroxyapatite (HAP) with hierarchical lamellar nanostructure can be formed and the aligned HAP assembly tightly bundled by 3-4 rod-like nanocrystals like an enamel prism. However, in the absence of EMPs, only a sheet-like structure of octacalcium phosphate (OCP) phase was presented. EMPs promote HAP formation and inhibit the growth of OCP on the (010) plane. It is discussed that the organized Amelogenin/Amorphous Calcium Phosphate might be the precursor to the bundled HAP crystal prism. The study benefits the understanding of biomineralization of tooth enamel. - Highlights: Black-Right-Pointing-Pointer An aligned hydroxyapatite crystal bundled by rod-like nanosize crystals was obtained. Black-Right-Pointing-Pointer An organized Amel/ACP would be the precursor of the bundled hydroxyapatite crystal prism. Black-Right-Pointing-Pointer EMPs inhibit the growth of octacalcium phosphate in a defined plane.

  20. Pyridoxamine protects proteins from damage by hypohalous acids in vitro and in vivo.

    Science.gov (United States)

    Madu, Hartman; Avance, Josh; Chetyrkin, Sergei; Darris, Carl; Rose, Kristie Lindsey; Sanchez, Otto A; Hudson, Billy; Voziyan, Paul

    2015-12-01

    Diabetes is characterized, in part, by activation of toxic oxidative and glycoxidative pathways that are triggered by persistent hyperglycemia and contribute to diabetic complications. Inhibition of these pathways may benefit diabetic patients by delaying the onset of complications. One such inhibitor, pyridoxamine (PM), had shown promise in clinical trials. However, the mechanism of PM action in vivo is not well understood. We have previously reported that hypohalous acids can cause disruption of the structure and function of renal collagen IV in experimental diabetes (K.L. Brown et al., Diabetes 64:2242-2253, 2015). In the present study, we demonstrate that PM can protect protein functionality from hypochlorous and hypobromous acid-derived damage via a rapid direct reaction with and detoxification of these hypohalous acids. We further demonstrate that PM treatment can ameliorate specific hypohalous acid-derived structural and functional damage to the renal collagen IV network in a diabetic animal model. These findings suggest a new mechanism of PM action in diabetes, namely sequestration of hypohalous acids, which may contribute to known therapeutic effects of PM in human diabetic nephropathy. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. THE ROLE OF PROTEIN OXIDATIVE MODIFICATION IN REDOX-REGULATION OF CASPASE-3 ACTIVITY IN BLOOD LYMPHOCYTES DURING OXIDATIVE STRESS IN VITRO

    Directory of Open Access Journals (Sweden)

    O. L. Nosareva

    2015-01-01

    Full Text Available The formation of oxidative stress lies at the heart of many frequent and socially-important diseases. Blood lymphocytes are the cells which provide immunological control of our organism. As a result of their function implementation blood lymphocytes contact with different endogenic and exogenic factors, which can lead to active oxygen species production activation, macromolecules oxidative modification and to cell survival alteration. At the present time it is essential to expand and deepen the fundamental knowledge of blood lymphocytes apoptosis regulation peculiarities. The research objective was to establish the interaction among alterations of glutathione system condition, carbonylation level, protein glutathionylation and caspase-3 activity in blood lymphocytes during oxidative stress in vitro.Material and Methods. The material for research was blood lymphocytes cultivated with addition of hydrogen peroxide in final concentration of 0,5 mmol and/or protein SH-group inhibitor N-ethylmaleimide – 5 mmol, protector – 5 mmol – 1,4-dithioerythritol. Reduced, oxidized and protein-bound glutathione concentration was measured by method of spectropho-tometry, additionally, the ratio size of reduced to oxidized thiol fraction was estimated. With help of enzymoimmunoassay the level of protein carbonyl derivatives was evaluated; caspase-3 activity was registered by spectrofluorometric method.Results. Protein SH-group blocking in blood lymphocytes during oxidative stress in vitro was accompanied by protein-bound glutathione concentration rapid decrease in connection with increase of protein carbonyl derivatives content and caspase-3 activity. Protein SH-group protection in blood lymphocytes during oxidative stress in vitro was accompanied by concentration increase of protein-bound glutathione and protein carbonyl derivatives under comparable values of enzyme activity under study.Conclusion. The carried out research shows that caspase-3 and protein

  2. Preliminary results on the effects of grape (Vitis vinifera) seed condensed tannins on in vitro intestinal digestibility of the lupin (Lupinus angustifolius) seed protein fraction in small ruminants.

    Science.gov (United States)

    Bruno-Soares, A M; Soares-Pereira, A L; Matos, T J S; Ricardo-da-Silva, J M

    2011-08-01

    Condensed tannins (CT) from grape seeds (Vitis vinifera L.) were added to complex the protein fraction of Lupinus angustifolius seeds. Three CT/protein ratios were used: 96 mg/g (T(1)), 180 mg/g (T(2)) and 0 mg/g (T(0)). The CP losses in the rumen were assessed by the nylon-bag technique and CP intestinal digestibility (CPID) was estimated using an in vitro assay applying a three-step procedure: samples were subject to rumen degradation (in situ, 16 h) and the remaining residues were subject to the digestive enzymes of the abomasum and pancreas in vitro. A positive effect (p 0.05) by the presence of CT. These findings suggest that the use of grape seed CT might have the potential to improve the efficiency of utilisation of the protein fraction from lupin seeds. © 2010 Blackwell Verlag GmbH.

  3. Synthesis of ubiquitylated histone H3 using a thiirane linker for chemical ligation.

    Science.gov (United States)

    Kawakami, Toru; Mishima, Yuichi; Hojo, Hironobu; Suetake, Isao

    2017-07-01

    Post-translational modifications of histone proteins, which form nucleosome cores, play an important role in gene regulation. Ubiquitin modification is one such modification. We previously reported on the use of a thiirane linker to introduce a 1,2-aminothiol moiety at a cysteine residue for native chemical ligation with peptide thioesters, which permitted isopeptide mimetics to be produced. In this report, we describe the preparation of the ubiquitylated full length histone H3 at the 18 position and the construction of tetranucleosomes with recombinant histones H2A, H2B, H4, and DNA, which are slightly more stable than those that are prepared without ubiquitin modification. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  4. Effects of different tannin-rich extracts and rapeseed tannin monomers on methane formation and microbial protein synthesis in vitro.

    Science.gov (United States)

    Wischer, G; Boguhn, J; Steingaß, H; Schollenberger, M; Rodehutscord, M

    2013-11-01

    Tannins, polyphenolic compounds found in plants, are known to complex with proteins of feed and rumen bacteria. This group of substances has the potential to reduce methane production either with or without negative effects on digestibility and microbial yield. In the first step of this study, 10 tannin-rich extracts from chestnut, mimosa, myrabolan, quebracho, sumach, tara, valonea, oak, cocoa and grape seed, and four rapeseed tannin monomers (pelargonidin, catechin, cyanidin and sinapinic acid) were used in a series of in vitro trials using the Hohenheim gas test, with grass silage as substrate. The objective was to screen the potential of various tannin-rich extracts to reduce methane production without a significant effect on total gas production (GP). Supplementation with pelargonidin and cyanidin did not reduce methane production; however, catechin and sinapinic acid reduced methane production without altering GP. All tannin-rich extracts, except for tara extract, significantly reduced methane production by 8% to 28% without altering GP. On the basis of these results, five tannin-rich extracts were selected and further investigated in a second step using a Rusitec system. Each tannin-rich extract (1.5 g) was supplemented to grass silage (15 g). In this experiment, nutrient degradation, microbial protein synthesis and volatile fatty acid production were used as additional response criteria. Chestnut extract caused the greatest reduction in methane production followed by valonea, grape seed and sumach, whereas myrabolan extract did not reduce methane production. Whereas chestnut extract reduced acetate production by 19%, supplementation with grape seed or myrabolan extract increased acetate production. However, degradation of fibre fractions was reduced in all tannin treatments. Degradation of dry matter and organic matter was also reduced by tannin supplementation, and no differences were found between the tannin-rich extracts. CP degradation and ammonia

  5. In vitro re-hardening of artificial enamel caries lesions using enamel matrix proteins or self-assembling peptides

    Directory of Open Access Journals (Sweden)

    Patrick Schmidlin

    2016-02-01

    Full Text Available ABSTRACT Objectives To assess the re-hardening potential of enamel matrix derivatives (EMD and self-assembling peptides in vitro, hypothesizing that these materials may increase the mineralization of artificial carious lesions and improve hardness profiles. Material and Methods Forty-eight enamel samples were prepared from extracted bovine lower central incisors. After embedding and polishing, nail varnish was applied, leaving a defined test area. One third of this area was covered with a flowable composite (non-demineralized control. The remaining area was demineralized in an acidic buffer solution for 18 d to simulate a carious lesion. Half the demineralized area was then covered with composite (demineralized control, while the last third was left open for three test and one control treatments: (A Application of enamel-matrix proteins (EMD - lyophilized protein fractions dissolved in acetic acid, Straumann, (B self-assembling peptides (SAP, Curodont, or (C amine fluoride solution (Am-F, GABA for 5 min each. Untreated samples (D served as control. After treatment, samples were immersed in artificial saliva for four weeks (remineralization phase and microhardness (Knoop depth profiles (25-300 µm were obtained at sections. Two-way ANOVA was calculated to determine differences between the areas (re-hardening or softening. Results Decalcification resulted in significant softening of the subsurface enamel in all groups (A-D. A significant re-hardening up to 125 µm was observed in the EMD and SAP groups. Conclusions This study showed that EMD and SAP were able to improve the hardness profiles when applied to deep demineralized artificial lesions. However, further research is needed to verify and improve this observed effect.

  6. In vivo and in vitro toxicity of nanogold conjugated snake venom protein toxin GNP-NKCT1

    Directory of Open Access Journals (Sweden)

    Partha Pratim Saha

    2014-01-01

    Full Text Available Research on nanoparticles has created interest among the biomedical scientists. Nanoparticle conjugation aims to target drug delivery, increase drug efficacy and imaging for better diagnosis. Toxicity profile of the nanoconjugated molecules has not been studied well. In this communication, the toxicity profile of snake venom cytotoxin (NKCT1, an antileukemic protein toxin, was evaluated after its conjugation with gold nanoparticle (GNP-NKCT1. Gold nanoparticle conjugation with NKCT1 was done with NaBH4 reduction method. The conjugated product GNP-NKCT1 was found less toxic than NKCT1 on isolated rat lymphocyte, mice peritoneal macrophage, in culture, which was evident from the MTT/Trypan blue assay. Peritoneal mast cell degranulation was in the order of NKCT1 > GNP-NKCT1. The in vitro cardiotoxicity and neurotoxicity were increased in case of NKCT1 than GNP-NKCT1. On isolated kidney tissue, NKCT1 released significant amount of ALP and γ-GT than GNP-NKCT1. Gold nanoconjugation with NKCT1 also reduced the lethal activity in mice. In vivo acute/sub-chronic toxicity studies in mice showed significant increase in molecular markers due to NKCT1 treatment, which was reduced by gold nanoconjugation. Histopathology study showed decreased toxic effect of NKCT1 in kidney tissue after GNP conjugation. The present study confirmed that GNP conjugation significantly decreased the toxicity profile of NKCT1. Further studies are in progress to establish the molecular mechanism of GNP induced toxicity reduction.

  7. In vitro bioaccessibility, bioavailability and plasma protein interaction of polyphenols from Annurca apple (M. pumila Miller cv Annurca).

    Science.gov (United States)

    Tenore, Gian Carlo; Campiglia, Pietro; Ritieni, Alberto; Novellino, Ettore

    2013-12-15

    The in vitro bioaccessibility, bioavailability and plasma protein interaction of polyphenols from Annurca apple and other conventional cultivars were evaluated. Salivary digestion concentrated into the medium 27-35% of native apple polyphenols, suggesting the potential bioavailability through the oral mucosal epithelium of significant amounts of bioactive compounds that could be gastric sensitive and/or poorly absorbed in the intestine. Annurca flesh revealed the highest content and provided the best intestinal bioaccessibility and bioavailability of oligomeric procyanidins among all of the apple peel and flesh tested. Since 49.4% of native procyanidins were not absorbed, they are expected to accumulate in the intestinal lumen where a potential inhibition capacity of cellular cholesterol uptake could be assumed. The permeated procyanidins (6.7% of their native pattern, 12.0% of intestinal procyanidins) significantly bound (58.7%) to plasma HDLs, suggesting a major role in cholesterol metabolism. Our results would indicate Annurca apple and its potential nutraceuticals as effective in the regulation of plasma cholesterol levels. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. In vitro study on digestion of pumpkin oil cake protein hydrolysate: evaluation of impact on bioactive properties.

    Science.gov (United States)

    Vaštag, Zužana; Popović, Ljiljana; Popović, Senka; Peričin-Starčević, Ivana; Krimer-Malešević, Vera

    2013-06-01

    In this work, a simulated gastrointestinal digestion of pumpkin oil cake protein hydrolysate prepared by alcalase (AH) was studied to evaluate the impact of the main gastrointestinal proteases on its antiradical and angiotensin I-converting enzyme (ACE) inhibitory activity. The in vitro digestion was performed in a model system under optimized reaction conditions, first by pepsin and then with α-chymotrypsin and trypsin, simultaneously. The treatment with the gastrointestinal proteases led to a significant increase of the degree of hydrolysis, up to 55.95 ± 3.1% in the final digest. After the digestion, the 2,2-azinobis3-ethylbenzo-thiazoline-6-sulphonic acid radical cation activity of AH was increased from 7.59 ± 0.1 to 10.25 ± 0.3 mM trolox equivalent antioxidant coefficient/mg (p 0.05) in the final digest. These results showed an advantage of AH to increase the antiradical and resist ACE inhibitory activity during digestion by main gastrointestinal proteases, appearing as promising bioactive food ingredient.

  9. Effects of electron beam irradiation on chemical composition, antinutritional factors, ruminal degradation and in vitro protein digestibility of canola meal

    Energy Technology Data Exchange (ETDEWEB)

    Taghinejad-Roudbaneh, M., E-mail: mtaghinejad@iaut.ac.i [Department of Animal Science, Faculty of Agriculture, Islamic Azad University, Tabriz Branch, P.O. Box 51589, Tabriz (Iran, Islamic Republic of); Ebrahimi, S.R. [Department of Animal Science, Faculty of Agriculture, Shahr-e-Qods Branch, Islamic Azad University, P.O. Box 37515-374, Shahr-e-Qods (Iran, Islamic Republic of); Azizi, S. [Department of Clinical Sciences, Faculty of Veterinary Medicine, Urmia University, P.O. Box 57155-1177, Urmia (Iran, Islamic Republic of); Shawrang, P. [Nuclear Science and Technology Research Institute, Agricultural, Medical and Industrial Research School, Atomic Energy Organization of Iran, P.O. Box 31485-498, Karaj (Iran, Islamic Republic of)

    2010-12-15

    The aim of the present study was to determine the impact of electron beam (EB) irradiation at doses of 15, 30 and 45 kGy on the nutritional value of canola meal. The phytic acid and total glucosinolate content of EB-irradiated canola meal decreased as irradiation doses increased (P<0.01). From in situ results, irradiation of canola meal at doses of 45 kGy decreased (P<0.05) the effective degradibility of crude protein (CP) by 14%, compared with an untreated sample. In vitro CP digestibility of EB-irradiated canola meal at doses of 15 and 30 kGy was improved (P<0.05). Electrophoresis results showed that napin and cruciferin sub-units of 30 and 45 kGy EB-irradiated canola meal were more resistant to degradation, compared with an untreated sample. Electron beam irradiation was effective in protecting CP from ruminal degradation and reducing antinutritional factors of irradiated canola meal.

  10. [Deinococcus radiodurans RecX and Escherichia coli RecX Proteins Are Able to Replace Each Other in vivo and in vitro].

    Science.gov (United States)

    Bakhlanova, I V; Baitin, D M

    2016-03-01

    A plasmid carrying the Deinococcus radiodurans recXgene under the control of a lactose promoter decreases the Escherichia coli cell resistance to UV irradiation and γ irradiation and also influences the conjugational recombination process. The D. radiodurans. RecX protein functions in the Escherichia coli cells similarly to the E. coli RecX protein. Isolated and purified D. radiodurans RecX and E. coli RecX proteins are able to replace each other interacting with the E. coli RecA and D. radiodurans RecA proteins in vitro. Data obtained demonstrated that regulatory interaction of RecA and RecX proteins preserves a high degree of conservatism despite all the differences in the recombination reparation system between E. coli and D. radiodurans.

  11. Entrapment of proteins and peptides in chitosan-polyphosphoric acid hydrogel beads: A new approach to achieve both high entrapment efficiency and controlled in vitro release.

    Science.gov (United States)

    Yuan, Dongdong; Jacquier, Jean Christophe; O'Riordan, E Dolores

    2018-01-15

    Bovine serum albumin (BSA), whey protein isolate (WPI), insulin and a casein hydrolysate were entrapped in chitosan-polyphosphoric acid (PPA) beads. The in vitro release of protein from the beads in simulated gastric fluid (SGF, pH 3) and simulated intestinal fluid (SIF, pH 7) was evaluated. High entrapment efficiencies were achieved for intact proteins (>95% in all cases) but entrapment was lower for the casein hydrolysate (circa 50%), possibly indicating a physical or steric entrapment of the proteins in these chitosan-PPA beads. Inhibited release of BSA, in both SGF and SIF, was achieved with low PPA concentration. Insulin and WPI were effectively retained in SGF and gradually released in SIF. Peptides from casein hydrolysate were partially (circa 35%) but quickly released in SGF with no further release in SIF. Overall, these results indicate that chitosan-PPA beads show potential for lower gastrointestinal delivery of bioactive protein material. Copyright © 2017. Published by Elsevier Ltd.

  12. Menstrual Pattern following Tubal Ligation: A Historical Cohort Study

    Directory of Open Access Journals (Sweden)

    Shahideh Jahanian Sadatmahalleh

    2016-12-01

    Full Text Available Background: Tubal ligation (TL is recommended for women who have completed their family planning. The existence of the menstrual disorders following this procedure has been the subject of debate for decades. This study was conducted to identify the relationship between tubal ligation and menstrual disorders. Materials and Methods: A historical cohort study was carried out on 140 women undergoing tubal ligation (TL group and on 140 women using condom as the main contraceptive method (Non-TL group. They aged between 20 and 40 years and were selected from a health care center in Rudsar, Guilan Province, Iran, during 2013-2014. The two groups were comparable in demographic characteristics, obstetrical features and menstrual bleeding pattern using a routine questionnaire. A validated pictorial blood loss assessment chart (PBLAC was also used to measure the menstrual blood loss. Results: Women with TL had more menstrual irregularity than those without TL (24.3 vs. 10%, P=0.002. Women with TL had more polymenorrhea (9.3 vs. 1.4%, P=0.006, hypermenorrhea (12.1 vs. 2.1%, P=0.002, menorrhagia (62.9 vs. 22.1%, P<0.0001 and menometrorrhagia (15.7 vs. 3.6%, P=0.001 than those without TL. There is a significant difference in the PBLAC score between women with and without TL (P<0.0001. According to logistic regression, age odds ratio [(OR=1.08, confidence interval (CI:1.07-1.17, P=0.03], TL (OR=5.95, CI:3.45-10.26, P<0.0001 and cesarean section (OR=2.72, CI:1.49-4.97, P=0.001 were significantly associated with menorrhagia. Conclusion: We found significant differences in menstrual disorders between women with and without TL. Therefore, women should be informed by the health providers regarding the advantages and disadvantages of TL before the procedures.

  13. Traditional elastic ligatures versus slide ligation system. A morphological evaluation.

    Science.gov (United States)

    Condò, R; Casaglia, A; Armellin, E; Condò, S G; Cerroni, L

    2013-01-01

    Elastomeric materials play an important role in the orthodontic practice, including the retraction force to move teeth into extraction sites, closing diastemas, selective shifting of the midline and generalized space closure. Frictional resistance and ligating strength of archwire-bracket-ligature complex occurs during utilization of elastomeric and metallic ligatures when orthodontic forces are applicated. The aim of this study was to analyze elastic deformation of three types of elastomeric ligatures, after clinical use. ELASTOMERIC LIGATURES: ring-shape, transparent, latex ligatures (Leone® S.p.A.), ring-shape, grey, polyurethane ligatures (Micerium® S.p.A.) and grey, polyurethane, Slide low-friction ligatures (Leone® S.p.A.). A total of 9 orthodontic patients undergoing fixed orthodontic therapy were selected. Three specimens were applied, one for each types of ligature, inside the oral cavity of each subject. Samples were kept in the oral cavity for 28 days, ligating 0.16 X 0.22 inches stainless steel archwires to stainless steel premolars brackets (Leone® S.p.A., Sesto Fiorentino, FI, Italy) for Bidimensional technique. After the pre-established time, the systems of ligature were removed and washed. Control group consisted of 9 unused specimens of each ligation type. Each elastomeric ligature was observed under the scanning electron microscope (SEM) to determine variations in size. The archwire-bracket-ligature complex was also analyzed. Transparent O-ring ligatures showed significant volumetric and structural changes. The external rounded shape was rather maintained, while the internal shape tended to appear square. Both external and internal diameter significantly increased (pO-ring ligatures endure significant volumetric and structural changes, after clinical use, index of a greater degree of friction and early loss in functionality. Grey, polyurethane Slide low-friction ligatures presented limited variation in size after clinical use.

  14. Self-ligating vs conventional brackets as perceived by orthodontists.

    Science.gov (United States)

    Prettyman, Chase; Best, Al M; Lindauer, Steven J; Tufekci, Eser

    2012-11-01

    To determine if there are significant clinical differences between self-ligating brackets (SLB) and conventional brackets (CB) during orthodontic treatment, as perceived by orthodontists. A survey was developed and distributed to evaluate how SLB compare to CB in terms of orthodontists' perceptions (n  =  430). SLB were preferred during the initial stage of treatment based on the shorter adjustment appointments and faster initial treatment progress they provided (P orthodontists' preference was significantly influenced by (1) the proportion of patients treated with SLB (P < .0001), (2) the number of cases it took them to become accustomed to SLB (P < .0001), and (3) the average appointment intervals associated with SLB (P < .0001).

  15. In vitro digestibility and nutritional characterization of distillers dried grains with solubles according to the Cornell Net Carbohydrate and Protein System

    Directory of Open Access Journals (Sweden)

    Luiz Juliano Valério Geron

    2017-08-01

    Full Text Available We evaluated the fractions of protein and carbohydrates in distillers dried grains with solubles (DDGS, corn grain (CG, soybean meal (SM, and corn silage (CS, as well as the in vitro digestibility (IVD of DDGS, CG, SM, CS, rations containing 0.0, 8.0, 16.0, and 24.0% DDGS, and in vitro fermentation parameters after 24 h of incubation. DDGS were obtained following microbial fermentation for ethanol production from a sugar and alcohol distillery located in the state of Mato Grosso - Brazil. The Cornell Net Carbohydrate and Protein System (CNCPS was used to determine the protein and carbohydrate fractions of experimental diets. For the in vitro nutrient digestion assay using the experimental foods and experimental diets, two sheep with an average body weight of 26 kg were used as inoculum donors. The in vitro digestibility of food and feed was assayed in three replicates. Fraction A of DDGS CP was 88, 71, and 37% lower in relation to fraction A of SM, CG, and CS, respectively. Fraction B2 of DDGS protein contained 21% CP, which represents 78.84% of DDGS protein in fraction B2, and is higher than the SM, which was 70.44%. The B3 fraction of CP, which is partly released during ruminal fermentation, was 18% lower for SM compared to DDGS, and is expressed in %CP. For carbohydrate fractionation, the DDGS presented 8.64% for the A + B1 fraction on a DM basis, which was 62, 86, and 74% lower compared to those obtained for SM, CG. and CS, respectively. The hemicellulose and cellulose contents of DDGS were higher than those of SM, as verified in fraction B2, with a value of 46.92%, expressed in DM. The in vitro digestibility coefficients (IVDC of the DDGS nutrients did not differ (p > 0.05 in relation to those of the other experimental foods. The inclusion of DDGS in rations formulated for sheep did not change (p > 0.05 the IVDC of DM, OM, CP. NDF, or ADF, with mean values of 70.93, 70.64, 59.58, 52.83, and 43.40%, respectively. Therefore, DDGS comprise a

  16. Adipose tissue-deprived stem cells acquire cementoblast features treated with dental follicle cell conditioned medium containing dentin non-collagenous proteins in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Wen, Xiujie; Nie, Xin; Zhang, Li [Department of Stomatology, Daping Hospital and Research Institute of Surgery, Third Military Medical University, 10 Daping Changjiang Branch Road, Yuzhong District, Chongqing 400042 (China); Liu, Luchuan, E-mail: liuluchuan1957@126.com [Department of Stomatology, Daping Hospital and Research Institute of Surgery, Third Military Medical University, 10 Daping Changjiang Branch Road, Yuzhong District, Chongqing 400042 (China); Deng, Manjing, E-mail: iradeng@163.com [Department of Stomatology, Daping Hospital and Research Institute of Surgery, Third Military Medical University, 10 Daping Changjiang Branch Road, Yuzhong District, Chongqing 400042 (China)

    2011-06-10

    Highlights: {yields} In this study we examine the effects of dental follicle cell conditioned medium (DFCCM) containing dentin non-collagenous proteins (dNCPs) on differentiation of ADSCs. {yields} We examined that ADSCs treated with dNCPs/DFCCM underwent morphological changes and significantly lost their proliferative capacity. {yields} dNCPs/DFCCM enhanced the mineralization behaviour and mineralization-related marker expression of ADSCs. {yields} ADSCs acquired cementoblast features in vitro with dNCPs/DFCCM treatment. -- Abstract: Adipose tissue-derived stem cells (ADSCs), which are easily harvested and show excellent pluripotency potential, have generated considerable interest in regenerative medicine. In this study, the differentiation of ADSCs was assessed after treatment with dental follicle cell conditioned medium (DFCCM) containing dentin non-collagenous proteins (dNCPs). ADSCs exhibited a fibroblast-like morphology and high proliferative capacity. However, after treatment with dNCPs/DFCCM, ADSCs changed from a fibroblast-like to cementoblast-like morphology and significantly lost their proliferative capacity. Alkaline phosphatase activity and in vitro mineralization behaviour of ADSCs were significantly enhanced. Mineralization-related markers including cementum attachment protein, bone sialoprotein, osteocalcin, osteopontin and osteonectin were detected at mRNA or protein levels, whereas dentin sialophosphoprotein and dentin sialoprotein were not detected, implying a cementoblast-like phenotype. These results demonstrate that ADSCs acquired cementoblast features in vitro with dNCPs/DFCCM treatment and could be a potential source of cementogenic cells for periodontal regeneration.

  17. Size-dependent studies of macromolecular crowding on the thermodynamic stability, structure and functional activity of proteins: in vitro and in silico approaches.

    Science.gov (United States)

    Shahid, Sumra; Hassan, Md Imtaiyaz; Islam, Asimul; Ahmad, Faizan

    2017-02-01

    The environment inside cells in which proteins fold and function are quite different from that of the dilute buffer solutions often used during in vitro experiments. The presence of large amounts of macromolecules of varying shapes, sizes and compositions makes the intracellular milieu extremely crowded. The overall concentration of macromolecules ranges from 50 to 400gl-1, and they occupy 10-40% of the total cellular volume. These differences in solvent conditions and the level of crowdedness resulting in excluded volume effects can have significant consequences on proteins' biophysical properties. A question that arises is: how important is it to examine the roles of shape, size and composition of macromolecular crowders in altering the biological properties of proteins? This review article aims at focusing, gathering and summarizing all of the research investigations done by means of in vitro and in silico approaches taking into account the size-dependent influence of the crowders on proteins' properties. Altogether, the internal architecture of macromolecular crowding environment including size, shape and concentration of crowders, appears to be playing an extremely important role in causing changes in the biological processes. Most often the small sized crowders have been found more effective crowding agents. However, thermodynamic stability, structure and functional activity of proteins have been governed by volume exclusion as well as soft (chemical) interactions. The article provides an understanding of importance of internal architecture of the cellular environment in altering the biophysical properties of proteins. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Evaluation of cerebral electrical activity and cardiac output after patent ductus arteriosus ligation in preterm infants.

    LENUS (Irish Health Repository)

    Leslie, A T F S

    2013-11-01

    To characterize and investigate the relationship between systemic blood flow and pre- and postoperative cerebral electrical activity in preterm neonates undergoing patent ductus arteriosus (PDA) ligation.

  19. Reformulating cereal bars: high resistant starch reduces in vitro digestibility but not in vivo glucose or insulin response; whey protein reduces glucose but disproportionately increases insulin.

    Science.gov (United States)

    Wolever, Thomas Ms; van Klinken, B Jan-Willem; Bordenave, Nicolas; Kaczmarczyk, Melissa; Jenkins, Alexandra L; Chu, YiFang; Harkness, Laura

    2016-10-01

    Resistant starch (RS) and whey protein are thought to be effective nutrients for reducing glycemic responses. We aimed to determine the effect of varying the sucrose, RS, and whey protein content of cereal bars on glucose and insulin responses. Twelve healthy subjects [mean ± SD age: 36 ± 12 y; mean ± SD body mass index (in kg/m(2)): 24.9 ± 2.7] consumed 40 g available-carbohydrate (avCHO) portions of 5 whole-grain cereal bars that contained varying amounts of RS and whey protein concentrate [WPC; 70% protein; RS:WPC, %wt:wt: 15:0 (Bar15/0); 15:0, low in sucrose (Bar15/0LS); 15:5 (Bar15/5); 10:5 (Bar10/5); and 10:10 (Bar10/10)] and 2 portion sizes of a control bar low in whole grains, protein, and RS [control 1 contained 40 g avCHO (Control1); control 2 contained total carbohydrate equal to Bar15/0LS (Control2)] on separate days by using a randomized crossover design. Glucose and insulin responses in vivo and carbohydrate digestibility in vitro were measured over 3 h. Incremental area under the curve (iAUC) over 0-3 h for glucose (min × mmol/L) differed significantly between treatments (P digested in vitro ∼30% more slowly than the control bars (P digestibility in vitro. Glucose and insulin responses elicited by high-RS, whey protein-free bars were similar to those elicited from control bars. The inclusion of RS in cereal bar formulations did not reduce glycemic responses despite slower starch digestion in vitro. Thus, caution is required when extrapolating in vitro starch digestibility to in vivo glycemic response. The inclusion of whey protein in cereal bar formulations to reduce glycemic response requires caution because this may be associated with a disproportionate increase in insulin as judged by an increased insulin-to-glucose iAUC ratio. This trial was registered at clinicaltrials.gov as NCT02537587. © 2016 American Society for Nutrition.

  20. Expression of kit ligand and insulin-like growth factor binding protein 3 during in vivo or in vitro development of ovarian follicles in sheep.

    Science.gov (United States)

    Srividya, D; Praveen Chakravarthi, V; Kona, Ssr; Siva Kumar, Avn; Brahmaiah, K V; Rao, V H

    2017-08-01

    Expression of Kit ligand (KL) and insulin-like growth factor binding protein (IGFBP3) genes was studied at different in vivo and corresponding in vitro stages of development of the ovarian follicles in sheep. The expression of both KL and IGFBP3 was significantly higher in the primordial follicles relative to any other stage of development. Compared to the other stages, the KL expression in the cumulus cells from in vivo grown large antral follicles and that of IGFBP3 in COCs' isolated from large antral follicles matured in vitro for 24 hr were significantly higher. In the oocytes from in vivo grown ovarian follicles, the expression of KL was the same at all the stages of development. Insulin-like growth factor binding protein 3 expression was also the same in the oocytes at all the stages of the development except for a significantly lower expression in those from antral follicles. The expression of KL in the cumulus cells decreased significantly in the in vitro grown early antral follicles but did not change further as the development progressed. The expression of IGFBP3 in the cumulus cells from in vitro grown ovarian follicles appeared to increase as the development progressed although the increase was not significant between any two consecutive stages of development. In the oocytes in in vitro grown ovarian follicles, the expression levels of KL and IGFBP3 genes did not change with development. It is concluded that (i) KL and IGFBP3 genes follow specific patterns of expression during ovarian folliculogenesis and (ii) in vitro culture of preantral follicles compromises the development potential through alterations in the stage-specific patterns of expression of these and other developmentally important genes. © 2017 Blackwell Verlag GmbH.

  1. Effect of pre-treatment on in vitro gastric digestion of quinoa protein (Chenopodium quinoa Willd.) obtained by wet and dry fractionation.

    Science.gov (United States)

    Opazo-Navarrete, M; Schutyser, M A I; Boom, R M; Janssen, A E M

    2018-02-01

    Quinoa protein was isolated from quinoa seeds using wet fractionation that resulted in a protein isolate (QPI) with a high protein purity of 87.1% (w/dw) and a protein yield of around 54%, and a dry fractionation method delivered a quinoa protein concentrate (QPC) with a purity of 27.8% (w/dw) and yield of around 47%. The dry fractionation process only involves milling and sieving and keeps the protein in its natural, native state. The aim was to study the in vitro gastric digestibility of both protein. Attention was paid to thermal pre-treatment of QPI and QPC. QPC showed significantly higher (p < .05) digestibility than QPI samples. The results were interpreted with a simple double exponential model. The fraction of easily digested protein in QPC is higher than for QPI. The better digestibility of the QPC was explained by the prevention of the formation of large aggregates during pre-heating of the protein.

  2. Longer treatment times with self-ligated orthodontic brackets.

    Science.gov (United States)

    O'Brien, Kevin

    2014-09-01

    The Medline, Cochrane Library, Biomed Central, BBO including LILACS, Ind Med, Sceilo, Clinical trials.gov, Conference paper Index, Digital Dissertations, German National Library of Medicine (ZB MED), Google Scholar, ISI Web of Knowledge, metaRegister of Controlled Trials, OpenSIGLE and Scirus databases were searched. Randomised controlled trials (RCTs) and quasi-RCTs in patients having fixed-appliance orthodontic treatments were considered. Study assessment data extraction and risk of bias assessment was carried out independently by two reviewers. Overall quality of evidence was based on the Grades of Recommendation Assessment, Development and Evaluation (GRADE) approach. Random-effects meta-analyses were performed where data could be pooled. Twenty five trials (1321 patients) were included. The majority (24) compared self-ligated (SL) and conventional brackets (CL). No trials primarily investigated the effect of bracket material and no indirect comparison was possible. Two trials assessed the bracket slot size but found no consistent difference between 0.022'' and 0.018'' brackets. Four studies contributed to a meta-analysis that showed overall duration of the orthodontic treatment be significantly longer in the SL group by 2.01 months (95%CI; 0.45 to 3.57). Based on existing evidence, no clinical recommendation can be made regarding the bracket material or different ligation modules. For Sl brackets, no conclusive benefits could be proven, while their use was associated with longer treatment durations.

  3. TLR9 ligation in pancreatic stellate cells promotes tumorigenesis.

    Science.gov (United States)

    Zambirinis, Constantinos P; Levie, Elliot; Nguy, Susanna; Avanzi, Antonina; Barilla, Rocky; Xu, Yijie; Seifert, Lena; Daley, Donnele; Greco, Stephanie H; Deutsch, Michael; Jonnadula, Saikiran; Torres-Hernandez, Alejandro; Tippens, Daniel; Pushalkar, Smruti; Eisenthal, Andrew; Saxena, Deepak; Ahn, Jiyoung; Hajdu, Cristina; Engle, Dannielle D; Tuveson, David; Miller, George

    2015-11-16

    Modulation of Toll-like receptor (TLR) signaling can have protective or protumorigenic effects on oncogenesis depending on the cancer subtype and on specific inflammatory elements within the tumor milieu. We found that TLR9 is widely expressed early during the course of pancreatic transformation and that TLR9 ligands are ubiquitous within the tumor microenvironment. TLR9 ligation markedly accelerates oncogenesis, whereas TLR9 deletion is protective. We show that TLR9 activation has distinct effects on the epithelial, inflammatory, and fibrogenic cellular subsets in pancreatic carcinoma and plays a central role in cross talk between these compartments. Specifically, TLR9 activation can induce proinflammatory signaling in transformed epithelial cells, but does not elicit oncogene expression or cancer cell proliferation. Conversely, TLR9 ligation induces pancreatic stellate cells (PSCs) to become fibrogenic and secrete chemokines that promote epithelial cell proliferation. TLR9-activated PSCs mediate their protumorigenic effects on the epithelial compartment via CCL11. Additionally, TLR9 has immune-suppressive effects in the tumor microenvironment (TME) via induction of regulatory T cell recruitment and myeloid-derived suppressor cell proliferation. Collectively, our work shows that TLR9 has protumorigenic effects in pancreatic carcinoma which are distinct from its influence in extrapancreatic malignancies and from the mechanistic effects of other TLRs on pancreatic oncogenesis. © 2015 Zambirinis et al.

  4. Impact of Processing on the Protein Quality of Pinto Bean (Phaseolus vulgaris) and Buckwheat (Fagopyrum esculentum Moench) Flours and Blends, As Determined by in Vitro and in Vivo Methodologies.

    Science.gov (United States)

    Nosworthy, Matthew G; Franczyk, Adam; Zimoch-Korzycka, Anna; Appah, Paulyn; Utioh, Alphonsus; Neufeld, Jason; House, James D

    2017-05-17

    Blending of protein sources can increase protein quality by compensating for limiting amino acids present in individual sources, whereas processing grain flours by extrusion or baking can also alter protein quality. To determine the effect of baking and extrusion on the protein quality of blended flours from buckwheat and pinto beans, a rodent bioassay was performed and compared to an in vitro method of protein quality determination. Overall, extruded products had higher protein efficiency ratio values, increased digestibility, and greater protein digestibility corrected amino acid score (PDCAAS) values than baked products, with the extruded buckwheat/pinto blend having the greatest PDCAAS value of the experimental diets investigated. A correlation was found between both digestibility and PDCAAS values generated from in vitro and in vivo methods. The use of in vitro digestibility analysis should be investigated as a potential replacement for the current rodent assay for nutrient content claim purposes.

  5. Single daily dosing of antibiotics: importance of in vitro killing rate, serum half-life, and protein binding.

    Science.gov (United States)

    Potel, G; Chau, N P; Pangon, B; Fantin, B; Vallois, J M; Faurisson, F; Carbon, C

    1991-10-01

    The relative importance of pharmacokinetic and pharmacodynamic parameters for the feasibility of a single daily dose (SDD) of antibiotics remains to be established. Therefore, we studied the relationship between in vitro bacteriological parameters (MIC, MBC, and killing rate [KR], defined as the reduction in the inoculum within 3 h), pharmacokinetic parameters (t1/2 and protein binding [PB], and in vivo antibacterial effect of a single antibiotic dose in an experimental rabbit model of Escherichia coli endocarditis. Nine antibiotics were investigated: two aminoglycosides, two quinolones, and five beta-lactams. For each drug, the minimal effective dose (MED) (in milligrams per kilogram) was defined as the lowest dose able to achieve a significant difference (P less than 0.05) of CFU in the vegetations in comparison with controls 24 h after a single intravenous injection. Aminoglycosides and quinolones had the lowest MEDs, followed by beta-lactams. Univariate regression analysis showed that KR was the major determinant of MED. A stepwise regression analysis showed that t1/2 significantly improved the predictive value of KR, while PB, MIC, and MBC did not. The final equation was MED = 1,586-238 KR-297 t1/2 (r = 0.90, P = 0.01). We concluded that the pharmacodynamic parameters (especially the high KR) of aminoglycosides and quinolones explained their low MEDs and might allow SDD. In contrast, the low KR of beta-lactams emphasized the critical importance of a long t1/2, as for ceftriaxone, allowing the use of this beta-lactam alone in SDD.

  6. Raf kinase inhibitor protein (RKIP) inhibits the cell migration and invasion in human glioma cell lines in vitro.

    Science.gov (United States)

    Lei, Xuhui; Chang, Liang; Ye, Wei; Jiang, Chuanlu; Zhang, Zhiren

    2015-01-01

    To investigate the effects and the potential mechanisms of RKIP on cell migration, invasion and proliferation in human glioma cell lines in vitro. The RKIP over-expressing and RKIP knockdown human U87 glioma cells were used to reveal the effects of RKIP on human glioma cells migration, invasion and proliferation. After the recombinant plasmid pcDNA3.0-RKIP or RKIP-shRNA was transfected into the cell lines U87 by the means of liposome assay, the cells migration, invasion and proliferation were detected by wound healing, Transwell and MTT assay. Then, the levels of RKIP, MMP-3, MMP-9 and HMGA2 mRNA transcription were measured by means of RT-qPCR and levels of proteins expressions were determined using Western blot. The results of MTT assay suggested that the PKIP have little inhibitive effects on glioma cells proliferation (P>0.05). The present paper showed that the migration distances in the group of RKIP-shRNA were markedly increased compared to the pcDNA3.0-RKIP and control. Similarly, the results showed that the numbers of invasion cells in RKIP-shRNA were remarkably increased than the pcDNA3.0-RKIP group and control group. Western blot and RT-qPCR suggested that over-expressions of RKIP lessened the MMP-2, MMP-9 and HMGA2 expression, however, turning down the RKIP expression showed the inverse effects. RKIP inhibits the cells migrations and invasions. Meanwhile, RKIP might inhibit the glioma cells through inhibiting MMPs and HMAG2 expression. Therefore, we demonstrated that RKIP is an underlying target for the treatment of glioma.

  7. In vitro screening for protein tyrosine phosphatase 1B and dipeptidyl peptidase IV inhibitors from selected Nigerian medicinal plants.

    Science.gov (United States)

    Saidu, Yusuf; Muhammad, Suleiman Alhaji; Abbas, Abdullahi Yahaya; Onu, Andrew; Tsado, Ibrahim Mohammed; Muhammad, Luba

    2017-01-01

    Protein tyrosine phosphatase 1B (PTP 1B) and dipeptidyl peptidase IV (DPP IV) have been identified as one of the drug targets for the treatment of Type-2 diabetes. This study was designed to screen for PTP 1B and DPP-IV inhibitors from some Nigerian medicinal plants. PTP 1B and DPP-IV drug discovery kits from Enzo Life Sciences were used to investigate in vitro inhibitory effect of crude methanolic extract of 10 plants; Mangifera indica, Moringa oleifera, Acacia nilotica, Arachis hypogaea, Senna nigricans, Azadirachta indica, Calotropis procera, Leptadenia hastata, Ziziphus mauritiana, and Solanum incanum. The results indicated PTP IB inhibition by S. nigricans (68.2 ± 2.29%), A. indica (67.4 ± 2.80%), A. hypogaea (57.2 ± 2.50%), A. nilotica (55.1 ± 2.19%), and M. oleifera (41.2 ± 1.87%) were significantly (P 0.05) different from that of sumarin. The DPP-IV inhibition by S. incanum (68.1 ± 2.71%) was significantly higher as compared with a known inhibitor, P32/98. S. nigrican (57.0±1.91%), Z. mauritiana (56.6±2.01%), A. hypogaea (51.0±1.30%), M. indica (44.6 ± 2.40%), C. procera (36.2 ± 2.00%), A. nilotica (35.4 ± 2.10%), and A. indica (33.6 ± 1.50%) show significantly (P < 0.05) lower inhibitions toward DPP-IV. The work demonstrated that these plant materials could serve as sources of lead compounds in the development of anti-diabetic agent(s) targeting PTP 1B and/or DPP-IV.

  8. In vitro gastrointestinal digestion of pea protein isolate as a function of pH, food matrices, autoclaving, high-pressure and re-heat treatments

    OpenAIRE

    Laguna, Laura; PICOUET, Pierre; Guàrdia, M. Dolors; Renard, Catherine; Sarkar, Anwesha

    2017-01-01

    This study investigated the influence of pH and processing conditions (autoclave at 93 °C/13 min or high pressure processing (HPP) at 600 MPa/5 min without/with follow-up reheating at 80 °C/30 min) on the digestibility of pea protein isolate. Both aqueous solutions and real food matrices (apple and carrot purees) containing pea protein was examined at 37 °C. In vitro gastrointestinal digestion was followed using sodium dodecyl sulphate polyacrylamide gel electrophoresis, titrimetric technique...

  9. In vitro reconstitution of flagellar filaments onto hooks of filamentless mutants of Salmonella typhimurium by addition of hook-associated proteins.

    OpenAIRE

    Homma, M; Iino, T; Kutsukake, K; Yamaguchi, S.

    1986-01-01

    An in vitro system for reconstituting flagellar filaments onto hooks of filamentless mutants of Salmonella typhimurium was used to investigate the role played in filament formation by the three hook-associated proteins (HAPs, products of the flaW, flaU, and flaV genes). These proteins--FlaW, FlaU, and FlaV--are believed to be assembled in this order at the distal end of the hook. When the recipient hooks were provided by flaU mutants, whose hook tips contained FlaW only, exogenous FlaU was es...

  10. Investigation on Secondary Structure Perturbations of Proteins Embedded in Solid Lipid Matrices as a Novel Indicator of their Biological Activity upon In Vitro Release

    DEFF Research Database (Denmark)

    Zeeshan, Farrukh; Tabbassum, Misbah; Jorgensen, Lene

    2018-01-01

    Protein biologics are prone to conformational changes during formulation development. Limited methods are available for conformational analysis of proteins in solid state and in the presences of formulation excipients. The aim of this study was to investigate the secondary structures of proteins...... encased in solid lipid matrices as a novel indicator of their stability upon in vitro release. Model proteins namely catalase and lysozyme were incorporated into lipid namely Precirol® AT05 (glycerol palmitostearate, melting point 58°C) at 30% w/w loading using melting and mixing and wet granulation...... methods. Attenuated total reflectance (ATR-FTIR) spectroscopy, size-exclusion chromatography (SEC) and biological activity analyses were performed. The information about secondary structure was acquired using second derivative analysis of amide-I band (1600-1700 cm(-1)). ATR analysis demonstrated...

  11. Magnolol causes alterations in the cell cycle in androgen insensitive human prostate cancer cells in vitro by affecting expression of key cell cycle regulatory proteins.

    Science.gov (United States)

    McKeown, Brendan T; McDougall, Luke; Catalli, Adriana; Hurta, Robert A R

    2014-01-01

    Prostate cancer, one of the most common cancers in the Western world, affects many men worldwide. This study investigated the effects of magnolol, a compound found in the roots and bark of the magnolia tree Magnolia officinalis, on the behavior of 2 androgen insensitive human prostate cancer cell lines, DU145 and PC3, in vitro. Magnolol, in a 24-h exposure at 40 and 80 μM, was found to be cytotoxic to cells. Magnolol also affected cell cycle progression of DU145 and PC3 cells, resulting in alterations to the cell cycle and subsequently decreasing the proportion of cells entering the G2/M-phase of the cell cycle. Magnolol inhibited the expression of cell cycle regulatory proteins including cyclins A, B1, D1, and E, as well as CDK2 and CDK4. Protein expression levels of pRBp107 decreased and pRBp130 protein expression levels increased in response to magnolol exposure, whereas p16(INK4a), p21, and p27 protein expression levels were apparently unchanged post 24-h exposure. Magnolol exposure at 6 h did increase p27 protein expression levels. This study has demonstrated that magnolol can alter the behavior of androgen insensitive human prostate cancer cells in vitro and suggests that magnolol may have potential as a novel anti-prostate cancer agent.

  12. Activation of Stat-3 is involved in the induction of apoptosis after ligation of major histocompatibility complex class I molecules on human Jurkat T cells

    DEFF Research Database (Denmark)

    Skov, S; Nielsen, M; Bregenholt, S

    1998-01-01

    . In addition, the transcription factor Stat-3 was tyrosine phosphorylated in the cytoplasm and subsequently translocated to the cell nucleus. Data obtained by electrophoretic mobility shift assay suggested that the activated Stat-3 protein associates with the human serum-inducible element (hSIE) DNA......-probe derived from the interferon-gamma activated site (GAS) in the c-fos promoter, a common DNA sequence for Stat protein binding. An association between hSIE and Stat-3 after MHC-I ligation was directly demonstrated by precipitating Stat-3 from nuclear extracts with biotinylated hSIE probe and avidin......Activation of Janus tyrosine kinases (Jak) and Signal transducers and activators of transcription (Stat) after ligation of major histocompatibility complex class I (MHC-I) was explored in Jurkat T cells. Cross-linking of MHC-I mediated tyrosine phosphorylation of Tyk2, but not Jak1, Jak2, and Jak3...

  13. Magnolol Affects Cellular Proliferation, Polyamine Biosynthesis and Catabolism-Linked Protein Expression and Associated Cellular Signaling Pathways in Human Prostate Cancer Cells in vitro

    Directory of Open Access Journals (Sweden)

    Brendan T. McKeown

    2015-01-01

    Full Text Available Background: Prostate cancer is the most commonly diagnosed form of cancer in men in Canada and the United States. Both genetic and environmental factors contribute to the development and progression of many cancers, including prostate cancer. Context and purpose of this study: This study investigated the effects of magnolol, a compound found in the roots and bark of the magnolia tree Magnolia officinalis, on cellular proliferation and proliferation-linked activities of PC3 human prostate cancer cells in vitro. Results: PC3 cells exposed to magnolol at a concentration of 80 μM for 6 hours exhibited decreased protein expression of ornithine decarboxylase, a key regulator in polyamine biosynthesis, as well as affecting the expression of other proteins involved in polyamine biosynthesis and catabolism. Furthermore, protein expression of the R2 subunit of ribonucleotide reductase, a key regulatory protein associated with DNA synthesis, was significantly decreased. Finally, the MAPK (mitogen-activated protein kinase, PI3K (phosphatidylinositol 3-kinase, NFκB (nuclear factor of kappa-light-chain-enhancer of activated B cells and AP-1 (activator protein 1 cellular signaling pathways were assayed to determine which, if any, of these pathways magnolol exposure would alter. Protein expressions of p-JNK-1 and c-jun were significantly increased while p-p38, JNK-1/2, PI3Kp85, p-PI3Kp85, p-Akt, NFκBp65, p-IκBα and IκBα protein expressions were significantly decreased. Conclusions: These alterations further support the anti-proliferative effects of magnolol on PC3 human prostate cancer cells in vitro and suggest that magnolol may have potential as a novel anti-prostate cancer agent.

  14. The use of an in vitro microneutralization assay to evaluate the potential of recombinant VP5 protein as an antigen for vaccinating against Grass carp reovirus

    Directory of Open Access Journals (Sweden)

    Xu Dan

    2011-03-01

    Full Text Available Abstract Background Grass carp reovirus (GCRV is the causative pathogen of grass carp hemorrhagic disease, one of the major diseases damaging grass carp Ctenopharyngon idellus breeding industry in China. Prevention and control of the disease is impeded largely due to the lack of research in economic subunit vaccine development. This study aimed to evaluate the potential of viral outer shell protein VP5 as subunit vaccine. Methods The vp5 gene was isolated from the viral genome through RT-PCR and genetically engineered to express the recombinant VP5 protein in E coli. The viral origin of the recombinant protein was confirmed by Western blot analysis with a monoclonal antibody against viral VP5 protein. Polyclonal antibody against the recombinant VP5 protein was prepared from mice. A microneutralization assay was developed to test its neutralizing ability against GCRV infection in cell culture. Results The GST-VP5 fusion protein (rVP5 was produced from E. Coli with expected molecular weight of 90 kDa. The protein was purified and employed to prepare anti-VP5 polyclonal antibody from mice. The anti-VP5 antibody was found to neutralize GCRV through in vitro microneutralization assay and viral progeny quantification analysis. Conclusions The present study showed that the viral VP5 protein was involved in viral infection and bacterially-expressed VP5 could be suitable for developing subunit vaccine for the control of GCRV infection.

  15. Chemical protein synthesis: Inventing synthetic methods to decipher how proteins work.

    Science.gov (United States)

    Kent, Stephen

    2017-09-15

    Total chemical synthesis of proteins has been rendered practical by the chemical ligation principle: chemoselective condensation of unprotected peptide segments equipped with unique, mutually reactive functional groups, enabled by formation of a non-native replacement for the peptide bond. Ligation chemistries are briefly described, including native chemical ligation - thioester-mediated, amide-forming reaction at Xaa-Cys sites - and its extensions. Case studies from the author's own works are used to illustrate the utility and applications of chemical protein synthesis. Selected recent developments in the field are briefly discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. In vitro and in vivo α-amylase and α-glucosidase inhibiting activities of the protein extracts from two varieties of bitter gourd (Momordica charantia L.).

    Science.gov (United States)

    Poovitha, Sundar; Parani, Madasamy

    2016-07-18

    α-amylase and α-glucosidase digest the carbohydrates and increase the postprandial glucose level in diabetic patients. Inhibiting the activity of these two enzymes can control postprandial hyperglycemia, and reduce the risk of developing diabetes. Bitter gourd or balsam pear is one of the important medicinal plants used for controlling postprandial hyperglycemia in diabetes patients. However, there is limited information available on the presence of α-amylase and α-glucosidase inhibiting compounds. In the current study, the protein extracts from the fruits of M. charantia var. charantia (MCC) and M. charantia var. muricata (MCM) were tested for α-amylase and α-glucosidase inhibiting activities in vitro, and glucose lowering activity after oral administration in vivo. The protein extract from both MCC and MCM inhibited the activity of α-amylase and α-glucosidase through competitive inhibition, which was on par with Acarbose as indicated by in vitro percentage of inhibition (66 to 69 %) and IC50 (0.26 to 0.29 mg/ml). Both the protein extracts significantly reduced peak blood glucose and area under the curve in Streptozotocin-induced diabetic rats, which were orally challenged with starch and sucrose. Protein extracts from the fruits of the two varieties of bitter gourd inhibited α-amylase and α-glucosidase in vitro and lowered the blood glucose level in vivo on par with Acarbose when orally administrated to Streptozotocin-induced diabetic rats. Further studies on mechanism of action and methods of safe and biologically active delivery will help to develop an anti-diabetic oral protein drug from these plants.

  17. Banding ligation versus beta-blockers as primary prophylaxis in esophageal varices

    DEFF Research Database (Denmark)

    Gluud, Lise L; Klingenberg, Sarah; Nikolova, Dimitrinka

    2007-01-01

    To compare banding ligation versus beta-blockers as primary prophylaxis in patients with esophageal varices and no previous bleeding.......To compare banding ligation versus beta-blockers as primary prophylaxis in patients with esophageal varices and no previous bleeding....

  18. Level of arterial ligation in total mesorectal excision (TME): An anatomical study

    NARCIS (Netherlands)

    M. Buunen (Mark); M.M. Lange (Marilyne); M. Ditzel (Max); G.J. Kleinrensink (Gert Jan); C.J.H. van de Velde (Cornelis); J.F. Lange (Johan)

    2009-01-01

    textabstractIntroduction: High-tie ligation is a common practice in rectal cancer surgery. However, it compromises perfusion of the proximal limb of the anastomosis. This anatomical study was designed to assess the value of low-tie ligation in order to obtain a tension-free anastomosis. Materials

  19. Tubal ligation and risk of ovarian cancer subtypes: a pooled analysis of case-control studies

    NARCIS (Netherlands)

    Sieh, W.; Salvador, S.; McGuire, V.; Weber, R.P.; Terry, K.L.; Rossing, M.A.; Risch, H.; Wu, A.H.; Webb, P.M.; Moysich, K.; Doherty, J.A.; Felberg, A.; Miller, D.; Jordan, S.J.; Goodman, M.T.; Lurie, G.; Chang-Claude, J.; Rudolph, A.; Kjaer, S.K.; Jensen, A.; Hogdall, E.; Bandera, E.V.; Olson, S.H.; King, M.G.; Rodriguez-Rodriguez, L.; Kiemeney, L.A.L.M.; Marees, T.; Massuger, L.F.A.G.; Altena, A.M. van; Ness, R.B.; Cramer, D.W; Pike, M.C.; Pearce, C.L.; Berchuck, A.; Schildkraut, J.M.; Whittemore, A.S.

    2013-01-01

    BACKGROUND: Tubal ligation is a protective factor for ovarian cancer, but it is unknown whether this protection extends to all invasive histological subtypes or borderline tumors. We undertook an international collaborative study to examine the association between tubal ligation and ovarian cancer

  20. En bloc ligation of renal vessels is safe and reduces duration of surgery

    DEFF Research Database (Denmark)

    Azawi, Nessn Htum; Hult, Mariam Annalisa Skibsted; Dahl, Claus

    2016-01-01

    INTRODUCTION: Conventionally, individual ligation of the renal vessels with clips is performed during laparoscopic nephrectomy (LN). Concomitant ligation of the vessels is not a standard procedure due to an expected risk of stapler dysfunction and the development of arteriovenous fistulas (AVF). ...

  1. The Effect of Various Ligation Methods on Friction in Sliding Mechanics

    Directory of Open Access Journals (Sweden)

    Amit Gupta

    2013-01-01

    Conclusion: Slide modules produce least friction followed by loose SS ligation, slick modules, regular modules, tight SS ligation and highest friction was produced by regular modules tied in a ′figure of 8′ pattern. Width of bracket had no influence on friction produced.

  2. A fast, visible-light-sensitive azobenzene for bioorthogonal ligation.

    Science.gov (United States)

    Poloni, Claudia; Szymański, Wiktor; Hou, Lili; Browne, Wesley R; Feringa, Ben L

    2014-01-20

    Azobenzenes have been used as photoresponsive units for the control of numerous biological processes. Primary prerequisites for such applications are site-selective incorporation of photoswitchable units into biomolecules and the possibility of using non-destructive and deep-tissue-penetrating visible light for the photoisomerization. Here we report a push-pull azobenzene that readily undergoes a Staudinger-Bertozzi ligation with azide groups, that can be addressed with visible light (>440 nm) and exhibits the solvato- and acidochromism typical for push-pull systems. The thermal relaxation in aqueous environment proceeds on the low-millisecond timescale, thus enabling control over biological processes on similar timescales. The approach is demonstrated in the modification of a quartz surface and in the incorporation of an azobenzene unit into a functional peptide, the third zinc finger in the mammalian factor Sp1. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Confinement-induced Molecular Templating and Controlled Ligation

    Science.gov (United States)

    Berard, Daniel; Shayegan, Marjan; Michaud, François; Henkin, Gil; Scott, Shane; Leith, Jason; Leslie, Sabrina; Leslie Lab Team

    Loading and manipulating long DNA molecules within sub-50 nm cross-section nanostructures for genomic and biochemical analyses, while retaining their structural integrity, present key technological challenges to the biotechnology sector, such as device clogging and molecular breakage. We overcome these challenges by using Convex Lens-induced Confinement (CLiC) technology to gently load DNA into nanogrooves from above. Here, we demonstrate single-fluorophore visualization of custom DNA barcodes as well as efficient top-loading of DNA into sub-50 nm nanogrooves of variable topographies. We study confinement-enhanced self-ligation of polymers loaded in circular nanogrooves. Further, we use concentric, circular nanogrooves to eliminate confinement gradient-induced drift of stretched DNA.

  4. In vitro incorporation of nonnatural amino acids into protein using tRNACys-derived opal, ochre, and amber suppressor tRNAs

    Science.gov (United States)

    Gubbens, Jacob; Kim, Soo Jung; Yang, Zhongying; Johnson, Arthur E.; Skach, William R.

    2010-01-01

    Amber suppressor tRNAs are widely used to incorporate nonnatural amino acids into proteins to serve as probes of structure, environment, and function. The utility of this approach would be greatly enhanced if multiple probes could be simultaneously incorporated at different locations in the same protein without other modifications. Toward this end, we have developed amber, opal, and ochre suppressor tRNAs derived from Escherichia coli, and yeast tRNACys that incorporate a chemically modified cysteine residue with high selectivity at the cognate UAG, UGA, and UAA stop codons in an in vitro translation system. These synthetic tRNAs were aminoacylated in vitro, and the labile aminoacyl bond was stabilized by covalently attaching a fluorescent dye to the cysteine sulfhydryl group. Readthrough efficiency (amber > opal > ochre) was substantially improved by eRF1/eRF3 inhibition with an RNA aptamer, thus overcoming an intrinsic hierarchy in stop codon selection that limits UGA and UAA termination suppression in higher eukaryotic translation systems. This approach now allows concurrent incorporation of two different modified amino acids at amber and opal codons with a combined apparent readthrough efficiency of up to 25% when compared with the parent protein lacking a stop codon. As such, it significantly expands the possibilities for incorporating nonnative amino acids for protein structure/function studies. PMID:20581130

  5. The effect of organochlorines and heavy metals on sex steroid-binding proteins in vitro in the plasma of nesting green turtles, Chelonia mydas.

    Science.gov (United States)

    Ikonomopoulou, Maria Petrou; Olszowy, Henry; Hodge, Mary; Bradley, Adrian J

    2009-07-01

    In this study on green turtles, Chelonia mydas, from Peninsular Malaysia, the effect of selected environmental toxicants was examined in vitro. Emphasis was placed on purported hormone-mimicking chemicals such as dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethylene, dieldrin, lead, zinc and copper. Five concentrations were used: high (1 mg/L), medium (10(-1) mg/L), low (10(-2) mg/L), very low (10(-6) mg/L) and control (diluted carrier solvent but no toxicants). The results suggest that environmental pesticides and heavy metals may significantly alter the binding of steroids [i.e. testosterone (T) and oestradiol] to the plasma proteins in vitro. Competition studies showed that only Cu competed for binding sites with testosterone in the plasma collected from nesting C. mydas. Dieldrin and all heavy metals competed with oestradiol for binding sites. Furthermore, testosterone binding affinity was affected at various DDT concentrations and was hypothesised that DDT in vivo may act to inhibit steroid-protein interactions in nesting C. mydas. Although the precise molecular mechanism is yet to be described, DDT could have an effect upon the protein conformation thus affecting T binding (e.g. the T binding site on the steroid hormone binding protein molecule).

  6. In Vitro Transcripts of Wild-Type and Fluorescent Protein-Tagged Triticum mosaic virus (Family Potyviridae) are Biologically Active in Wheat.

    Science.gov (United States)

    Tatineni, Satyanarayana; McMechan, Anthony J; Bartels, Melissa; Hein, Gary L; Graybosch, Robert A

    2015-11-01

    Triticum mosaic virus (TriMV) (genus Poacevirus, family Potyviridae) is a recently described eriophyid mite-transmitted wheat virus. In vitro RNA transcripts generated from full-length cDNA clones of TriMV proved infectious on wheat. Wheat seedlings inoculated with in vitro transcripts elicited mosaic and mottling symptoms similar to the wild-type virus, and the progeny virus was efficiently transmitted by wheat curl mites, indicating that the cloned virus retained pathogenicity, movement, and wheat curl mite transmission characteristics. A series of TriMV-based expression vectors was constructed by engineering a green fluorescent protein (GFP) or red fluorescent protein (RFP) open reading frame with homologous NIa-Pro cleavage peptides between the P1 and HC-Pro cistrons. We found that GFP-tagged TriMV with seven or nine amino acid cleavage peptides efficiently processed GFP from HC-Pro. TriMV-GFP vectors were stable in wheat for more than 120 days and for six serial passages at 14-day intervals by mechanical inoculation and were transmitted by wheat curl mites similarly to the wild-type virus. Fluorescent protein-tagged TriMV was observed in wheat leaves, stems, and crowns. The availability of fluorescent protein-tagged TriMV will facilitate the examination of virus movement and distribution in cereal hosts and the mechanisms of cross protection and synergistic interactions between TriMV and Wheat streak mosaic virus.

  7. Extraction, partial characterization and evaluation of in vitro digestibility of the protein associated with the exoskeleton of white shrimp (Litopenaeus vannamei

    Directory of Open Access Journals (Sweden)

    Escobedo-Lozano, A.Y.

    2014-01-01

    Full Text Available The need of new food sources to satisfy human requirements forces researchers to study any possible alternative supplies. Therefore this study aimed to explore the quality and digestibility evaluation of the protein fractions obtained as a by-product of processing the shrimp exoskeleton Litopenaeus vannamei. Shrimp exoskeletons were washed, they were dried, mincedand they were brought under acid hydrolysis and alkaline hydrolysis; released proteins were then precipitated and characterized. 496 g of demineralized exoskeleton were obtained from 1 kg of shrimp shell; out of these, 376 g corresponded to chitin and 120 g corresponded to protein Total protein content was 33.80 ± 0.34 %. The digestible protein fractions were 26.7 g and 92.1 g were insoluble proteins. Of this latter fraction, the largest proportion belonged to the scleroprotein type. The amino acid profile analysis from the recovered proteins indicated that there were present nine amino acids out of the ten essential ones, being leucine the one with the highest proportion. In vitro digestibility was shown to be up to an 83.7 %. Therefore, it was possible to conclude that the recovery of digestibleproteins from shrimp exoskeleton may be useful in diet formulation.

  8. Universal ligation-detection-reaction microarray applied for compost microbes

    Directory of Open Access Journals (Sweden)

    Romantschuk Martin

    2008-12-01

    Full Text Available Abstract Background Composting is one of the methods utilised in recycling organic communal waste. The composting process is dependent on aerobic microbial activity and proceeds through a succession of different phases each dominated by certain microorganisms. In this study, a ligation-detection-reaction (LDR based microarray method was adapted for species-level detection of compost microbes characteristic of each stage of the composting process. LDR utilises the specificity of the ligase enzyme to covalently join two adjacently hybridised probes. A zip-oligo is attached to the 3'-end of one probe and fluorescent label to the 5'-end of the other probe. Upon ligation, the probes are combined in the same molecule and can be detected in a specific location on a universal microarray with complementary zip-oligos enabling equivalent hybridisation conditions for all probes. The method was applied to samples from Nordic composting facilities after testing and optimisation with fungal pure cultures and environmental clones. Results Probes targeted for fungi were able to detect 0.1 fmol of target ribosomal PCR product in an artificial reaction mixture containing 100 ng competing fungal ribosomal internal transcribed spacer (ITS area or herring sperm DNA. The detection level was therefore approximately 0.04% of total DNA. Clone libraries were constructed from eight compost samples. The LDR microarray results were in concordance with the clone library sequencing results. In addition a control probe was used to monitor the per-spot hybridisation efficiency on the array. Conclusion This study demonstrates that the LDR microarray method is capable of sensitive and accurate species-level detection from a complex microbial community. The method can detect key species from compost samples, making it a basis for a tool for compost process monitoring in industrial facilities.

  9. MANAGEMENT OF INTERNAL HEMORRHOID WITH RUBBER BAND LIGATION PROCEDURE

    Directory of Open Access Journals (Sweden)

    I Made Arya Winangun

    2013-10-01

    Full Text Available Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4 Hemorrhoid is regarded as the cases most seen in population. The prevalence of this cases about 4,4% with the incidence 12 of 1.000 patient. The current management of hemorrhoid is lifestyle modification, conservative management such as farmacology, minimally invasive treatment and more aggressive therapy using surgical procedure. Rubber band ligation was one of the minimally invasive procedures. This procedure was easy, inexpensive, and can be done outpatient using simple tools without complicated procedure like hemorrhoidectomy. Some studies explained rubber band ligation effectively done in internal hemorrhoid grade II and grade III even this procedure still had minimal complication such as bleeding and unpleasentness /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

  10. Regulation of high mobility group box protein 1 expression following mechanical loading by orthodontic forces in vitro and in vivo

    National Research Council Canada - National Science Library

    Wolf, Michael; Lossdörfer, Stefan; Küpper, Katharina; Jäger, Andreas

    2014-01-01

    ...) cells challenged by mechanical loading similar to force levels being applied in orthodontic treatment in vitro and to transfer these findings to an in vivo microenvironment in an animal model of tooth movement in rats...

  11. The mycotoxin patulin induces intra- and intermolecular protein crosslinks in vitro involving cysteine, lysine, and histidine side chains, and alpha-amino groups.

    Science.gov (United States)

    Fliege, R; Metzler, M

    1999-11-30

    As previous studies have indicated a multiple electrophilic reactivity of patulin (PAT) towards simple thiol nucleophiles, we have methodically investigated the ability of PAT to covalently crosslink proteins in vitro. By means of sodium dodecylsulphate polyacrylamide gel electrophoresis, the formation of PAT-induced intermolecular protein-protein crosslinks was clearly demonstrated for bovine serum albumin containing one thiol group per molecule, but also for the thiol-free hen egg lysozyme. Characterization of the crosslink sites was carried out by (1) modulation of the thiol groups with N-ethylimaleimide and 2-iminothiolane; (2) comparison with various known crosslinking agents, i.e. phenylenedimaleimide, glutardialdehyde, and dimethylsuberimidate, and (3) fluorescence incorporation studies using dansyl-labeled amino acids and a fluorescent glutathione derivative. The thiol group of cysteine was preferred for PAT-mediated crosslink reactions, but the side chains of lysine and histidine, and alpha-amino groups also exhibited reactivity. PAT can act both as a homobifunctional as well as a heterobifunctional crosslinking agent. The initial formation of a monoadduct with a thiol group appears to activate PAT for the subsequent reaction with an amino group, but also leads to rapid loss of further electrophilic properties when no second nucleophile for crosslink completion is available. Studies using microtubule proteins as a protein with experimentally controllable quarternary structure and a proposed cellular target for PAT toxicity emphasized the influence of specific sterical conditions on crosslink formation at low protein concentrations. Non-polymerized microtubule proteins, i.e. tubulin alpha,beta-dimers, formed a defined product with PAT consisting of an intramolecularly crosslinked beta-tubulin, whereas guanosine triphosphate- or paclitaxel-induced polymerization to microtubule-like quarternary structures prior to treatment with PAT gave rise to

  12. Lack of Detectable Allergenicity in Genetically Modified Maize Containing “Cry” Proteins as Compared to Native Maize Based on In Silico & In Vitro Analysis

    Science.gov (United States)

    Mathur, Chandni; Kathuria, Pooran C.; Dahiya, Pushpa; Singh, Anand B.

    2015-01-01

    Background Genetically modified, (GM) crops with potential allergens must be evaluated for safety and endogenous IgE binding pattern compared to native variety, prior to market release. Objective To compare endogenous IgE binding proteins of three GM maize seeds containing Cry 1Ab,1Ac,1C transgenic proteins with non GM maize. Methods An integrated approach of in silico & in vitro methods was employed. Cry proteins were tested for presence of allergen sequence by FASTA in allergen databases. Biochemical assays for maize extracts were performed. Specific IgE (sIgE) and Immunoblot using food sensitized patients sera (n = 39) to non GM and GM maize antigens was performed. Results In silico approaches, confirmed for non sequence similarity of stated transgenic proteins in allergen databases. An insignificant (p> 0.05) variation in protein content between GM and non GM maize was observed. Simulated Gastric Fluid (SGF) revealed reduced number of stable protein fractions in GM then non GM maize which might be due to shift of constituent protein expression. Specific IgE values from patients showed insignificant difference in non GM and GM maize extracts. Five maize sensitized cases, recognized same 7 protein fractions of 88-28 kD as IgE bindng in both GM and non-GM maize, signifying absence of variation. Four of the reported IgE binding proteins were also found to be stable by SGF. Conclusion Cry proteins did not indicate any significant similarity of >35% in allergen databases. Immunoassays also did not identify appreciable differences in endogenous IgE binding in GM and non GM maize. PMID:25706412

  13. Lack of detectable allergenicity in genetically modified maize containing "Cry" proteins as compared to native maize based on in silico & in vitro analysis.

    Directory of Open Access Journals (Sweden)

    Chandni Mathur

    Full Text Available Genetically modified, (GM crops with potential allergens must be evaluated for safety and endogenous IgE binding pattern compared to native variety, prior to market release.To compare endogenous IgE binding proteins of three GM maize seeds containing Cry 1Ab,1Ac,1C transgenic proteins with non GM maize.An integrated approach of in silico & in vitro methods was employed. Cry proteins were tested for presence of allergen sequence by FASTA in allergen databases. Biochemical assays for maize extracts were performed. Specific IgE (sIgE and Immunoblot using food sensitized patients sera (n = 39 to non GM and GM maize antigens was performed.In silico approaches, confirmed for non sequence similarity of stated transgenic proteins in allergen databases. An insignificant (p> 0.05 variation in protein content between GM and non GM maize was observed. Simulated Gastric Fluid (SGF revealed reduced number of stable protein fractions in GM then non GM maize which might be due to shift of constituent protein expression. Specific IgE values from patients showed insignificant difference in non GM and GM maize extracts. Five maize sensitized cases, recognized same 7 protein fractions of 88-28 kD as IgE bindng in both GM and non-GM maize, signifying absence of variation. Four of the reported IgE binding proteins were also found to be stable by SGF.Cry proteins did not indicate any significant similarity of >35% in allergen databases. Immunoassays also did not identify appreciable differences in endogenous IgE binding in GM and non GM maize.

  14. Optimization of in vitro conditions to study the arachidonic acid induction of 4R isoforms of the microtubule-associated protein tau.

    Science.gov (United States)

    Mutreja, Yamini; Gamblin, Truman C

    2017-01-01

    The microtubule-associated protein tau exists in six different isoforms that accumulate as filamentous aggregates in a wide spectrum of neurodegenerative diseases classified as tauopathies. One potential source of heterogeneity between these diseases could arise from differential tau isoform aggregation. in vitro assays employing arachidonic acid as an inducer of aggregation have been pivotal in gaining an understanding of the longest four repeat tau isoform (2N4R). These approaches have been less successful for modeling the shorter 1N4R and 0N4R tau isoforms in vitro. Through a careful analysis of in vitro conditions for aggregation, we found that the differences in the acidity of tau isoform N-terminal projection domains determine whether tau filaments cluster into larger assemblies in solution. Beyond the potential biological implications of filament clustering, we provide optimized conditions for the arachidonic acid induction of shorter 4R tau isoforms aggregation in vitro that greatly reduce filament clustering and improved modeling results. © 2017 Elsevier Inc. All rights reserved.

  15. Studies in vitro on infectivity and sensitivity to antileishmanial drugs in New World Leishmania species transfected with the green fluorescent protein [pIR3(-)-eGFP].

    Science.gov (United States)

    Palacios, Genesis; Parodi, Adriana; Upegui, Yulieth A; Montoya, Andres; Pulido, Sergio; Vélez, Iván D; Robledo, Sara M

    2017-11-01

    Current chemotherapeutic agents for leishmaniasis have several disadvantages interfering with the effective treatment and therefore more and better antileishmanial drugs are needed. Discovery of candidates for leishmaniasis treatment requires not only accurate and precise methodologies but also well-known biological system to measure infectivity of parasites and antileishmanial activity of the new compounds. Significant variation in the in vitro and in vivo infectivity and sensitivity to established and experimental drugs in Leishmania strains are reported. This work reports the in vitro biological behavior and antileishmanial drugs sensitivity of different green fluorescent protein transfectant Leishmanias strains. The in vitro growth kinetic and infectivity to U937 cells vary slightly in the Leishmania transfectant strains in comparison with their correspondant wild-type. However, the insertion of the pIR3(-)-eGFP may affect the sensitivity of the parasites to meglumine antimoniate (MA) and miltefosine but not to amphotericin B (AMB) and pentamidine isethionate. In consequence, AMB or pentamidine isethionate but not MA or miltefosine should be used as antileishmanial control drugs during in vitro assays of antileishmanial activity. Furthermore, is recommended to test compounds against more than one Leishmania strain in order to verify that the antileihmanial activity of these compound is similar among species.

  16. Cat and dog primordial follicles enclosed in ovarian cortex sustain viability after in vitro culture on agarose gel in a protein-free medium.

    Science.gov (United States)

    Fujihara, M; Comizzoli, P; Wildt, D E; Songsasen, N

    2012-12-01

    Our objective was to examine the influences of differing media, protein supplementation and the microenvironment on cat vs dog primordial follicle viability in vitro. Ovarian cortical slices were cultured for 3, 9 or 15 days in α-minimum essential medium (α-MEM) or MEM supplemented with 10% fetal bovine serum (FBS), 10% knock-out serum replacement (KSR) or 0.1% polyvinyl alcohol (protein free). In a separate study, cat and dog ovarian tissues were cultured in protein-free α-MEM and MEM, respectively, in cell culture inserts, on 1.5% agarose gel or in 24-well cell culture plates (control). Follicle viability was assessed in both studies using calcein AM/ethidium homodimer and histological evaluation with haematoxylin/eosin staining. No cat follicle sustained viability beyond 9 days of in vitro culture in α-MEM compared to 37.5% of those incubated for 15 days in MEM in protein-free condition (p free condition at 15 days. Serum was detrimental (p free condition supported cat follicle viability, whereas the latter was superior (p culture insert and control groups after 3 and 9 days of culture. For the cat, the agarose gel better (p culture insert. Therefore, sustaining primordial follicle survival from intracortical ovarian slices requires a different in vitro microenvironment for the cat vs the dog. A key factor to enhancing survival of these early stage follicles in culture appears to be the use of agarose gel, which enhances follicle viability, perhaps by promoting gas exchange. © 2012 Blackwell Verlag GmbH.

  17. In vitro digestion of rice bran proteins produces peptides with potent inhibitory effects on α-glucosidase and angiotensin I converting enzyme.

    Science.gov (United States)

    Uraipong, Chatchaporn; Zhao, Jian

    2018-01-01

    The bioactivities of peptides released from the digestion of rice bran protein under in vitro simulated human digestive conditions were investigated. Four protein fractions extracted from rice bran were digested and the hydrolysates were fractionated by ultrafiltration and anion exchange chromatography. α-Glucosidase and angiotensin converting enzyme (ACE) inhibitory activities of the crude hydrolysates and their fractions were determined. Peptides with molecular weight (MW) digests, while the ACE inhibitory activities of the MW digestive tract with substantial health benefits. ACE and α-glucosidase inhibitory activities of the digests of rice bran proteins, and albumin and glutelin in particular, were especially strong, comparable to that of the standard drugs. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  18. Peptides Derived from Soy and Lupin Protein as Dipeptidyl-Peptidase IV Inhibitors: In Vitro Biochemical Screening and in Silico Molecular Modeling Study.

    Science.gov (United States)

    Lammi, Carmen; Zanoni, Chiara; Arnoldi, Anna; Vistoli, Giulio

    2016-12-28

    Dipeptidyl peptidase IV (DPP-IV) is a new molecular target correlated with the development of type 2 diabetes. Literature describes the identification of some inhibitory peptides from the hydrolysis of different food proteins. This article reports a study on six peptides from soybean and lupin proteins, i.e., Soy 1 (IAVPTGVA), Soy 2 (YVVNPDNDEN), Soy 3 (YVVNPDNNEN), Lup 1 (LTFPGSAED), Lup 2 (LILPKHSDAD), and Lup 3 (GQEQSHQDEGVIVR), which were screened for their capacity to inhibit the activity of DPP-IV, using an in vitro bioassay against human recombinant DPP-IV. Two peptides Soy 1 and Lup 1 resulted to be efficient inhibitors with IC50 values equal to 106 and 228 μM, respectively. A molecular docking analysis predicted the key molecular interactions, stabilizing the active peptides within DPP-IV enzyme. Soy and lupin proteins may be sources of DPP-IV inhibitory peptides potentially useful for the prevention of type 2 diabetes.

  19. Cationic lipids (lipofectamine) and disturbance of cellular cholesterol and sphingomyelin distribution modulates gamma-secretase activity within amyloid precursor protein in vitro.

    Science.gov (United States)

    Urmoneit, B; Turner, J; Dyrks, T

    1998-04-01

    To study beta-amyloid protein generation we expressed different amyloid precursor protein (APP) isoforms in the human neuroblastoma cell line SY5Y (for details see (1)). Treatment with lipofectamine, an cationic lipid for eucaryotic cell transfection, inhibits gamma-secretase activity and stimulates the physiological APP cleavage by alpha-secretase activity. Beside the MDL inhibitor (2), this is the second agent that shows modulation of gamma-secretase activity in vitro. Further, we show that disturbance of cellular cholesterol and sphingomyelin distribution in transfected SY5Y cells results in an overproduction of beta-amyloid protein. This provides experimental evidence that membrane instability influenced the proteolytic activity of gamma-secretase within the APP molecule.

  20. Application of a pig ligated intestinal loop model for early Lawsonia intracellularis infection

    DEFF Research Database (Denmark)

    Boutrup, Torsten Snogdal; Schauser, Kirsten Hallundbæk; Agerholm, Jørgen Steen

    2010-01-01

    BACKGROUND: Porcine proliferative enteropathy in pigs is caused by the obligate, intracellular bacterium Lawsonia intracellularis. In vitro studies have shown close bacterium-cell interaction followed by cellular uptake of the bacterium within 3 h post inoculation (PI). However, knowledge......-enterocyte interactions. METHODS: A ligated small intestinal loop model using three different L. intracellularis inocula was applied to 10-11-week-old pigs. The inocula were 1) wild type bacteria derived from overnight incubation of L. intracellularis bacteria from spontaneous disease, 2) crude vaccine bacteria...... (Enterisol Ileitis Vet), and 3) vaccine bacteria propagated in cell culture. The bacteria-enterocyte interaction was visualised using immunohistochemistry on specimens derived 1, 3 and 6 h PI respectively. RESULTS: Although at a low level, close contact between bacteria and the enterocyte brush border...

  1. Chemical Ligation and Isotope Labeling to Locate Dynamic Effects during Catalysis by Dihydrofolate Reductase.

    Science.gov (United States)

    Luk, Louis Y P; Ruiz-Pernía, J Javier; Adesina, Aduragbemi S; Loveridge, E Joel; Tuñón, Iñaki; Moliner, Vincent; Allemann, Rudolf K

    2015-07-27

    Chemical ligation has been used to alter motions in specific regions of dihydrofolate reductase from E. coli and to investigate the effects of localized motional changes on enzyme catalysis. Two isotopic hybrids were prepared; one with the mobile N-terminal segment containing heavy isotopes ((2) H, (13) C, (15) N) and the remainder of the protein with natural isotopic abundance, and the other one with only the C-terminal segment isotopically labeled. Kinetic investigations indicated that isotopic substitution of the N-terminal segment affected only a physical step of catalysis, whereas the enzyme chemistry was affected by protein motions from the C-terminal segment. QM/MM studies support the idea that dynamic effects on catalysis mostly originate from the C-terminal segment. The use of isotope hybrids provides insights into the microscopic mechanism of dynamic coupling, which is difficult to obtain with other studies, and helps define the dynamic networks of intramolecular interactions central to enzyme catalysis. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

  2. The bile duct ligated rat: A relevant model to study muscle mass loss in cirrhosis.

    Science.gov (United States)

    Bosoi, Cristina R; Oliveira, Mariana M; Ochoa-Sanchez, Rafael; Tremblay, Mélanie; Ten Have, Gabriella A; Deutz, Nicolaas E; Rose, Christopher F; Bemeur, Chantal

    2017-04-01

    Muscle mass loss and hepatic encephalopathy (complex neuropsychiatric disorder) are serious complications of chronic liver disease (cirrhosis) which impact negatively on clinical outcome and quality of life and increase mortality. Liver disease leads to hyperammonemia and ammonia toxicity is believed to play a major role in the pathogenesis of hepatic encephalopathy. However, the effects of ammonia are not brain-specific and therefore may also affect other organs and tissues including muscle. The precise pathophysiological mechanisms underlying muscle wasting in chronic liver disease remains to be elucidated. In the present study, we characterized body composition as well as muscle protein synthesis in cirrhotic rats with hepatic encephalopathy using the 6-week bile duct ligation (BDL) model which recapitulates the main features of cirrhosis. Compared to sham-operated control animals, BDL rats display significant decreased gain in body weight, altered body composition, decreased gastrocnemius muscle mass and circumference as well as altered muscle morphology. Muscle protein synthesis was also significantly reduced in BDL rats compared to control animals. These findings demonstrate that the 6-week BDL experimental rat is a relevant model to study liver disease-induced muscle mass loss.

  3. A protein isolated from human oviductal tissue in vitro secretion, identified as human lactoferrin, interacts with spermatozoa and oocytes and modulates gamete interaction.

    Science.gov (United States)

    Zumoffen, C M; Gil, R; Caille, A M; Morente, C; Munuce, M J; Ghersevich, S A

    2013-05-01

    Is lactoferrin (LF) (detected in oviductal secretion) able to bind to oocytes and sperm and modulate gamete interaction? LF binds to zona pellucida (ZP) and spermatozoa (depending upon the capacitation stage and acrosome status) and inhibits gamete interaction in vitro. Proteins from human oviductal tissue secretion modulate gamete interaction and parameters of sperm function in vitro and some of them bind to sperm, but they remain to be isolated and identified. Proteins were isolated from human oviductal tissue secretion using their sperm membrane binding ability. One of the isolated proteins was identified as human LF and immunolocalized in tubal tissues. LF expression was analyzed in native oviductal fluid and oviduct epithelial cells (at different phases of the menstrual cycle: proliferative, periovulatory and secretory). In addition, the LF binding sites on spermatozoa (at different capacitation and acrosome reaction stages) and on ZP and the dose-dependent effect of LF on gamete interaction were investigated. All experiments were performed at least three times. Tubal tissues obtained from premenopausal patients (scheduled for hysterectomy, n = 23) were cultured in DMEM/Ham's F12 medium and conditioned media (CM) were collected. Motile spermatozoa were obtained by swim-up from normozoospermic semen samples from healthy donors (n = 4). An affinity chromatography with sperm membrane extracts was used to isolate proteins from CM. Isolated proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophresis and further identified by nano liquid chromatography tandem mass spectrometry peptide sequencing. The presence of LF in oviductal tissue was investigated by immunohistochemistry and immunofluorescence and was detected in native oviductal fluid and oviduct epithelial cells homogenates by western blot. LF binding sites on gametes were investigated by incubating gametes with the protein coupled to fluorescein isothiocyanate (FITC). The acrosome

  4. An in vivo Study on Bacterial Colonization with Metal, Ceramic and Self-ligating Brackets: A Scanning Electron Microscopy Study

    Directory of Open Access Journals (Sweden)

    Aravind S Raju

    2013-01-01

    Conclusion: This study highlights that higher retention of plaque in ceramic brackets ligated with elastomeric ring followed with metal brackets ligated with steel ligatures and comparatively less plaque retention in self-ligating brackets. Excess composite around the bracket base is the critical site of plaque accumulation associated with fixed appliances due to its rough surface texture.

  5. Rice MEL2, the RNA recognition motif (RRM) protein, binds in vitro to meiosis-expressed genes containing U-rich RNA consensus sequences in the 3'-UTR.

    Science.gov (United States)

    Miyazaki, Saori; Sato, Yutaka; Asano, Tomoya; Nagamura, Yoshiaki; Nonomura, Ken-Ichi

    2015-10-01

    Post-transcriptional gene regulation by RNA recognition motif (RRM) proteins through binding to cis-elements in the 3'-untranslated region (3'-UTR) is widely used in eukaryotes to complete various biological processes. Rice MEIOSIS ARRESTED AT LEPTOTENE2 (MEL2) is the RRM protein that functions in the transition to meiosis in proper timing. The MEL2 RRM preferentially associated with the U-rich RNA consensus, UUAGUU[U/A][U/G][A/U/G]U, dependently on sequences and proportionally to MEL2 protein amounts in vitro. The consensus sequences were located in the putative looped structures of the RNA ligand. A genome-wide survey revealed a tendency of MEL2-binding consensus appearing in 3'-UTR of rice genes. Of 249 genes that conserved the consensus in their 3'-UTR, 13 genes spatiotemporally co-expressed with MEL2 in meiotic flowers, and included several genes whose function was supposed in meiosis; such as Replication protein A and OsMADS3. The proteome analysis revealed that the amounts of small ubiquitin-related modifier-like protein and eukaryotic translation initiation factor3-like protein were dramatically altered in mel2 mutant anthers. Taken together with transcriptome and gene ontology results, we propose that the rice MEL2 is involved in the translational regulation of key meiotic genes on 3'-UTRs to achieve the faithful transition of germ cells to meiosis.

  6. In vitro assessment of the combined effect of eicosapentaenoic acid, green tea extract and curcumin C3 on protein loss in C2C12 myotubes.

    Science.gov (United States)

    Mirza, Kamran A; Luo, Menghua; Pereira, Suzette; Voss, Anne; Das, Tapas; Tisdale, Michael J

    2016-09-01

    EPA has been clinically shown to reduce muscle wasting during cancer cachexia. This study investigates whether curcumin or green tea extract (GTE) enhances the ability of low doses of eicosapentaenoic acid (EPA) to reduce loss of muscle protein in an in vitro model. A low dose of EPA with minimal anti-cachectic activity was chosen to evaluate any potential synergistic effect with curcumin or GTE. Depression of protein synthesis and increase in degradation was determined in C2C12 myotubes in response to tumour necrosis factor-α (TNF-α) and proteolysis-inducing factor (PIF). EPA (50 μM) or curcumin (10 μg ml(-1)) alone had little effect on protein degradation caused by PIF but the combination produced complete inhibition, as did the combination with GTE (10 μg ml(-1)). In response to TNF-α (25 ng ml(-1))-induced protein degradation, EPA had a small, but not significant effect on protein degradation; however, when curcumin and GTE were combined with EPA, the effect was enhanced. EPA completely attenuated the depression of protein synthesis caused by TNF-α, but not that caused by PIF. The combination of EPA with curcumin produced a significant increase in protein synthesis to both agents. GTE alone or in combination with EPA had no effect on the depression of protein synthesis by TNF-α, but did significantly increase protein synthesis in PIF-treated cells. Both TNF-α and PIF significantly reduced myotube diameter from 17 to 13 μm for TNF-α (23.5%) and 15 μm (11.8%) for PIF However the triple combination of EPA, curcumin and GTE returned diameters to values not significantly different from the control. These results suggest that either curcumin or GTE or the combination could enhance the anti-catabolic effect of EPA on lean body mass.

  7. Purification and in vitro Chaperone Activity of a Class I Small Heat-Shock Protein Abundant in Recalcitrant Chestnut Seeds

    National Research Council Canada - National Science Library

    Carmen Collada; Luis Gomez; Rosa Casado; Cipriano Aragoncillo

    1997-01-01

    .... This protein, termed Cs smHSP 1, forms homododecameric complexes under nondenaturing conditions and appears to be homologous to cytosolic class I small heat-shock proteins (smHSPs) from plant sources...

  8. In vitro and in vivo comparisons of the effects of the fruiting body and mycelium of Antrodia camphorata against amyloi