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Sample records for 7b protein in-vitro

  1. Interaction of protein with DNA in vitro

    One of the important means to control eukaryotic gene expression involves the binding of proteins to specific sites in the promoter and other regulatory regions of the gene. This chapter is devoted to methodology for identifying DNA sequences that are bound in vitro by proteins in crude nuclear extracts. The authors present four methods of detection: nitrocellulose filter binding, mobility shift, exonuclease III protection, and DNAse I protection. In addition to providing assay conditions, they discuss some applications of the different methods. Very often data from protein-DNA interaction studies permit ambiguous interpretations. They there discuss control experiments designed to help the researcher to evaluate data

  2. In vitro hydroxyapatite adsorbed salivary proteins

    In spite of the present knowledge about saliva components and their respective functions, the mechanism(s) of pellicle and dental plaque formation have hitherto remained obscure. This has prompted recent efforts on in vitro studies using hydroxyapatite (HA) as an enamel model. In the present study salivary proteins adsorbed to HA were extracted with TFA and EDTA and resolved by 2D electrophoresis over a pH range between 3 and 10, digested, and then analysed by MALDI-TOF/TOF mass spectrometry and tandem mass spectrometry. Nineteen different proteins were identified using automated MS and MS/MS data acquisition. Among them, cystatins, amylase, carbonic anhydrase, and calgranulin B, were identified

  3. Wilson Disease Protein ATP7B Utilizes Lysosomal Exocytosis to Maintain Copper Homeostasis

    Polishchuk, Elena V.; Concilli, Mafalda; Iacobacci, Simona; Chesi, Giancarlo; Pastore, Nunzia; Piccolo, Pasquale; Paladino, Simona; Baldantoni, Daniela; van IJzendoorn, Sven C.D.; Chan, Jefferson; Chang, Christopher J.; Amoresano, Angela; Pane, Francesca; Pucci, Piero; Tarallo, Antonietta; Parenti, Giancarlo; Brunetti-Pierri, Nicola; Settembre, Carmine; Ballabio, Andrea; Polishchuk, Roman S.

    2014-01-01

    Summary Copper is an essential yet toxic metal and its overload causes Wilson disease, a disorder due to mutations in copper transporter ATP7B. To remove excess copper into the bile, ATP7B traffics toward canalicular area of hepatocytes. However, the trafficking mechanisms of ATP7B remain elusive. Here, we show that, in response to elevated copper, ATP7B moves from the Golgi to lysosomes and imports metal into their lumen. ATP7B enables lysosomes to undergo exocytosis through the interaction with p62 subunit of dynactin that allows lysosome translocation toward the canalicular pole of hepatocytes. Activation of lysosomal exocytosis stimulates copper clearance from the hepatocytes and rescues the most frequent Wilson-disease-causing ATP7B mutant to the appropriate functional site. Our findings indicate that lysosomes serve as an important intermediate in ATP7B trafficking, whereas lysosomal exocytosis operates as an integral process in copper excretion and hence can be targeted for therapeutic approaches to combat Wilson disease. PMID:24909901

  4. Nanoparticle-protein corona in invertebrate in vitro testing

    Hayashi, Yuya; Miclaus, Teodora; Scavenius, Carsten;

    2013-01-01

    We have previously established an in vitro model of the earthworm Eisenia fetida and highlighted a considerable involvement of phagocytes in the cellular responses to silver nanoparticles exposure. Our in vitro model was tested in a conventional cell culture environment with serum supplements, an...... underscore the need for evaluation of the protein corona in invertebrate in vitro setting....

  5. Problem-Solving Test: "In Vitro" Protein Kinase A Reaction

    Szeberenyi, Jozsef

    2009-01-01

    Phosphorylation of proteins by protein kinases is an important mechanism in the regulation of protein activity. Among hundreds of protein kinases present in human cells, PKA, the first kinase discovered, belongs to the most important and best characterized group of these enzymes. The author presents an experiment that analyzes the "in vitro"…

  6. Variation in In Vitro Digestibility of Barley Protein

    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...

  7. PROTEIN FRACTIONATION AND IN VITRO DIGESTIBILITY OF AZOLLA IN RUMINANTS

    S. PARASHURAMULU

    2013-05-01

    Full Text Available A study was undertaken to evaluate the nutritive value and digestibility of Azolla in ruminants by in vitro techniques. The crude protein, crude fibre and ether extract contents were at a level of 21.37%, 12.5% and 2.3%, respectively. The neutral and acid detergent fibre levels were about 35.4 and 23.9%, respectively. The average in vitro dry matter digestibility, in vitro organic matter digestibility and metabolozable energy contents were 79.5%, 63.8 mg/200mg and 7.36 MJ/kg DM (1759 kcal/kg, respectively. The various protein fractions A, B1, B2, B3 and C estimated by Cornell net crude protein solubility system were 18.22, 42.56, 15.15, 7.47 and 16.61% of total protein, respectively. The Azolla contained significantly higher B1 fraction followed by A, B2 and C and lowest fraction of C. Thus in view of above, present study indicated Azolla to be a good source protein supplement with 21.37% crude protein with highest B protein fractions, moderate source of energy (1759 kcal ME/kg, high dry matter and organic matter digestibilities and rich in trace minerals thus could be used as an alternate protein supplement or as supplementary protein supplement to ruminants.

  8. In vitro biosynthesis of complement protein D

    Barnum, S.R.

    1985-01-01

    The aim of this study was twofold: to determine site(s) of complement protein D biosynthesis and to examine D biosynthesis with respect to the kinetics of D secretion, the post-translational modification of D and the tissue-specific differences in D secretion and processing. Antigenic D was detected in the culture supernatants of two cell lines, U937 and HepG2, and adherent blood monocytes by a solid-phase radioimmunoassay. D secreted by U937 cells was hemolytically active with a specific activity comparable to D in serum. De novo synthesis of D by U937 cells was demonstrated with the use of cycloheximide. Biosynthetic labeling using /sup 35/S labeled methionine or cysteine, followed by immunoprecipitation demonstrated a single d band intra- and extra-cellularly in all three cell types as analyzed by SDS-PAGE and auto-radiography. Elevated serum D levels in individuals with IgA nephropathy led to studies on the D levels in serum and urine of individuals with chronic renal failure and an individual with Fanconi's syndrome. The former group had elevated serum D levels, compared to normals, and insignificant levels of D in their urine while the patient with Fanconi's syndrome had normal serum D levels but markedly elevated urinary D levels. These studies demonstrate that the monocyte and hepatocyte are both sites of D synthesis and that there are no apparent differences in the secretion rates and processing of D produced by these cell types. The results also suggest that D is not synthesized or secreted as a precursor molecule. Additionally, these studies suggest that the kidney is a major site of D catabolism.

  9. Directed Evolution of Proteins through In Vitro Protein Synthesis in Liposomes

    Takehiro Nishikawa; Takeshi Sunami; Tomoaki Matsuura; Tetsuya Yomo

    2012-01-01

    Directed evolution of proteins is a technique used to modify protein functions through “Darwinian selection.” In vitro compartmentalization (IVC) is an in vitro gene screening system for directed evolution of proteins. IVC establishes the link between genetic information (genotype) and the protein translated from the information (phenotype), which is essential for all directed evolution methods, by encapsulating both in a nonliving microcompartment. Herein, we introduce a new liposome-based I...

  10. Effects of experiences on synaptic protein phosphorylation in vitro

    In vitro transfer of 32P from [gamma-32P]-ATP into proteins of particulate fractions from osmotically shocked preparations enriched in rat brain synaptosomes was studied. Phosphate incorporation into protein bands of apparent molecular weights (MW) 44,000, 24,000, 21,000, and 19,000 was affected by the prior experiences of the rats from which the particulate fractions were prepared. Incorporation into all four proteins was increased in particulate fraction from previously naive rats that received active avoidance training. Handling of the subjects prior to training prevented the response of the 24,000 MW protein to training. Phosphate incorporation into 24,000 and 19,000 MW proteins was increased in preparations from previously naive rats that underwent a yoked experience, while incorporation into the 21,000 MW protein was slightly decreased. The yoked experience did not affect in vitro phosphate incorporation into any of these proteins in particulate fractions from previously handled rats

  11. Expression of ATP7B in normal human liver

    D Fanni

    2009-06-01

    Full Text Available ATP7B is a copper transporting P-type ATPase, also known as Wilson disease protein, which plays a key role in copper distribution inside cells. Recent experimental data in cell culture have shown that ATP7B putatively serves a dual function in hepatocytes: when localized to the Golgi apparatus, it has a biosynthetic role, delivering copper atoms to apoceruloplasmin; when the hepatocytes are under copper stress, ATP7B translocates to the biliary pole to transport excess copper out of the cell and into the bile canaliculus for subsequent excretion from the body via the bile. The above data on ATP7B localization have been mainly obtained in tumor cell systems in vitro. The aim of the present work was to assess the presence and localization of the Wilson disease protein in the human liver. We tested immunoreactivity for ATP7B in 10 human liver biopsies, in which no significant pathological lesion was found using a polyclonal antiserum specific for ATP7B. In the normal liver, immunoreactivity for ATP7B was observed in hepatocytes and in biliary cells. In the hepatocytes, immunoreactivity for ATP7B was observed close to the plasma membrane, both at the sinusoidal and at the biliary pole. In the biliary cells, ATP7B was localized close to the cell membrane, mainly concentrated at the basal pole of the cells. The data suggest that, in human liver, ATP7B is localized to the plasma membrane of both hepatocytes and biliary epithelial cells.

  12. Recombinant Human Prion Protein Inhibits Prion Propagation in vitro

    Jue Yuan; Yi-An Zhan; Romany Abskharon; Xiangzhu Xiao; Manuel Camacho Martinez; Xiaochen Zhou; Geoff Kneale; Jacqueline Mikol; Sylvain Lehmann; Surewicz, Witold K.; Joaquín Castilla; Jan Steyaert; Shulin Zhang; Qingzhong Kong; Petersen, Robert B.

    2013-01-01

    Prion diseases are associated with the conformational conversion of the cellular prion protein (PrPC) into the pathological scrapie isoform (PrPSc) in the brain. Both the in vivo and in vitro conversion of PrPC into PrPSc is significantly inhibited by differences in amino acid sequence between the two molecules. Using protein misfolding cyclic amplification (PMCA), we now report that the recombinant full-length human PrP (rHuPrP23-231) (that is unglycosylated and lacks the glycophosphatidylin...

  13. Antioxidant properties of wheat germ protein hydrolysates evaluated in vitro

    CHENG Yun-hui; WANG Zhang; XU Shi-ying

    2006-01-01

    Wheat germ protein hydrolysates were prepared by protease hydrolysis, ultrafiltration and dynamical adsorption of resin. The total amount of amino acids in 100 g wheat germ protein hydrolysates is 93.95 g. Wheat germ protein hydrolysates are primarily composed of 4 fractions: 17.78 % in the relative molecular mass range of 11 563 -1 512, 17.50% in 1512 -842, 27.38% in 842- 372 and 30.65% in 372- 76, respectively. The antioxidant properties of wheat germ protein hydrolysates were evaluated by using different antioxidant tests in vitro. 1.20 g/L wheat germ protein hydrolysates exhibit 78.75% inhibition of peroxidation in linolei acid system; and 1.6 g/L wheat germ protein hydrolysates show 81.11% scavenging effect on the 1,1-diphenyl-2-picrylhrazyl radical. The reducing power of 2.50 g/L wheat germ protein hydrolysates is 0. 84. Furthermore, the scavenging activity of 0.60 g/L wheat germ protein hydrolysates against superoxide radical is 75. 40%; 0. 50 g/L wheat germ protein hydrolysates exhibit63.35 % chelating effect on ferrous ion. These antioxidant activities of wheat germ protein hydrolsates increase with the increase of its concentration. Experimental results suggest that wheat germ protein hydrolysate is a suitable natural antioxidant rich in nutrition and nontoxic.

  14. Retroviral DNA Integration Directed by HIV Integration Protein in Vitro

    Bushman, Frederic D.; Fujiwara, Tamio; Craigie, Robert

    1990-09-01

    Efficient retroviral growth requires integration of a DNA copy of the viral RNA genome into a chromosome of the host. As a first step in analyzing the mechanism of integration of human immunodeficiency virus (HIV) DNA, a cell-free system was established that models the integration reaction. The in vitro system depends on the HIV integration (IN) protein, which was partially purified from insect cells engineered to express IN protein in large quantities. Integration was detected in a biological assay that scores the insertion of a linear DNA containing HIV terminal sequences into a λ DNA target. Some integration products generated in this assay contained five-base pair duplications of the target DNA at the recombination junctions, a characteristic of HIV integration in vivo; the remaining products contained aberrant junctional sequences that may have been produced in a variation of the normal reaction. These results indicate that HIV IN protein is the only viral protein required to insert model HIV DNA sequences into a target DNA in vitro.

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

    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 (pfeed proteins 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. PMID:26954145

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

    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

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

    Gautier Virginie W

    2009-05-01

    Full Text Available Abstract 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

  18. Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression.

    Focke, Paul J; Hein, Christopher; Hoffmann, Beate; Matulef, Kimberly; Bernhard, Frank; Dötsch, Volker; Valiyaveetil, Francis I

    2016-08-01

    Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K(+) channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and in vitro folding to show the structural similarity to the cellular expressed KcsA channel and to establish the feasibility of using this two-step approach for membrane protein production for structural studies. Our studies show that the correct folding of these membrane proteins with complex topologies can take place in vitro without the involvement of the cellular machinery for membrane protein biogenesis. This indicates that the folding instructions for these complex membrane proteins are contained entirely within the protein sequence. PMID:27384110

  19. Techniques for Monitoring Protein Misfolding and Aggregation in Vitro and in Living Cells

    Gregoire, Simpson; Irwin, Jacob; Kwon, Inchan

    2012-01-01

    Protein misfolding and aggregation have been considered important in understanding many neurodegenerative diseases and recombinant biopharmaceutical production. Therefore, various traditional and modern techniques have been utilized to monitor protein aggregation in vitro and in living cells. Fibril formation, morphology and secondary structure content of amyloidogenic proteins in vitro have been monitored by molecular probes, TEM/AFM, and CD/FTIR analyses, respectively. Protein aggregation i...

  20. Processing Pisum sativum seed storage protein precursors in vitro

    YANGLIJUN; CDOMONEY; 等

    1990-01-01

    The profile of polypeptides separated by SDS-PAGE from seed of major crop species such as pea(Pisum sativum) is complex,resulting from cleavage (processing) of precursors expressed from multiple copies of genes encoding vicilin and legumin,the major storage globulins.Translation in vitro of mRNAs hybridselected from mid-maturation pea seed RNAs by defined vicilin and legumin cDNA clones provided precursor molecules that were cleaved in vitro by a cell-free protease extract obtained from similar stage seed;the derived polypeptides were of comparable sizes to those observed in vivo.The feasibility of transcribing mRNA in vitro from a cDNA clone and cleavage in vitro of the derived translation products was established for a legumin clone,providing a method for determining polypeptide products of an expressed sequence.This approach will also be useful for characterising cleavage site requirements since modifications an readily be introduced at the DNA level.

  1. CYP7B1

    Roos, P; Svenstrup, K; Danielsen, E R;

    2014-01-01

    UNLABELLED: The SPG5A subtype of Hereditary Spastic Paraplegia (HSP) is a rare autosomal recessive neurodegenerative disorder caused by mutations in the CYP7B1 gene, which encodes a steroid cytochrome P450 7α-hydroxylase. This enzyme provides the primary metabolic route for neurosteroids. Clinica......UNLABELLED: The SPG5A subtype of Hereditary Spastic Paraplegia (HSP) is a rare autosomal recessive neurodegenerative disorder caused by mutations in the CYP7B1 gene, which encodes a steroid cytochrome P450 7α-hydroxylase. This enzyme provides the primary metabolic route for neurosteroids...

  2. Improving the in vitro protein digestibility of sorghum with reducing agents

    Hamaker, B. R.; Kirleis, A. W.; Butler, L G; Axtell, J. D.; Mertz, E T

    1987-01-01

    We have shown in previous reports that cooked sorghum protein is less digestible than other cooked cereal proteins. The pepsin-indigestible proteins in sorghum were found to be mainly prolamin proteins. Cooking sorghum in the presence of 2-mercaptoethanol increased protein digestibility (in vitro with pepsin or trypsin/chymotrypsin) to a level comparable with other cereals. At a concentration of 100 mM, other reducing agents (dithiothreitol, sodium bisulfite, and L-cysteine) were equally effe...

  3. Protein oxidation and proteolysis during storage and in vitro digestion of pork and beef patties.

    Rysman, Tine; Van Hecke, Thomas; Van Poucke, Christof; De Smet, Stefaan; Van Royen, Geert

    2016-10-15

    The effect of protein oxidation on proteolysis during meat digestion was investigated following storage and subsequent in vitro digestion of beef and pork patties. Protein oxidation was evaluated as thiol oxidation, total carbonylation, and specific carbonylation (α-amino adipic and γ-glutamic semialdehyde). Furthermore, 4-hydroxyphenylalanine, a hydroxylation product of phenylalanine, was identified and quantified as a new protein oxidation marker. After 7days of chilled illuminated storage (4°C), significant oxidative modifications were quantified and the oxidative degradation was continued during in vitro digestion. The observed effects were more abundant in beef patties. Protein oxidation before digestion resulted in impaired proteolysis during digestion. PMID:27173550

  4. Kepler-7b

    Latham...[], David W.; Borucki, W.J.; Koch, D.G.;

    2010-01-01

    We report on the discovery and confirmation of Kepler-7b, a transiting planet with unusually low density. The mass is less than half that of Jupiter, M P = 0.43 M J, but the radius is 50% larger, R P = 1.48 R J. The resulting density, ¿P = 0.17 g cm–3, is the second lowest reported so far for an...

  5. In vitro chloroplast protein synthesis by the chromophytic alga Olisthodiscus luteus

    The chloroplasts of chlorophytic and chromophytic plants exhibit significant morphological and biochemical differences. Presently, it is impossible to compare the influence of ctDNA on the structure and function of organelles within these two phylogenetic groups for no data exist in the literature on the profile of protein products synthesized by a chromophytic plastid. In this paper, the chloroplast DNA coded proteins of the chromophytic plant Olisthodiscus luteus are investigated by labeling isolated chloroplasts in vitro. Isolated plastids of excellent morphological condition are pulse labeled with [35S]methionine. Approximately 100 proteins are detected by two-dimensional gel electrophoresis and fluorography. However, these isolated plastids have a number of unusual characteristics: (1) they are photosynthetically inactive; (2) in vitro protein synthesis is light independent; (3) all proteins synthesized in vitro are membrane associated

  6. Expression of Green Fluorescent Protein (GFP using In Vitro translation cell free system

    M Mohamadipoor

    2009-03-01

    Full Text Available ABSTRACT Background and the purpose of the study: One of the major concerns about recombinant protein production is its possible toxicity for the organism. Purification of the recombinant protein is another challenge in this respect. Recently In Vitro translation cell free system that provides a coupled transcription-translation reaction for protein synthesis to overcome the above mentioned problems has been emerged. The aim of this study was expression of GFP as a marker for gene expression and protein in In Vitro translation system. Methods: pIVEX2.3-GFP plasmid was cloned to E. coli   and the plasmid DNA extracted. In Vitro translation was performed with RTS 100 E. coli Hy kit according to manufacture's instructions. Expression of recombinant fusion protein, His- GFP, was determined by SDS-PAGE, ELISA and western blot analysis. Results: Expected size of recombinant protein was detected in SDS-PAGE and further confirmed by western blot analysis and ELISA. Major conclusion: Results showed that In Vitro translation is suitable for expression of recombinant protein and fusion of the recombinant protein with His-tag facilitates the purification.

  7. Protein Adsorption of Calcium Phosphate Ceramics in vitro

    2005-01-01

    In order to provide valuable information for the design of new calcium phosphate bone repair materials, bone tissue engineering scaffold materials, and other clinical application, the interaction between calcium phosphate materials and proteins were investigated. The adsorption of the calcium phosphate ceramic to the protein was investigated by using FT- IR, XPS, SEM, and SDS- PAGE. As the results shown, the proteins were strongly adsorbed by the CPC, and a shift of the feature peak of the protein and also a chemical shift in the Ca2p and O1s bind energy of CPC was observed. This indicated that the acidic amino-group and alkaline amino- residue on the proteins' surface bonded to the Ca2 + in the β- TCP crystal by ionic bond and the proteins' alkaline amino groups to the oxygen in PO3-4 by hydrogen bond and electrostatic attraction. The adsorption mechanism of the protein in the CPC can be described as three ndsorption layers: irreversible chemical adsorption layer, physical adsorption layer and biomineralized adsorption layer.

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

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

    2016-11-01

    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)protein(s) was susceptible to proteolysis. Thus the compact structure of pasta protects the starch and proteins in the interior of the whole pasta, reducing the enzymatic degradation of starch molecules, especially for molecules with Rh>100nm. PMID:27516291

  9. Applications of phasor plots to in vitro protein studies

    James, N. G.; Ross, J. A.; Štefl, Martin; Jameson, D. M.

    2011-01-01

    Roč. 410, č. 1 (2011), s. 70-76. ISSN 0003-2697 R&D Projects: GA MŠk(CZ) LC06063 Institutional research plan: CEZ:AV0Z40400503 Keywords : protein fluorescence * kinetics * lifetimes Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.996, year: 2011

  10. In vitro antithrombotic activities of peanut protein hydrolysates.

    Zhang, Shao Bing

    2016-07-01

    The antithrombotic activities of peanut protein hydrolysates were investigated using a microplates assay. When peanut proteins were hydrolyzed to a limited extent by various enzymes, their thrombin inhibitory abilities were significantly enhanced. However, the resultant hydrolysates showed significantly different activities even at the same degrees of hydrolysis. The hydrolysates generated by Alcalase 2.4L displayed the best antithrombotic activities and the hydrolysis process was further optimized by response surface methodology. The antithrombotic activities were increased to 86% based on a protein concentration of 50mg/ml under the optimal conditions: pH 8.5, enzyme concentration of 5000IU/g of peanut proteins, and 2h hydrolysis time at 50°C. The Alcalase 2.4L crude hydrolysates were then fractionated successively by preparative and semi-preparative reverse-phase high-performance liquid chromatography (RP-HPLC). The peptide fraction collected inhibited thrombin-catalyzed coagulation of fibrinogen completely at a concentration of 0.4mg/ml, with an antithrombotic activity close to that of heparin at quite a low concentration (0.2mg/ml). This peptide fraction was further analyzed by online reverse-phase ultra-performance liquid chromatography (RP-UPLC) coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and three new peptides were identified as Ser-Trp-Ala-Gln-Leu, Gly-Asn-His-Glu-Ala-Gly-Glu and Cys-Phe-Asn-Glu-Tyr-Glu, respectively. This research provided an effective way to produce antithrombotic peptides from peanut proteins, and also helped to elucidate the structure-function relationships of peanut peptides. PMID:26920259

  11. Signal transduction protein PII from Synechococcus elongatus PCC 7942 senses low adenylate energy charge in vitro.

    Fokina, Oleksandra; Herrmann, Christina; Forchhammer, Karl

    2011-01-01

    Abstract PII proteins belong to a family of highly conserved signal transduction proteins widely spread in bacteria, archaea and plants. They respond to the central metabolites ATP, ADP and 2-oxoglutarate (2-OG) and control enzymes, transcription factors and transport proteins involved in nitrogen metabolism. Here we studied the effect of ADP on in vitro PII signalling properties from the cyanobacterium Synechococcus elongatus, a model for oxygenic phototrophic organisms. Different...

  12. In vitro Immunogenicity Risk-assessment of Therapeutic Proteins in Preclinical Setting

    Gaitonde, Puneet; Balu-Iyer, Sathy V.

    2011-01-01

    Immunogenicity against therapeutic proteins is a clinical problem in the successful treatment of many diseases and as such, immunogenicity risk assessment in preclinical setting would be useful to improve safety and efficacy of protein based therapeutics in the product development stages. Here, we attempted a mechanism based in vitro studies as screening tool to capture clinically observed antibody based immune response against two representative therapeutic proteins; recombinant human Erythr...

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

    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.

  14. Effects of enzymatic dephosphorylation on infant in vitro gastrointestinal digestibility of milk protein concentrate.

    Liu, Dasong; Wang, Yuanyuan; Yu, Yun; Hu, Jinhua; Lu, Naiyan; Regenstein, Joe M; Wang, Miao; Zhou, Peng

    2016-04-15

    This study investigated the effects of dephosphorylation extent on infant in vitro gastric clotting property and gastrointestinal digestibility of milk protein concentrate. Dephosphorylation was affected by phosphatase type and incubation pH. A series of milk protein concentrate with 0-69% dephosphorylation were obtained by incubation with calf intestinal alkaline phosphatase at pH 6.5 for 0-420 min. Both β- and αs1-caseins in the modified milk protein concentrate showed multiply dephosphorylated isoforms with different numbers of phosphate groups depending on the extent of dephosphorylation. With increased dephosphorylation of milk protein concentrate, the gastric clotting extent decreased and the gastrointestinal digestibility increased under infant in vitro conditions. These results suggested the potential of developing a dephosphorylated milk protein concentrate, with improved gastric clotting property and gastrointestinal digestibility, to simulate the multiply phosphorylated patterns of human casein and hence to further the humanization of infant formula on a molecular level. PMID:26617031

  15. Comparing protein VEGF inhibitors: In vitro biological studies

    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.

  16. In vitro polymerization of mussel polyphenolic proteins catalyzed by mushroom tyrosinase.

    Burzio, L A; Burzio, V A; Pardo, J; Burzio, L O

    2000-07-01

    The in vitro enzymatic polymerization of the polyphenolic protein purified from the mussels Aulacomya ater, Mytilus edulis chilensis and Choromytilus chorus was studied. Mushroom tyrosinase was used to oxidize the dopa residues present in these proteins, and polymerization was monitored by acid-urea polyacrylamide gel electrophoresis. The protein from A. ater polymerized at a faster rate than the other two. Amino acid analysis of the crosslinked protein showed a notable decrease in the content of dopa, but no significant change of other amino acids. This suggests that crosslink formation may be limited to the oxidized dopa derivatives of the protein molecules. PMID:11007180

  17. In vitro binding of selenium by rat liver mitochondrial selenium-binding protein

    Last year the authors reported that upon freezing and thawing mitochondria from rats injected with [75Se]Na2SeO3 (75Se-selenite), a 75Se-binding protein (SeBP) was released. They have studied further in vitro labelling of SeBP. This matrix protein was labelled in vitro when lysed mitochondria (containing non-matrix material) were incubated with 75Se-selenite but not when matrix material alone was incubated with 75Se-selenite. Thus, there are one or more promoters of in vitro SeBP labelling in the non-matrix fraction. SeBP was also labelled in vitro when 75Se-selenite was added to matrix alone and dialyzed. Dialysis tubing, and not the dialysis process, promoted labelling by affecting SeBP and not by affecting 75Se-selenite. Labelling did not occur when matrix alone and 75Se-selenite were incubated (not dialyzed) in a glass test tube but did occur in a polystyrene test tube. They hypothesize that non-covalent interactions occur between SeBP and dialysis tubing or polystyrene that expose Se binding sites on the protein. A similar mechanism involving mitochondrial non-matrix material may function in vivo. Non-denaturing disc gel electrophoresis of partially purified SeBP labelled in vivo or in vitro suggested that the same protein was labelled in both conditions. Using in vitro binding techniques, SeBP was also found in sheep liver mitochondrial matrix. This supports the theory that SeBP is important in Se metabolism

  18. Photoregulation of protein plasmid expression in vitro and in vivo using BHQ caging group

    Zhi Ping Zhang; Yi Ming Li; Xiao Yun Chen; Qing Xiang Guo

    2011-01-01

    Green fluorescent protein (GFP) plasmid was caged by 8-bromo-7-hydroxyquinolinyl chromophore (BHQ) for controlling its expression with exact spatiotemporal resolution. In vitro and in vivo experiments clearly verified that, comparing with Bhc caging, the expression level of caged GFP plasmid was dramatically decreased and then efficiently restored after subsequent photolysis.

  19. Neurotrophic effects of amyloid precursor protein peptide 165 in vitro.

    Yao, Jie; Ma, Lina; Wang, Rong; Sheng, Shuli; Ji, Zhijuan; Zhang, Jingyan

    2016-01-01

    Diabetic encephalopathy is one of the risk factors for Alzheimer's disease. Our previous findings indicated that animals with diabetic encephalopathy exhibit learning and memory impairment in addition to hippocampal neurodegeneration, both of which are ameliorated with amyloid precursor protein (APP) 17-mer (APP17) peptide treatment. Although APP17 is neuroprotective, it is susceptible to enzymatic degradation. Derived from the active sequence structure of APP17, we have previously structurally transformed and modified several APP5-mer peptides (APP328-332 [RERMS], APP 5). We have developed seven different derivatives of APP5, including several analogs. Results from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on human neuroblastoma SH-SY5Y cells in the present study showed that P165 was the most neuroprotective APP5 derivative. Furthermore, we tested the effects of APP5 and P165 on the number of cells and the release of lactate dehydrogenase. Western immunoblot analyses were also performed. The digestion rates of P165 and APP5 were determined by the pepsin digestion test. P165 resisted pepsin digestion significantly more than APP5. Therefore, P165 may be optimal for oral administration. Overall, these findings suggest that P165 may be a potential drug for the treatment of diabetic encephalopathy. PMID:26551064

  20. Significance of "extravascular" protein binding for antimicrobial pharmacodynamics in an in vitro capillary model of infection.

    Dudley, M N; Blaser, J; D. Gilbert; Zinner, S H

    1990-01-01

    The effect of protein binding in an "extravascular" space on antimicrobial pharmacodynamics was studied in an in vitro capillary model of infection. Simulated 500-mg oral doses of dicloxacillin (approximately 96% bound) or cephalexin (less than 5% bound) were administered every 6 h for four doses. A 10-fold-higher dose of dicloxacillin was also studied to determine the effect of drug concentration on the reduction of bacterial killing in the presence of protein. Staphylococcus aureus ATCC 259...

  1. Incidence of various process parameters on in vitro protein digestibility of beef meat

    Hassoun, Ahmad; Sante-Lhoutellier, Veronique; Lebert, André; Kondjoyan, Alain; Daudin, Jean-Dominique

    2011-01-01

    Protein in vitro digestion was characterized by pepsin proteolysis of myofibrillar proteins extracted from processed beef samples using 2 descriptors of the kinetics: maximum value (ODmax) and half life time (t 1/2). An experimental fractional factorial design with 32 trials was used to investigate the effect of processes variables; it consists of 5 factors each taking 2 levels (muscle type, mincing, pH, NaCl content, cooking time) and 1 factor taking 4 levels (cooking temperature). The stati...

  2. 中华鲟神经内分泌多肽7B2基因克隆及组织特异性表达%Cloning and expression analysis of a neuroendocrine secretory protein 7B2 in Acipenser sinensis

    刘婷; 章龙珍; 张涛; 庄平; 冯广朋; 赵峰; 马境

    2011-01-01

    In order to clone full-length 7B2 gene and characterize its' tissue expression profile in Chinese sturgeon (Acipenser sinensis ), RT-PCR and rapid-amplification of cDNA ends (RACE) were adopted to obtain full-length cDNA. A full-length 986 bp cDNA sequence in A. sinensis was cloned, including a 711 bp complete ORF encoding a 236 amino acids peptide, a 14 bp long 5′-untranslate region and a 261 bp long 3′-untranslate region. The first 43 of the 236 amino acids were 7B2 signal peptide. The 7B2 in A. sinens showed high similarity to that of Danio rerio. The protein analysis showed that the A. sinensis 7B2 had the closest relationship with the D. redo. Futhermore, 7B2 mRNA tissue expressed profile was assessed by semiquantitative RT-PCR. In the nine examined tissues, the 7B2 gene was expressed strongly in brain, weakly in heart, gonad and pancreas, and little in kidney, bowel and skin, but hardly expressed in gill and liver.%以中华鲟(Acipenser sinensis)脑垂体总RNA为模板,采用RT-PCR和RACE方法,获得中华鲟神经内分泌多肽(782)基因的3个重叠片段,测序后拼接得到986 bp全长基因序列,其中包括5端非翻译区(5'-UTR)14 bp、3'端非翻译区(3'-UTR)261 bp和开放阅读框711 bp.翻译编码236个氨基酸.其中前43个氨基酸为782的信号肽.经BLAST比对发现中华鲟782蛋白的同源性与斑马鱼(Danio rerio)的相似性最高为82%.系统发育分析表明,中华鲟与斑马鱼亲缘关系最近.半定量RT-PCR分析表明:在脑中782 mRNA表达量最高,心脏、性腺、胰等组织中表达次之,肠、肾、皮肤等组织中少量表达,肝和鳃几乎不表达.

  3. Auxin effects on in vitro and in vivo protein phosphorylation in pea. [Pisum sativum

    Gallagher, S.R.; Ray, P.M.

    1987-04-01

    Terminal 8mm sections from the third internode of dark grown 7 day old Pisum sativum cv Alaska seedlings were separated into membrane and soluble fractions. SDS gradient PAGE identified approximately 50 in vivo phosphorylated proteins and proved superior to 2-D SDS PAGE in terms of resolution and repeatability. Addition of indoleacetic acid (IAA), fusicoccin, or 2,4 dichlorophenoxyacetic acid to membranes resulted in no detectable change in the number or phosphorylation level of the labeled proteins during in vitro phosphorylation in the presence of submicromolar concentrations of calcium. Similar results were obtained with soluble proteins. In the absence of calcium, the level of in vitro protein phosphorylation was much less, but not auxin effects could be identified. Furthermore, treatment of the sections with IAA in vivo followed by cell fractionation and in vitro phosphorylation failed to identify auxin responsive proteins. Lastly, when sections were labeled with /sup 32/P inorganic phosphate in the presence of 17 uM IAA, no auxin specific changes were found in the level of phosphorylation or in the number of phosphorylated proteins. Auxin effects on phosphorylation are thus slight or below their detection limit.

  4. Phosphorylation of human hnRNP protein A1 abrogates in vitro strand annealing activity.

    Cobianchi, F; Calvio, C; Stoppini, M; Buvoli, M; Riva, S

    1993-01-01

    In HeLa cells metabolically labeled in vivo with [32P] orthophosphate in the presence of okadaic acid the concentration of phosphorylated A1 protein was increased significantly as compared to controls. Purified recombinant hnRNP protein A1 served as an excellent substrate in vitro for the catalytic subunit of cAMP-dependent protein kinase (PKA) and for casein kinase II (CKII). Thin layer electrophoresis of A1 acid hydrolysates showed the protein to be phosphorylated exclusively on serine resi...

  5. IN VITRO RUMINAL PROTEIN DEGRADABILITY OF LEAVES FROM THREE TREE SPECIES HARVESTED AT TWO CUTTING INTERVALS

    A. EDWARDS

    2012-05-01

    Full Text Available In vitro ruminal protein degradation characteristics of protein supplements represent an accurate measure of the quality of protein for ruminant animals. As such, crude protein disappearance of Gliricidia sepium, Leucaena leucocephala and Trichanthera gigantea leaves, which are potential sources of supplemental protein for ruminants, was determined using the ANKOM in vitro ruminal degradability technique. Dry matter (DM and crude protein (CP disappearance were measured after 0, 2, 4, 6, 12, 24, 36, 48 and 72 h of incubation. Degradation kinetics were described using the Ørskov and McDonald equation  cx  y a b e   1 . The degradable part of the insoluble DM fraction (b was highest (P<0.05 in G. sepium leaves (27% at the 12 week cutting interval. Effective dry matter degradability (EDMD was highest (P < 0.05 in the leaves of G. sepium (74.9% at the 12-week cutting interval. CP washing losses was highest (P < 0.05 in the leaves of L. leucocephala (46.8% and lowest in T. gigantea leaves (16.3% at the 6-week cutting interval. Crude protein disappearance was highest (P<0.05 in the leaves of G. sepium and lowest in T. gigantea leaves at both the 6 and 12-week cutting intervals after incubation at 48 h. It is concluded that in vitro ruminal protein degradability is more pronounced in the leaves of G. sepium and L. leucocephala. Approximately 50% of their protein is degraded in the rumen suggesting that they would be useful as sources of readily available nitrogen for rumen microbes challenged with low nitrogen, fibrous basal diets. Trichanthera gigantea leaves have higher levels of rumen undegradable protein suggesting that they can be used to supply by-pass protein for animal.

  6. In vitro assembly of polymorphic virus-like particles from the capsid protein of a nodavirus.

    Bajaj, Saumya; Banerjee, Manidipa

    2016-09-01

    Viral capsid proteins are programmed to assemble into homogeneous structures in native environments; but the molecular details of these assembly pathways are seldom clearly understood. In order to define the chain of events in the construction of a minimal system, we attempted controlled assembly of the capsid protein of a small insect nodavirus, Flock House Virus (FHV). Bacterial expression of the FHV capsid protein, and subsequent in vitro assembly, generated a heterogeneous population of closed particles. We show that in spite of the altered structure, these particles are capable of membrane disruption, like native viruses, and of incorporating and delivering foreign cargo to specific locations. The unique structure and characteristics of these particles extends our understanding of nodavirus assembly. Additionally, the establishment of a bacterial production system, and methods for in vitro assembly and packaging are of considerable benefit for biotechnological applications of FHV. PMID:27289029

  7. Digestion kinetics of potato protein isolates in vitro and in vivo.

    He, Tao; Spelbrink, Robin E J; Witteman, Ben J; Giuseppin, Marco L F

    2013-11-01

    Recently, an industrial process was developed to isolate native protein fractions from potato: a high (HMW) and a low (LMW) molecular weight fraction. Digestion kinetics of HMW and LMW was studied in vitro and in vivo and compared with reference proteins. Under simulated conditions, highest digestion was found for whey protein, followed by soy, pea, HMW, casein and LMW. Ingestion of 20 g of proteins by eight healthy subjects (following a randomized, double-blind, cross-over design) induced a slow and moderate increase with HMW and LMW, while a peaked and high increase with whey protein, in postprandial plasma amino acid levels. Casein gave a similar profile as HMW, with higher levels. Contrary to whey and casein, HMW and LMW did not result in any changes in plasma insulin or glucose levels. This study provides insights in digestion of native potato protein isolates to assist their use as protein sources in food applications. PMID:23713493

  8. NUTRALYS® pea protein: characterization of in vitro gastric digestion and in vivo gastrointestinal peptide responses relevant to satiety

    Joost Overduin; Laetitia Guérin-Deremaux; Daniel Wils; Lambers, Tim T.

    2015-01-01

    Background: Pea protein (from Pisum sativum) is under consideration as a sustainable, satiety-inducing food ingredient. Objective: In the current study, pea-protein-induced physiological signals relevant to satiety were characterized in vitro via gastric digestion kinetics and in vivo by monitoring post-meal gastrointestinal hormonal responses in rats. Design: Under in vitro simulated gastric conditions, the digestion of NUTRALYS® pea protein was compared to that of two dairy proteins, slo...

  9. Analysis of green fluorescent protein bioluminescence in vivo and in vitro using a glow discharge

    Hernández, L.; Mandujano, L. A.; Cuevas, J.; Reyes, P. G.; Osorio-González, D.

    2015-03-01

    The discovery of fluorescent proteins has been a revolution in cell biology and related sciences because of their many applications, mainly emphasizing their use as cellular markers. The green fluorescent protein (GFP) is one of the most used as it requires no cofactors to generate fluorescence and retains this property into any organism when it is expressed by recombinant DNA techniques, which is a great advantage. In this work, we analyze the emission spectra of recombinant green fluorescent protein in vivo and in vitro exposed to a glow discharge plasma of nitrogen in order to relate electron temperature to fluorescence intensity.

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

    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.

  11. Serotonin binding in vitro by releasable proteins from human blood platelets

    Among the substances released from human blood platelets are serotonin and various proteins. It was hypothesized that one of these proteins binds serotonin and that serotonin might be important to the protein's function or that the protein might be important to serotonin's function. Two platelet-specific proteins, platelet factor 4 (PF4) and β-thromboglobulin (βTG) were found to bind serotonin in vitro. Endogenous PF4 was isolated by serotonin-affinity chromatography and was identified by radioimmunoassay. Purified [125I] -PF4 and native PF4 bound to and eluted from a serotonin-affinity column similarly. Ultrafiltration of the homologous protein, βTG, with [14C]-serotonin demonstrated binding of about 8 moles serotonin per mole tetrameric βTG with a dissociation constant of about 4 X 10(sup-8) M. Equilibrium dialysis of PF4 with radiolabelled serotonin was attempted, but no binding constant values were obtained because serotonin apparently bound to the dialysis membrane. Since EDTA was one of the two agents that eluted PF4 from the serotonin-affinity gel, calcium binding by PF4 was investigated by equilibrium dialysis. Evidence was obtained for positively cooperative binding of calcium ions by PF4. It is concluded that PF4 and βTG bind serotonin in vitro, that they may also bind in vivo when platelets undergo release, and that the functions of serotonin, PF4 and βTG may be mediated in part by serotonin-protein associations

  12. Production of Borreliacidal Antibody to Outer Surface Protein A In Vitro and Modulation by Interleukin-4

    Munson, Erik L.; Du Chateau, Brian K.; Jobe, Dean A.; Lovrich, Steven D.; Callister, Steven M.; Schell, Ronald F.

    2000-01-01

    Borreliacidal antibody production is one of several parameters for establishing the effectiveness of Borrelia burgdorferi vaccines. The production of borreliacidal antibody was studied in vitro by culturing immune lymph node cells with macrophages and B. burgdorferi. We showed that borreliacidal antibody, directed primarily against outer surface protein A (OspA), was readily produced by lymph node cells obtained from C3H/HeJ mice vaccinated with formalin-inactivated B. burgdorferi in aluminum...

  13. In Vitro Binding Capacity of Bile Acids by Defatted Corn Protein Hydrolysate

    Pierre Claver Irakoze

    2011-02-01

    Full Text Available Defatted corn protein was digested using five different proteases, Alcalase, Trypsin, Neutrase, Protamex and Flavourzyme, in order to produce bile acid binding peptides. Bile acid binding capacity was analyzed in vitro using peptides from different proteases of defatted corn hydrolysate. Some crystalline bile acids like sodium glycocholate, sodium cholate and sodium deoxycholate were individually tested using HPLC to see which enzymes can release more peptides with high bile acid binding capacity. Peptides from Flavourzyme defatted corn hydrolysate exhibited significantly (p

  14. TPX2 Protein of Arabidopsis Activates Aurora Kinase 1, But Not Aurora Kinase 3 In Vitro

    Tomaštíková, Eva; Demidov, D.; Jeřábková, Hana; Binarová, Pavla; Houben, A.; Doležel, Jaroslav; Petrovská, Beáta

    2015-01-01

    Roč. 33, č. 6 (2015), s. 1988-1995. ISSN 0735-9640 R&D Projects: GA ČR(CZ) GA14-28443S; GA MŠk(CZ) LO1204; GA ČR GAP501/12/2333 Institutional support: RVO:61389030 ; RVO:61388971 Keywords : Aurora kinase * Targeting protein for Xklp2 * In vitro kinase assay Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.656, year: 2014

  15. A double-emulsion microfluidic platform for in vitro green fluorescent protein expression

    Microfluidic droplet technology has gained popularity due to the advantages over conventional emulsion techniques and capabilities for a wide range of applications. In this paper, the development of a simple microfluidic-based double-emulsion system is reported. Such a system could be potentially used for in vitro protein synthesis. The system involves a two-step process to make water-in-oil-in-water (W/O/W) emulsions. A PMMA microchip is used for the formation of water-in-oil (W/O) single-emulsion droplets. Then, the single-emulsion droplets are transported to a PDMS/glass microchip to make the W/O/W double-emulsion droplets. The system was first characterized by detecting fluorescein sodium salt as a model dye in the internal aqueous droplets using laser-induced fluorescence. The effect of the flow rates of the internal aqueous phase and outer continuous aqueous phase on the formation of the double-emulsion droplets is investigated to provide information for system optimization. On-chip storage of double-emulsion droplets is also investigated to allow for protein synthesis from a PCR-generated DNA template using either commercial in vitro transcription and translation kits or crude Escherichia coli S30 extracts. In vitro expression of the green fluorescent protein is successfully demonstrated in this system

  16. Cyclin A-Cdk2 Phosphorylates BH3 only Protein Bad in vitro and in vivo

    HE Kan; CHEN Yue; LI Jing-hua; ZHAN Zhuo; WU Yong-ge; KONG Wei; JIN Ying-hua

    2007-01-01

    Increasing evidence suggests that Cyclin A-Cdk2 activity is required in the apoptosis process induced by various stimuli. To determine a specific substrate of Cyclin A-Cdk2 for apoptosis, in this study, we carried out anin vitro kinase assay using immunoprecipitated complex Cyclin A-Cdk2 as an enzyme source, and recombinant protein GST-Bad as a substrate. Our study showed that Bad was clearly phosphorylated by Cyclin A-Cdk2 in vitro. To examine whether protein Bad can also be phosphorylated by Cyclin A-Cdk2 kinase in vivo, we transiently overexpressed protein Bad with Cyclin A or Cdk2-dn, a dominant negative version of Cdk2, in Hela cells and determined the phosphorylation status of protein Bad. The test showed that protein Bad was clearly phosphorylated in Cyclin A overexpressed cells,but not in Cdk2-dn or mock transfectent. Moreover, etoposide also caused the phosphorylation of endogenetic Bad. In conclusion, here we provide first time evidence that protein Bad can be a substrate of Cyclin A-Cdk2 apoptosis for in vitro and in vivo.

  17. Genes and Proteins Differentially Expressed during In Vitro Malignant Transformation of Bovine Pancreatic Duct Cells

    R. Jesnowski

    2007-02-01

    Full Text Available Pancreatic carcinoma has an extremely bad prognosis due to lack of early diagnostic markers and lack of effective therapeutic strategies. Recently, we have established an in vitro model recapitulating the first steps in the carcinogenesis of the pancreas. SV40 large T antigen-immortalized bovine pancreatic duct cells formed intrapancreatic adenocarcinoma tumors on k-rasmut transfection after orthotopic injection in the nude mouse pancreas. Here we identified genes and proteins differentially expressed in the course of malignant transformation using reciprocal suppression subtractive hybridization and 2D gel electrophoresis and mass spectrometry, respectively. We identified 34 differentially expressed genes, expressed sequence tags, and 15 unique proteins. Differential expression was verified for some of the genes or proteins in samples from pancreatic carcinoma. Among these genes and proteins, the majority had already been described either to be influenced by a mutated ras or to be differentially expressed in pancreatic adenocarcinoma, thus proving the feasibility of our model. Other genes and proteins (e.g., BBC1, GLTSCR2, and rhoGDlα, up to now, have not been implicated in pancreatic tumor development. Thus, we were able to establish an in vitro model of pancreatic carcinogenesis, which enabled us to identify genes and proteins differentially expressed during the early steps of malignant transformation.

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

    Vojtíšková, Petra; Kráčmar, Stanislav

    2013-01-01

    The aim of this study was to determine crude protein, fi bre 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 modifi ed 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 conte...

  19. In vitro assessment of zinc binding to protein foods as a potential index of zinc bioavailability. Comparison of in vitro and in vivo data

    An in vitro equilibrium dialysis test for estimating the strength of zinc binding to protein foods was developed for predicting zinc bioavailability. Soy flour, soy concentrate, casein, and dried egg white were labeled with 65ZnCl2 before dialysis. The conditions included 24-h dialysis at pH 7.4 against 0.05 M tris(hydroxymethyl)aminomethane buffer (Tris), Tris plus 0.01 M L-histidine hydrochloride (Tris-His), and Tris plus 0.01 M Na2EDTA (Tris-EDTA). Dialyzate and retentate 65Zn were measured. The protein foods retained 65Zn in the following decreasing order according to treatment: Tris > Tris-His > Tris-EDTA. The bioavailability of residual 65Zn in casein, egg white, soy concentrate, and soy flour after each buffer treatment was determined by giving single doses of the protein foods to young Japanese quail. For these protein foods, the best agreement between in vitro and in vivo data was with Tris-His-dialyzable 65Zn values and the whole-body 65Zn retentions from the labeled casein and egg white (no treatment). The data suggest that this in vitro test could be useful for preliminary assessment of zinc bioavailability of protein foods

  20. Rice proteins, extracted by alkali and α-amylase, differently affect in vitro antioxidant activity.

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

    2016-09-01

    Alkali treatment and α-amylase degradation are different processes for rice protein (RP) isolation. The major aim of this study was to determine the influence of two different extraction methods on the antioxidant capacities of RPA, extracted by alkaline (0.2% NaOH), and RPE, extracted by α-amylase, during in vitro digestion for 2h with pepsin and for 3h with pancreatin. Upon pepsin-pancreatin digestion, the protein hydrolysates (RPA-S, RPE-S), which were the supernatants in the absence of undigested residue, and the whole protein digests (RPA, RPE), in which undigested residue remained, were measured. RPE exhibited the stronger antioxidant responses to free radical scavenging activity, metal chelating activity, and reducing power, whereas the weakest antioxidant capacities were produced by RPE-S. In contrast, no significant differences in antioxidant activity were observed between RPA and RPA-S. The present study demonstrated that the in vitro antioxidant responses induced by the hydrolysates and the protein digests of RPs could be affected differently by alkali treatment and α-amylase degradation, suggesting that the extraction is a vital processing step to modify the antioxidant capacities of RPs. The results of the current study indicated that the protein digests, in which undigested residues remained, could exhibit more efficacious antioxidant activity compared to the hydrolysates. PMID:27041309

  1. In vitro effect of dietary protein level and nondigestible oligosaccharides on feline fecal microbiota.

    Pinna, C; Stefanelli, C; Biagi, G

    2014-12-01

    The aim of the present study was to evaluate in vitro the effect of some prebiotic substances and 2 dietary protein levels on the composition and activity of feline fecal microbiota. Two in vitro studies were conducted. First, 6 nondigestible oligosaccharides were studied; treatments were control diet (CTRL), gluconic acid (GA), carrot fiber (CF), fructooligosaccharides (FOS), galactooligosaccharides (GOS), lactitol (LAC), and pectins from citrus fruit (PEC). Substrates were added to feline fecal cultures at 2 g/L for 24 h incubation. Compared with the CTRL, ammonia had been reduced (PFOS (+90%), GOS (+96%), and LAC (+87%). Compared with the CTRL, total VFA were higher (PFOS were selected to be tested in the presence of 2 diets differing in their protein content. There were 6 treatments: low-protein (LP) CTRL with no addition of prebiotics (CTRL-LP), high-protein (HP) CTRL with no addition of prebiotics (CTRL-HP), LP diet plus FOS, CTRL-HP plus FOS, LP diet plus LAC, and CTRL-HP plus LAC. Both FOS and LAC were added to feline fecal cultures at 2 g/L for 24 h incubation. Ammonia at 24 h was affected (PFOS. Total VFA were influenced (Pprebiotics exert different effects on the composition and activity of feline intestinal microbiota and that high dietary protein levels in a cat's diet can have negative effects on the animal intestinal environment. PMID:25367521

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

    Bjerregaard, Bolette; Wrenzycki, Christine; Strejcek, Frantisek;

    2004-01-01

    The expression of nucleolar-related proteins was studied as an indirect marker of the ribosomal RNA (rRNA) gene activation in porcine embryos up to the blastocyst stage produced in vivo and in vitro. A group of the in vivo-developed embryos were cultured with alpha-amanitin to block the de novo...... pocket proteins pRb and p130, which are involved in cell-cycle regulation, was assessed by semiquantitative RT-PCR up to the blastocyst stage. Toward the end of third cell cycle, the nuclei in non-alpha-amanitin-treated, in vivo-produced embryos displayed different stages of transformation of the nuclear...... 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...

  3. NUTRALYS® pea protein: characterization of in vitro gastric digestion and in vivo gastrointestinal peptide responses relevant to satiety

    Joost Overduin

    2015-04-01

    Design: Under in vitro simulated gastric conditions, the digestion of NUTRALYS® pea protein was compared to that of two dairy proteins, slow-digestible casein and fast-digestible whey. In vivo, blood glucose and gastrointestinal hormonal (insulin, ghrelin, cholecystokinin [CCK], glucagon-like peptide 1 [GLP-1], and peptide YY [PYY] responses were monitored in nine male Wistar rats following isocaloric (11 kcal meals containing 35 energy% of either NUTRALYS® pea protein, whey protein, or carbohydrate (non-protein. Results: In vitro, pea protein transiently aggregated into particles, whereas casein formed a more enduring protein network and whey protein remained dissolved. Pea-protein particle size ranged from 50 to 500 µm, well below the 2 mm threshold for gastric retention in humans. In vivo, pea-protein and whey-protein meals induced comparable responses for CCK, GLP-1, and PYY, that is, the anorexigenic hormones. Pea protein induced weaker initial, but equal 3-h integrated ghrelin and insulin responses than whey protein, possibly due to the slower gastric breakdown of pea protein observed in vitro. Two hours after meals, CCK levels were more elevated in the case of protein meals compared to that of non-protein meals. Conclusions: These results indicate that 1 pea protein transiently aggregates in the stomach and has an intermediately fast intestinal bioavailability in between that of whey and casein; 2 pea-protein- and dairy-protein-containing meals were comparably efficacious in triggering gastrointestinal satiety signals.

  4. PIG11 protein binds to DNA in sequence-independent manner in vitro

    PIG11 (p53-induced protein 11), one of early transcriptional targets of tumor suppressor p53, was up-regulated in the induction of apoptosis or cell growth inhibition by multiple chemopreventive agents. However, its biological role remains unclear. Here, we expressed His6-tagged PIG11 protein in Escherichia coli and demonstrated the recombinant His6-tagged PIG11 protein could bind to supercoiled and relaxed closed circular plasmid DNA or linear DNA with different length using gel retardation assays in vitro. The interaction between DNA and PIG11 protein was sequence-independent and related to charge effect. The reducing thiol group in PIG11 protein was involved in the binding activity of PIG11 to DNA. Furthermore, the images of atomic force microscopy directly confirmed the binding of DNA and PIG11 protein and showed the PIG11-DNA complex formed a beads-on-a-string appearance in which PIG11 protein associated with DNA as polymer. These findings suggest that PIG11 protein may play an important role by interaction with other biological molecules in the regulation of apoptosis and provided us a novel angel of view to explore the possible function of PIG11 in vivo

  5. In vitro protein binding of liraglutide in human plasma determined by reiterated stepwise equilibrium dialysis

    Plum, Anne; Jensen, Lisbeth Bjerring; Kristensen, Jesper Bøggild

    2013-01-01

    Liraglutide is a human glucagon-like peptide-1 (GLP-1) analogue approved for the treatment of type 2 diabetes. It is based on human GLP-1 with the addition of a 16-carbon fatty acid, which facilitates binding to plasma proteins, thus prolonging the elimination half-life and allowing once-daily administration. It has not been possible to quantify liraglutide protein binding by ultrafiltration (the usual method of choice), as the lipophilic molecule becomes trapped in the filter membrane. Therefore, the aim of this study was to develop a methodology that could determine the extent of liraglutide binding to plasma proteins in vitro. We report here the details of a novel reiterated stepwise equilibrium dialysis assay that has successfully been used to quantify liraglutide plasma protein binding. The assay allowed quantification of liraglutide binding to proteins in purified plasma protein solutions and human plasma samples and was effective at plasma dilutions as low as 5%. At a clinically relevant liraglutide concentration (104 pM), greater than 98.9% of liraglutide was bound to protein. Specific binding to human serum albumin and α1-acid glycoprotein was 99.4% and 99.3%, respectively. The novel methodology described herein could have an application in the quantification of plasma protein binding of other highly lipophilic drug molecules. PMID:23853127

  6. In vitro thermodynamic dissection of human copper transfer from chaperone to target protein.

    Moritz S Niemiec

    Full Text Available Transient protein-protein and protein-ligand interactions are fundamental components of biological activity. To understand biological activity, not only the structures of the involved proteins are important but also the energetics of the individual steps of a reaction. Here we use in vitro biophysical methods to deduce thermodynamic parameters of copper (Cu transfer from the human copper chaperone Atox1 to the fourth metal-binding domain of the Wilson disease protein (WD4. Atox1 and WD4 have the same fold (ferredoxin-like fold and Cu-binding site (two surface exposed cysteine residues and thus it is not clear what drives metal transfer from one protein to the other. Cu transfer is a two-step reaction involving a metal-dependent ternary complex in which the metal is coordinated by cysteines from both proteins (i.e., Atox1-Cu-WD4. We employ size exclusion chromatography to estimate individual equilibrium constants for the two steps. This information together with calorimetric titration data are used to reveal enthalpic and entropic contributions of each step in the transfer process. Upon combining the equilibrium constants for both steps, a metal exchange factor (from Atox1 to WD4 of 10 is calculated, governed by a negative net enthalpy change of ∼10 kJ/mol. Thus, small variations in interaction energies, not always obvious upon comparing protein structures alone, may fuel vectorial metal transfer.

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

    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. PMID:1237403

  8. Synthetic peptide homologous to β protein from Alzheimer's disease forms amyloid-like fibrils in vitro

    Progressive amyloid deposition in senile plaques and cortical blood vessels may play a central role in the pathogenesis of Alzheimer's disease. The authors have used x-ray diffraction and electron microscopy to study the molecular organization and morphology of macromolecular assemblies formed by three synthetic peptides homologous to β protein of brain amyloid: β-(1-28), residues 1-28 of the β protein; [Ala1-β-(1-28), β-(1-28) with alanine substituted for lysine at position 16; and β-(18-28), residues 18-28 of the β protein. β-(1-28) readily formed fibrils in vitro that were similar in ultrastructure to the in vivo amyloid and aggregated into large bundles resembling those of senile plaque cores. X-ray patterns from partially dried, oriented pellets showed a cross-β-conformation. [Ala16]β-(1-28) formed β-pleated sheet assemblies that were dissimilar to in vivo fibrils. The width of the 10-A spacing indicated stacks of about six sheets. Thus, substitution of the uncharged alanine for the positively charged lysine in the β-strand region enhances the packing of the sheets and dramatically alters the type of macromolecular aggregate formed. Β-(18-28) formed assemblies that had even a greater number of stacked sheets. The findings on these homologous synthetic assemblies help to define the specific sequence that is required to form Alzheimer's-type amyloid fibrils, thus providing an in vitro model of age-related cerebral amyloidogenesis

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

    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.

  10. Danthron activates AMP-activated protein kinase and regulates lipid and glucose metabolism in vitro

    Rong ZHOU; Ling WANG; Xing XU; Jing CHEN; Li-hong HU; Li-li CHEN; Xu SHEN

    2013-01-01

    Aim:To discover the active compound on AMP-activated protein kinase (AMPK) activation and investigate the effects of the active compound 1,8-dihydroxyanthraquinone (danthron) from the traditional Chinese medicine rhubarb on AMPK-mediated lipid and glucose metabolism in vitro.Methods:HepG2 and C2C12 cells were used.Cell viability was determined using MTT assay.Real-time PCR was performed to measure the gene expression.Western blotting assay was applied to investigate the protein phosphorylation level.Enzymatic assay kits were used to detect the total cholesterol (TC),triglyceride (TG) and glucose contents.Results:Danthron (0.1,1,and 10 μmol/L) dose-dependently promoted the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC)in both HepG2 and C2C12 cells.Meanwhile,danthron treatment significantly reduced the lipid synthesis related sterol regulatory element-binding protein 1c (SREBP1c) and fatty acid synthetase (FAS) gene expressions,and the TC and TG levels.In addition,danthron treatment efficiently increased glucose consumption.The actions of danthron on lipid and glucose metabolism were abolished or reversed by co-treatment with the AMPK inhibitor compound C.Conclusion:Danthron effectively reduces intracellular lipid contents and enhanced glucose consumption in vitro via activation of AMPK signaling pathway.

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

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

    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 kinas...

  12. AMP-activated protein kinase (AMPK) activation regulates in vitro bone formation and bone mass

    Shah, M; Kola, B; Bataveljic, A.; Arnett, T. R.; Viollet, B.; Saxon, L.; Korbonits, M.; C. Chenu

    2010-01-01

    Adenosine 5′-monophosphate-activated protein kinase (AMPK), a regulator of energy homeostasis, has a central role in mediating the appetite-modulating and metabolic effects of many hormones and antidiabetic drugs metformin and glitazones. The objective of this study was to determine if AMPK can be activated in osteoblasts by known AMPK modulators and if AMPK activity is involved in osteoblast function in vitro and regulation of bone mass in vivo. ROS 17/2.8 rat osteoblast-like cells were cult...

  13. Association of the adenovirus DNA-binding protein with RNA both in vitro and in vivo.

    Cleghon, V G; Klessig, D F

    1986-01-01

    The multifunctional DNA-binding protein (DBP) encoded by human adenovirus binds RNA. The association of purified DBP with RNA in vitro was demonstrated by using either a gel filtration or a filter binding assay. This association is sensitive to ionic strength and exhibits no apparent sequence specificity. DBP also interacts with RNA in vivo; it can be crosslinked to polyadenylylated RNA by UV-irradiation of intact cells during the late phase of adenovirus infections. The 46-kDa carboxyl-termi...

  14. Poly(A)-binding-protein-mediated regulation of hDcp2 decapping in vitro

    Khanna, Richie; Kiledjian, Megerditch

    2004-01-01

    Regulation of mRNA decapping is a critical determinant for gene expression. We demonstrate that the poly(A) tail-mediated regulation of mRNA decapping observed in humans can be recapitulated in vitro by the cytoplasmic poly(A)-binding protein PABP through a direct and specific binding to the 5′ end of capped mRNA. The specific association of PABP with the cap occurred only within the context of the RNA whereby a cap attached to an RNA moiety served as the high-affinity substrate but not the c...

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

    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.

  16. Differential expression of in vivo and in vitro protein profile of outer membrane of Acidovorax avenae subsp. avenae.

    Muhammad Ibrahim

    Full Text Available Outer membrane (OM proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium.

  17. Physicochemical properties and in vitro starch digestibility of potato starch/protein blends.

    Lu, Zhan-Hui; Donner, Elizabeth; Yada, Rickey Y; Liu, Qiang

    2016-12-10

    This study aimed to investigate effects of starch-protein interactions on physicochemical properties and in vitro starch digestibility of composite potato starch/protein blends (0, 5, 10, or 15% protein) during processing (cooking, cooling and reheating). The effect on recrystallization and short-range ordering in starch was studied by light microscopy, differential scanning calorimetry and Fourier transform infrared spectroscopy. The results show that protein in the blend proportionally restricted starch granule swelling during cooking and facilitated amylopectin recrystallization during cold-storage. The facilitating effect of protein diminished with increasing blend ratio. Resistant starch content in the processed blends was positively correlated to intensity ratio of 1053/1035cm(-1) in FTIR spectra arising from slow retrogradation of amylopectin (r(2)>0.88, P≤0.05), whose formation was favored by the presence of protein in the blends and further enhanced by cooling of cooked blends. As a conclusion, starch-protein interaction reduced starch digestibility of the processed blends. PMID:27577912

  18. Gastrointestinal Endogenous Protein-Derived Bioactive Peptides: An in Vitro Study of Their Gut Modulatory Potential

    Dave, Lakshmi A.; Hayes, Maria; Mora, Leticia; Montoya, Carlos A.; Moughan, Paul J.; Rutherfurd, Shane M.

    2016-01-01

    A recently proposed paradigm suggests that, like their dietary counterparts, digestion of gastrointestinal endogenous proteins (GEP) may also produce bioactive peptides. With an aim to test this hypothesis, in vitro digests of four GEP namely; trypsin (TRYP), lysozyme (LYS), mucin (MUC), serum albumin (SA) and a dietary protein chicken albumin (CA) were screened for their angiotensin-I converting (ACE-I), renin, platelet-activating factor-acetylhydrolase (PAF-AH) and dipeptidyl peptidase-IV inhibitory (DPP-IV) and antioxidant potential following simulated in vitro gastrointestinal digestion. Further, the resultant small intestinal digests were enriched to obtain peptides between 3–10 kDa in size. All in vitro digests of the four GEP were found to inhibit ACE-I compared to the positive control captopril when assayed at a concentration of 1 mg/mL, while the LYS < 3-kDa permeate fraction inhibited renin by 40% (±1.79%). The LYS < 10-kDa fraction inhibited PAF-AH by 39% (±4.34%), and the SA < 3-kDa fraction inhibited DPP-IV by 45% (±1.24%). The MUC < 3-kDa fraction had an ABTS-inhibition antioxidant activity of 150 (±24.79) µM trolox equivalent and the LYS < 10-kDa fraction inhibited 2,2-Diphenyl-1-picrylhydrazyl (DPPH) by 54% (±1.62%). Moreover, over 190 peptide-sequences were identified from the bioactive GEP fractions. The findings of the present study indicate that GEP are a significant source of bioactive peptides which may influence gut function. PMID:27043546

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

    Regina Stoltenburg

    Full Text Available 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.

  20. Cooking temperature is a key determinant of in vitro meat protein digestion rate: investigation of underlying mechanisms.

    Bax, Marie-Laure; Aubry, Laurent; Ferreira, Claude; Daudin, Jean-Dominique; Gatellier, Philippe; Rémond, Didier; Santé-Lhoutellier, Véronique

    2012-03-14

    The present study aimed to evaluate the digestion rate and nutritional quality of pig muscle proteins in relation to different meat processes (aging, mincing, and cooking). Under our experimental conditions, aging and mincing had little impact on protein digestion. Heat treatments had different temperature-dependent effects on the meat protein digestion rate and degradation potential. At 70 °C, the proteins underwent denaturation that enhanced the speed of pepsin digestion by increasing enzyme accessibility to protein cleavage sites. Above 100 °C, oxidation-related protein aggregation slowed pepsin digestion but improved meat protein overall digestibility. The digestion parameters defined here open new insights on the dynamics governing the in vitro digestion of meat protein. However, the effect of cooking temperature on protein digestion observed in vitro needs to be confirmed in vivo. PMID:22335241

  1. Quantitative determination of in vitro immunoglobulin secretion with protein A from Staphylococcus aureus

    A micromethod for the quantitative determination of Ig secreted in vitro by mice lymphocytes isolated from the spleen of normal animals is described. The indicator system consists in sheep erythrocytes radiolabelled with sodium chromate (51Cr) and coated with protein A of Staphylococcus aureus (51Cr-labelled ES). When splenocytes were incubated in fluid phase at 370C for 7/2 h with rabbit antisera to mouse Ig (IgM and IgG) and with guinea pig complement, the immune complexes formed between the secreted Ig and its specific IgG antibody are bound to protein A on the erythrocyte surface allowing the complement-mediated lysis of 51Cr-labelled ES. The degree of haemolysis produced in this experimental system, which reflects the amount of in vitro secreted Ig, was quantitatively measured by radioactive determination of 51Cr release. In combination with the ES plaque assay the method also gives information as immunoglobulin secretion per plaque forming cell. (Auth.)

  2. Effects of Osseointegration by Bone Morphogenetic Protein-2 on Titanium Implants In Vitro and In Vivo.

    Teng, Fu-Yuan; Chen, Wen-Cheng; Wang, Yin-Lai; Hung, Chun-Cheng; Tseng, Chun-Chieh

    2016-01-01

    This study designed a biomimetic implant for reducing healing time and achieving early osseointegration to create an active surface. Bone morphogenetic protein-2 (BMP-2) is a strong regulator protein in osteogenic pathways. Due to hardly maintaining BMP-2 biological function and specificity, BMP-2 efficient delivery on implant surfaces is the main challenge for the clinic application. In this study, a novel method for synthesizing functionalized silane film for superior modification with BMP-2 on titanium surfaces is proposed. Three groups were compared with and without BMP-2 on modified titanium surfaces in vitro and in vivo: mechanical grinding; electrochemical modification through potentiostatic anodization (ECH); and sandblasting, alkali heating, and etching (SMART). Cell tests indicated that the ECH and SMART groups with BMP-2 markedly promoted D1 cell activity and differentiation compared with the groups without BMP-2. Moreover, the SMART group with a BMP-2 surface markedly promoted early alkaline phosphatase expression in the D1 cells compared with the other surface groups. Compared with these groups in vivo, SMART silaning with BMP-2 showed superior bone quality and created contact areas between implant and surrounding bones. The SMART group with BMP-2 could promote cell mineralization in vitro and osseointegration in vivo, indicating potential clinical use. PMID:26977141

  3. Effects of Osseointegration by Bone Morphogenetic Protein-2 on Titanium Implants In Vitro and In Vivo

    Fu-Yuan Teng

    2016-01-01

    Full Text Available This study designed a biomimetic implant for reducing healing time and achieving early osseointegration to create an active surface. Bone morphogenetic protein-2 (BMP-2 is a strong regulator protein in osteogenic pathways. Due to hardly maintaining BMP-2 biological function and specificity, BMP-2 efficient delivery on implant surfaces is the main challenge for the clinic application. In this study, a novel method for synthesizing functionalized silane film for superior modification with BMP-2 on titanium surfaces is proposed. Three groups were compared with and without BMP-2 on modified titanium surfaces in vitro and in vivo: mechanical grinding; electrochemical modification through potentiostatic anodization (ECH; and sandblasting, alkali heating, and etching (SMART. Cell tests indicated that the ECH and SMART groups with BMP-2 markedly promoted D1 cell activity and differentiation compared with the groups without BMP-2. Moreover, the SMART group with a BMP-2 surface markedly promoted early alkaline phosphatase expression in the D1 cells compared with the other surface groups. Compared with these groups in vivo, SMART silaning with BMP-2 showed superior bone quality and created contact areas between implant and surrounding bones. The SMART group with BMP-2 could promote cell mineralization in vitro and osseointegration in vivo, indicating potential clinical use.

  4. HBV X PROTEIN (HBX) INTERACTS WITH GENERAL TRANSCRIPTION FACTOR TFIIB BOTH IN VITRO AND IN VIVO

    1999-01-01

    Objective.In order to demonstrate the binding of HBV X protein (HBX) with the general transcription factor TFIIB.Methods.In vitro glutathion S-transferase (GST)resin Pull-Down assay and Far-Western Blotting assay, in vivo Co-immunoprecipition assay were used.Results.The X199(51-99)domain of HBX is reponsible for HBX binding to TFIIB.While the d10 domain (125-295)of TFIIB is required for TFIIB binding to HBX.When the two basic amino acids(K) at position 178 and 189 of TFIIB were substituted by neutral amino acids(L),the binding of TFIIBK178L and K189L to HBX was siginificantly reduced. When the the basic amino acids were substituted by the acidic amino acids(E),the binding of TFIIB K178E and K189E to HBX were almost lost.In vitro results of HBX binding to TFIIB were further confirmed by in vivo co-immunoprecipitation assay.Our results also indicated that the Woodchuck hepatitis virus X protein (WHX)interacts with TFIIB.Conclusion.These results suggested that the communication between HBX and general transcription factor TFIIB is one of the mechanisms which account for its transcriptional transactivation.

  5. In Vitro Cytotoxicity and Protein Drug Release Properties of Chitosan/Heparin Microspheres

    2007-01-01

    Chitosan/heparin microspheres were prepared using the water-in-oil emulsification solvent evaporation technique. The microsphere diameters were controlled by selecting the fabrication process parameters. Scanning electron micrographs showed that the chitosan/heparin microspheres were regular and the surface morphology was smooth. Fourier transform infrared showed that the chitosan amino groups reacted with heparin carboxylic groups to form acylamides in the microspheres. Analysis of the microsphere cytotoxicity showed that they had no cytotoxic effect and behaved very similar to the negative control (polystyrene).To analyze the protein drug release profiles of the microspheres, bovine serum albumin was loaded as a model drug into the microspheres and released in vitro. Marked retardation was observed in the BSA release profiles. The results show that chitosan/heparin microspheres may provide a useful controlled release protein drug system for used in pharmaceutics.

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

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

    2013-01-01

    -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 was......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...

  7. In vitro and in vivo assays of protein kinase CK2 activity.

    Prudent, Renaud; Sautel, Céline F; Moucadel, Virginie; Laudet, Béatrice; Filhol, Odile; Cochet, Claude

    2010-01-01

    Protein kinase CK2 (formerly casein kinase 2) is recognized as a central component in the control of the cellular homeostasis; however, much remains unknown regarding its regulation and its implication in cellular transformation and carcinogenesis. Moreover, study of CK2 function and regulation in a cellular context is complicated by the dynamic multisubunit architecture of this protein kinase. Although a number of robust techniques are available to assay CK2 activity in vitro, there is a demand for sensitive and specific assays to evaluate its activity in living cells. We hereby provide a detailed description of several assays for monitoring the CK2 activity and its subunit interaction in living cells. The guidelines presented herein should enable researchers in the field to establish strategies for cellular screenings of CK2 inhibitors. PMID:21050938

  8. In vitro estimation of rumen protein degradability using 35S to label the bacterial mass

    An experiment was carried out in order to simplify a previously developed 15N-method for in vitro estimation of rumen protein degradability. Casein (Cas), whole soybeans (Sb) heated at 120oC for 20 min (SbTherm) and sunflower (Sfl) were incubated at 39oC for 4 hours in a water bathshaker with the following media: McDougall's buffer, strained and enriched with particle associated bacteria rumen fluid (2:1), rapidly (maltose, sucrose, glucose) and more slowly (pectin, soluble starch) degradable carbohydrates with final concentration of 815 mg/100 ml and 21.7 μCi/100 ml of35S (from Na235SO4). After the incubation had been ceased, a bacterial fraction was isolated through differential centrifugation and specific activity of bacterial (Bac) and high speed total solids (TS) nitrogen was measured. The ratio was used to calculate bacterial mass in TS and through the Kjeldahl nitrogen concentration in TS - the net bacterial growth (against control vessels without protein). The level of ammonia-N in the supernate after blank correction was used to find the ammonia-N released from protein degradation. The data showed that the rate (and extend) of degradation for the Cas (as a standard protein) was lower compared to those obtained through the 15N-method but it was higher than the rate derived through another in vitro method. The Cas equivalent of the Sb was higher than the figure we found in a previous experiment with solvent extracted soybean meal suggesting that the 35S-method underestimated the degradability of the Cas. After being tested on a wider range of foodstuffs, the proposed 35S-method might be considered as an alternative procedure which is less laborous than the 15N-method. (author)

  9. Two proteins of the Dictyostelium spore coat bind to cellulose in vitro.

    Zhang, Y; Brown, R D; West, C M

    1998-07-28

    The spore coat of Dictyostelium contains nine different proteins and cellulose. Interactions between protein and cellulose were investigated using an in vitro binding assay. Proteins extracted from coats with urea and 2-mercaptoethanol could, after removal of urea by gel filtration, efficiently bind to particles of cellulose (Avicel), but not Sephadex or Sepharose. Two proteins, SP85 and SP35, were enriched in the reconstitution, and they retained their cellulose binding activities after purification by ion exchange chromatography under denaturing conditions to suppress protein--protein interactions. Neither protein exhibited cellulase activity, though under certain conditions SP85 copurified with a cellulase activity which appeared after germination. Amino acid sequencing indicated that SP85 and SP35 are encoded by the previously described pspB and psvA genes. This was confirmed for SP85 by showing that natural M(r) polymorphisms correlated with changes in the number of tetrapeptide-encoding sequence repeats in pspB. Using PCR to reconstruct missing elements from the recombinogenic middle region of pspB, SP85 was shown to consist of three sequence domains separated by two groups of the tetrapeptide repeats. Expression of partial pspB cDNAs in Escherichia coli showed that cellulose-binding activity resided in the Cys-rich COOH-terminal domain of SP85. This cellulose-binding activity can explain SP85's ultrastructural colocalization with cellulose in vivo. Amino acid composition and antibody binding data showed that SP35 is derived from the Cys-rich N-terminal region of the previously described psvA protein. SP85 and SP35 may link other proteins to cellulose during coat assembly and germination. PMID:9692967

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

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

    2016-01-01

    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 ...

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

    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

  12. Functional properties of the two redox-active sites in yeast protein disulphide isomerase in vitro and in vivo

    Westphal, V; Darby, N J; Winther, Jakob R.

    1999-01-01

    Protein folding catalysed by protein disulphide isomerase (PDI) has been studied both in vivo and in vitro using different assays. PDI contains a CGHC active site in each of its two catalytic domains (a and a'). The relative importance of each active site in PDI from Saccharomyces cerevisiae (y...

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

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

    2016-02-01

    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

  14. Intestinal microbes influence the survival, reproduction and protein profile of Trichinella spiralis in vitro.

    Jiang, Hai-yan; Zhao, Na; Zhang, Qiao-ling; Gao, Jiang-ming; Liu, Li-li; Wu, Teng-Fei; Wang, Ying; Huang, Qing-hua; Gou, Qiang; Chen, Wei; Gong, Peng-tao; Li, Jian-hua; Gao, Ying-jie; Liu, Bo; Zhang, Xi-chen

    2016-01-01

    The interactions between intestinal microbes and parasitic worms play an essential role in the development of the host immune system. However, the effects of gut microbes on Trichinella spiralis are unknown. The aim of this work was to explore microbe-induced alterations in the survival and reproduction of T. spiralis in vitro. To further identify the proteins and genes involved in the response of nematodes to microbes, quantitative proteomic analysis of T. spiralis was conducted by iTRAQ-coupled LCMS/MS technology and quantitative real-time-PCR was used to measure changes in mRNA expression. The results showed Lactobacillus acidophilus, and especially Lactobacillus bulgaricus, significantly enhanced the survival and reproductive rates of nematodes. Salmonella enterica, and especially Escherichia coli O157:H7 (EHEC), had opposite effects. Genetic responses were activated mainly by EHEC. A total of 514 proteins were identified and quantified, and carbohydrate metabolism-related proteins existed in a higher proportion. These findings indicated that some gut bacteria are friendly or harmful to humans and in addition they may have similar beneficial or detrimental effects on parasites. This may be due to the regulation of expression of specific genes and proteins. Our studies provide a basis for developing therapies against parasitic infections from knowledge generated by studying the gut microbes of mammals. PMID:26432293

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

    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.

  16. In Vitro Determination of Wheat Dry Matter Solubility and Protein Digestibility

    Rodica Căpriţă

    2012-10-01

    Full Text Available The objectives of the present study were to determine the in vitro dry matter (DM solubility and protein digestibility (PD of wheat grains. Two experiments were conducted. In experiment 1, samples were incubated for different time periods with pepsin, simulating gastric digestion, and in experiment 2, samples were digested following an in vitrotwo-step procedure, simulating gastric and small intestine digestion. DM solubility in gastric digestion showed an increase with the incubation time. DM solubility of wheat ranged in experiment 1 from 0.1532 g/g at 30 minutes digestion time, to 0.1714 g/g at 120 minutes digestion time. Samples showed higher DM solubility and PD after small intestine incubation than after gastric incubation. DM solubility increased with 16.67% and PD increased with 24.87% when gastric digestion was followed by 240 minutes intestinal digestion.

  17. Tp17 membrane protein of Treponema pallidum activates endothelial cells in vitro.

    Zhang, Rui-Li; Wang, Qian-Qiu; Zhang, Jing-Ping; Yang, Li-Jia

    2015-04-01

    Tp17, a membrane immunogen of Treponema pallidum subsp. pallidum, was initially recognized as an inflammatory mediator of syphilis. Because the histopathology of syphilis is characterized by endothelial cell abnormalities, we investigated the effects of recombinant Tp17 (rTp17) on endothelial cell activation in vitro. Using real-time reverse transcription-PCR and whole-cell ELISA, we found that rTp17 activated endothelial cells, as demonstrated by the up-regulated expression and increased gene transcription of intercellular adhesion molecule 1 (ICAM-1), E-selectin, and monocyte chemoattractant protein-1 (MCP-1). rTp17 also enhanced the migration and subsequent adhesion of monocytes to endothelial cells as well as increased transendothelial migration of monocytes. These data suggest that the ability of Tp17 to activate endothelial cells may play an important role in the immunopathogenesis of syphilis. PMID:25744604

  18. Determination of possible effects of mineral concentration on protein synthesis by rumen microbes in vitro

    The aim of the present investigation was to determine the effect of different concentrations of sulphide, magnesium and zinc on protein synthesis by rumen micro-organisms in vitro. Rumen content was taken from a young bull fed a diet based on maize and dried sugar beet pulp (2/1) supplemented with urea. The rate of incorporation of 35S from Na235SO4 in relation to the mean specific radioactivity of the sulphide pool was used to estimate the overall rate of microbial protein synthesis. It was found that the rate of protein synthesis and the net rate of utilization of ammonia-N were not affected by differences in mean sulphide concentration from 3.6-8.0 mg/litre. The rate of reduction of sulphate appeared not to be affected by the addition of sodium sulphide to the medium. The rate and efficiency of protein synthesis by rumen micro-organisms were not significantly affected by increasing the concentration of total magnesium from 8.4-15.3 mg/100 ml. The values for soluble magnesium varied widely (1.2-7.8 mg/100 ml), and appeared to be partly dependent on the pH of the medium. Zinc concentrations varying from 5.2-12.4 mg/litre did not influence the overall rate of protein synthesis, although the efficiency tended to be higher when the concentration of zinc was greater. Concentrations of soluble zinc were low (0.3-1.15 mg/litre), and not influenced by changes in the concentration of total zinc. It was concluded that increasing the concentrations of the examined elements above the basic values did not lead consistently to an improved production of microbial protein but, on the other hand, had no obvious detrimental effect on microbial metabolic activity within the limits studied. (author)

  19. In vitro study of protein release from AFCo1 and implications in mucosal immunisation

    Reinaldo Acevedo

    2012-04-01

    Full Text Available Adjuvant Finlay Cochleate 1 (AFCo1 is a Proteoliposome-derived cochleate obtained from Neisseria meningitidis serogroup B. Transformation of proteoliposomes into AFCo1 potentiates the immune response on Neisseria antigens when it is administered by intranasal or intragastric (i.g routes. However, the i.n route has been demonstrated to be more effective. The aim of this work is to evaluate in vitro the protein release from AFCo1, in simulated gastric fluid (SGF or simulated nasal fluid (SNF using a microdissolution test and to provide support for the results found when AFCo1 was administered by i.g or i.n routes in BALB/c mice. Results showed that dilution of AFCo1 in simulated gastric fluid affects the delivery of Neisseria protein antigens because they were released from cochleate structures faster than when simulated nasal fluid was used. In conclusion, conditions simulating gastric environment affect the delivery of protein antigens from AFCo1 and this result could partially explain why i.n administration is more effective in vivo than i.g immunisation.

  20. Proteins upregulated by mild and severe hypoxia in squamous cell carcinomas in vitro identified by proteomics

    Background: Solid malignant tumours are characterised by an inadequate vascular system, which can give rise to micro-regional hypoxic areas. As the negative impact of tumour hypoxia is believed largely to depend on dynamic changes in gene expression, it is important to identify the genes regulated by hypoxia to further enlighten the biology behind the cellular response to hypoxia. Previous studies have demonstrated that hypoxia has an impact not only on the gene transcription, but also on gene-specific mRNA translation. Therefore, proteomics is a suitable approach to understand the complexity of gene regulation under hypoxia at protein level. In this in vitro study we have studied the proteome of cells under intermediate hypoxia (1% O2) and anoxia and compared these to normoxic (21% O2) cells to identify proteins upregulated by mild and severe hypoxia. Materials and methods: A human cervix cancer cell line (SiHa) and a human head and neck cancer cell line (FaDuDD) were used. Total cell lysate from hypoxic and normoxic cells was separated by 2-dimensional gel electrophoresis, and images were analysed using Quantity One software. Proteins from significant spots (difference in intensity by more than a factor 2) were identified by Liquid chromatography-mass spectrometry (LC-MS/MS). In order to confirm the hypoxic regulation of the identified proteins, immunoblotting and qPCR were employed when possible. Results: All together 32 spots were found to be upregulated in the hypoxic gels. Of these, 11 different proteins were successfully identified and largely confirmed by Western blotting and qPCR. Amongst these proteins are protein disulfide isomerase family A, member 6 (PDIA6) and dynein light chain roadblock-type 1 (DynLRB1). Both 2D gels and Western blots revealed that PDAI6 exhibited a cell line specific pattern; in FaDuDD there was upregulation at 1% and further upregulated at 0% compared to atmospheric air, whereas there was no upregulation in SiHa cells. DynLRB1 was

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

    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 600MPa) treatment of squids (Todarodes pacificus). The soluble protein nitrogen content and in vitro protein digestibility increased significantly (psquids 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 3.60-3.79ppm. 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. PMID:26948613

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

    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 45CaCl2 (5 microCi/ml) for 24 hr at various stages of a ten-day culture period or [3H]-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 45Ca 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. [3H]-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

  3. An in vitro reconstitution system for the assessment of chromatin protein fluidity during Xenopus development

    Research highlights: → An in vitro reconstitution system was established with isolated nuclei and cytoplasm. → Chromatin fluidities were measured in the system using FRAP. → Chromatin fluidities were higher in the cytoplasm of earlier-stage embryos. → Chromatin fluidities were higher in the earlier-stage nuclei with egg-extract. → Chromatin fluidity may decrease during embryonic development. -- Abstract: Chromatin fluidity, which is one of the indicators of higher-order structures in chromatin, is associated with cell differentiation. However, little is known about the relationships between chromatin fluidity and cell differentiation status in embryonic development. We established an in vitro reconstitution system that uses isolated nuclei and cytoplasmic extracts of Xenopus embryos and a fluorescence recovery after photobleaching assay to measure the fluidities of heterochromatin protein 1 (HP1) and histone H1 during development. The HP1 and H1 fluidities of nuclei isolated from the tailbuds of early tadpole stage (stage 32) embryos in the cytoplasmic extracts of eggs and of late blastula stage (stage 9) embryos were higher than those in the cytoplasmic extracts of mid-neurula stage (stage 15) embryos. The HP1 fluidities of nuclei isolated from animal cap cells of early gastrula stage (stage 10) embryos and from the neural plates of neural stage (stage 20) embryos were higher than those isolated from the tailbuds of stage 32 embryos in egg extracts, whereas the HP1 fluidities of these nuclei were the same in the cytoplasmic extracts of stage 15 embryos. These results suggest that chromatin fluidity is dependent upon both cytoplasmic and nuclear factors and decreases during development.

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

    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.

  5. An in vitro reconstitution system for the assessment of chromatin protein fluidity during Xenopus development

    Aoki, Ryuta; Inui, Masafumi; Hayashi, Yohei; Sedohara, Ayako [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 (Japan); Okabayashi, Koji [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 (Japan); ICORP Organ Regeneration Project, Japan Science and Technology Agency (JST), 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 (Japan); Ohnuma, Kiyoshi, E-mail: kohnuma@vos.nagaokaut.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 (Japan); Murata, Masayuki [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 (Japan); Asashima, Makoto, E-mail: asashi@bio.c.u-tokyo.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 (Japan); ICORP Organ Regeneration Project, Japan Science and Technology Agency (JST), 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 (Japan); Organ Development Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan)

    2010-09-17

    Research highlights: {yields} An in vitro reconstitution system was established with isolated nuclei and cytoplasm. {yields} Chromatin fluidities were measured in the system using FRAP. {yields} Chromatin fluidities were higher in the cytoplasm of earlier-stage embryos. {yields} Chromatin fluidities were higher in the earlier-stage nuclei with egg-extract. {yields} Chromatin fluidity may decrease during embryonic development. -- Abstract: Chromatin fluidity, which is one of the indicators of higher-order structures in chromatin, is associated with cell differentiation. However, little is known about the relationships between chromatin fluidity and cell differentiation status in embryonic development. We established an in vitro reconstitution system that uses isolated nuclei and cytoplasmic extracts of Xenopus embryos and a fluorescence recovery after photobleaching assay to measure the fluidities of heterochromatin protein 1 (HP1) and histone H1 during development. The HP1 and H1 fluidities of nuclei isolated from the tailbuds of early tadpole stage (stage 32) embryos in the cytoplasmic extracts of eggs and of late blastula stage (stage 9) embryos were higher than those in the cytoplasmic extracts of mid-neurula stage (stage 15) embryos. The HP1 fluidities of nuclei isolated from animal cap cells of early gastrula stage (stage 10) embryos and from the neural plates of neural stage (stage 20) embryos were higher than those isolated from the tailbuds of stage 32 embryos in egg extracts, whereas the HP1 fluidities of these nuclei were the same in the cytoplasmic extracts of stage 15 embryos. These results suggest that chromatin fluidity is dependent upon both cytoplasmic and nuclear factors and decreases during development.

  6. In vivo and in vitro phosphorylation and subcellular localization of trypanosomatid cytoskeletal giant proteins.

    Baqui, M M; Milder, R; Mortara, R A; Pudles, J

    2000-09-01

    Promastigote forms of Phytomonas serpens, Leptomonas samueli, and Leishmania tarentolae express cytoskeletal giant proteins with apparent molecular masses of 3,500 kDa (Ps 3500), 2,500 kDa (Ls 2500), and 1,200 kDa (Lt 1200), respectively. Polyclonal antibodies to Lt 1200 and to Ps 3500 specifically recognize similar polypeptides of the same genera of parasite. In addition to reacting with giant polypeptides of the Leptomonas species, anti-Ls 2500 also cross reacts with Ps 3500, and with a 500-kDa polypeptide of Leishmania. Confocal immunofluorescence and immunogold electron microscopy showed major differences in topological distribution of these three proteins, though they partially share a common localization at the anterior end of the cell body skeleton. Furthermore, Ps 3500, Ls 2500, and Lt 1200 are in vivo phosphorylated at serine and threonine residues, whereas, in vitro phosphorylation of cytoskeletal fractions reveal that only Ps 3500 and Ls 2500 are phosphorylated. Heat treatment (100 degrees C) of high salt cytoskeletal extracts demonstrates that Ps 3500 and Ls 2500 remain stable in solution, whereas Lt 1200 is denatured. Kinase assays with immunocomplexes of heat-treated giant proteins show that only Ps 3500 and Ls 2500 are phosphorylated. These results demonstrate the existence of a novel class of megadalton phosphoproteins in promastigote forms of trypanosomatids that appear to be genera specific with distinct cytoskeletal functions. In addition, there is also evidence that Ps 3500 and Ls 2500, in contrast to Lt 1200, seem to be autophosphorylating serine and threonine protein kinases, suggesting that they might play regulatory roles in the cytoskeletal organization. PMID:11002308

  7. A protein-based hydrogel for in vitro expansion of mesenchymal stem cells.

    Jingyu Wang

    Full Text Available Hydrogels are widely used as scaffolds in tissue engineering because they can provide excellent environments for bioactive components including growth factors and cells. We reported in this study on a physical hydrogel formed by a specific protein-peptide interaction, which could be used for the three dimensional (3D cell culture of murine mesenchymal stem cells (mMSC. The mMSC kept dividing during the 7-day culture period and the metabolic-active cell number at day 7 was 359% more than that at day 1. This kind of physical hydrogel could be converted to a homogeneous solution by firstly adding an equal volume of culture medium and then pipeting for several times. Therefore, mMSC post culture could be easily separated from cell-gel constructs. We believed that the protein-based hydrogel system in this study could be developed into a promising scaffold for in vitro expansion of stem cells and cell therapy. This work would be in the general interests of researchers in the fields of biomaterials and supramolecular chemistry.

  8. Effect of fluoride on protein and collagen biosynthesis in rabbit dental pulp in vitro

    The time course for incorporation of 14C-proline into various fractions of rabbit dental pulp in vitro has been measured. In the TCA-soluble precursor pool a steady state level of activity was indicated upon incubation after 3 h, whereas incorporation into protein and 14C-hydroxyproline, i.e. collagen formation, increased linearly for 9 h, leveling off upon further incubation. A lag period of about 3 h was indicated for the appearance of high molecular weight 14C-activity, including 14C-hydroxyproline, in the medium, increasing linearly from 3 h to the end of the incubation period (22 h). In this system, fluoride exhibited a dose-dependent inhibitory effect. At 5.3 mM fluoride the uptake of 14C-proline into the TCA-soluble pool was inhibited by about 50 percent, and the incorporation into protein and the subsequent conversion to hydroxyproline by about 90 and 60 percent, respectively. Release of collagen, i.e. 14C-hydroxyproline containing material, seemed to be the process most sensitive to fluoride; it was inhibited by about 50 percent at the lowest concentration (1.3 mM) tested. (author)

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

    Anthony Antonellis

    Full Text Available BACKGROUND: 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. METHODOLOGY: 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. PRINCIPAL FINDINGS: 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. CONCLUSIONS: 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.

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

    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.

  11. Axonal transport of labelled proteins and increased functional activity in sciatic nerve of the frog Rana hexadactyla in vitro

    In vitro speed of fast moving labelled protein fraction was investigated in the lumbar 8 nerve of R.hexadactyla during normal and electrical stimulation conditions. 3H-leucine labelled oroteins moved in a proximo-distal direction at a speed of 144 mm/day at 25 deg C. No change was observed in the rate of proteins transported in stimulated nerves but the amount of protein bound radioactivity increased over stimulation. In ligature experiments, amount of labelled proteins accumulating at a ligature was higher in stimulated nerves. Electrical stimulation of nerve resulted in an increase in protein synthetic rate in the respective ganglion. (author)

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

    Yang, Yong; Wang, Zhongjiang; Wang, Rui; Sui, Xiaonan; Qi, Baokun; Han, Feifei; Li, Yang; Jiang, Lianzhou

    2016-01-01

    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. PMID:27298825

  13. In vitro availability of iron and zinc: Effects of the type, concentration and fractions of digestion products of the protein

    Pérez-Llamas, F.; Diepenmaat-Wolters, M.G.E.; Zamora, S.

    1996-01-01

    An in vitro dialysis method was employed to determine the effect on the Fe and Zn absorption of the type (beef, pork and soyabean) and the amount (10 and 30 g/kg) of protein present. In addition, the effects of low- and high-molecular-weight (LMW and HMW respectively) digestion products were investi

  14. Energy and protein relations in the broiler chicken. 4. Role of sex, line and substrate on in vitro lipogenesis

    Experiments were conducted with dwarf (dw) and normal lines of chickens to determine the effect of sex, diet and line on lipogenesis in the 28-day-old chick. The chicks were fed diets containing 12, 18, 23 and 30% protein. In the first experiment, in vitro lipogenesis (incorporation of [2-14C] sodium acetate into hepatic fatty acids) as well as growth from 7 to 28 days of age were determined in males and females of both lines. In the second experiment, only males and females of the dwarf line were fed to determine the relative contribution of acetate and pyruvate to in vitro lipogenesis (incorporation of either [2-14C] sodium acetate or [2-14C) pyruvate into hepatic fatty acids). Chicks of the dwarf line were smaller than were those of the normal line. Females of both lines were smaller than males. In vitro lipogenesis was lower in the dwarf line; however the rate for both sexes within a given line was equal. An increase in the dietary protein decreased in vitro lipogenesis in both lines. The use of pyruvate as an in vitro precursor indicated that the regulation of lipid and carbohydrate metabolism may be an integrated process involving pyruvate carboxylation and subsequent flux of pyruvate carbon into either glucose or fatty acids. Based on the data presented, there is no evidence to assume, that the dwarf gene per se influences lipogenesis

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

    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.

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

    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 ( 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.

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

    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

  18. Comparison of protein patterns after two-dimensional gel electrophoresis from leaves of in vitro cultures and seedlings of Rubus chamaemorus L.

    Barbara Thiem

    2014-02-01

    Full Text Available Proteins from leaves of Rubus chamaemorus propagated in vitro were subjected to miniaturized 2-D electrophoresis. The 2-DE patterns of proteins showed qualitative differences between plants propagated in vitro and control seedlings. More proteins of a high molecular weight were observed in leaves of plants from in vitro culture. A two-dimensional map of proteins from leaves provides detailed data concerning both polymorphism and protein patterns of this species. This makes it possible to start constructing a protein map of R. chamaemorus. The reasons for qualitative differences are discussed.

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

    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.

  20. AMP-activated protein kinase (AMPK) activation regulates in vitro bone formation and bone mass.

    Shah, M; Kola, B; Bataveljic, A; Arnett, T R; Viollet, B; Saxon, L; Korbonits, M; Chenu, C

    2010-08-01

    Adenosine 5'-monophosphate-activated protein kinase (AMPK), a regulator of energy homeostasis, has a central role in mediating the appetite-modulating and metabolic effects of many hormones and antidiabetic drugs metformin and glitazones. The objective of this study was to determine if AMPK can be activated in osteoblasts by known AMPK modulators and if AMPK activity is involved in osteoblast function in vitro and regulation of bone mass in vivo. ROS 17/2.8 rat osteoblast-like cells were cultured in the presence of AMPK activators (AICAR and metformin), AMPK inhibitor (compound C), the gastric peptide hormone ghrelin and the beta-adrenergic blocker propranolol. AMPK activity was measured in cell lysates by a functional kinase assay and AMPK protein phosphorylation was studied by Western Blotting using an antibody recognizing AMPK Thr-172 residue. We demonstrated that treatment of ROS 17/2.8 cells with AICAR and metformin stimulates Thr-172 phosphorylation of AMPK and dose-dependently increases its activity. In contrast, treatment of ROS 17/2.8 cells with compound C inhibited AMPK phosphorylation. Ghrelin and propranolol dose-dependently increased AMPK phosphorylation and activity. Cell proliferation and alkaline phosphatase activity were not affected by metformin treatment while AICAR significantly inhibited ROS 17/2.8 cell proliferation and alkaline phosphatase activity at high concentrations. To study the effect of AMPK activation on bone formation in vitro, primary osteoblasts obtained from rat calvaria were cultured for 14-17days in the presence of AICAR, metformin and compound C. Formation of 'trabecular-shaped' bone nodules was evaluated following alizarin red staining. We demonstrated that both AICAR and metformin dose-dependently increase trabecular bone nodule formation, while compound C inhibits bone formation. When primary osteoblasts were co-treated with AICAR and compound C, compound C suppressed the stimulatory effect of AICAR on bone nodule formation

  1. Effect of radiation processing on in vitro protein digestibility and availability of calcium, phosphorus and iron of peanut

    Hassan, Amro B.; Diab, Eiman E.; Mahmoud, Nagat S.; Elagib, Randa A. A.; Rushdi, Mohamed A. H.; Osman, Gammaa A. M.

    2013-10-01

    The effect of gamma irradiation of two peanut cultivars (Sodari and Madani) on protein content, in vitro protein digestibility and availability of calcium, phosphorus and iron was determined. Seeds were treated with gamma irradiation at dose levels of 1.0, 1.5 and 2.0 kGy. Total protein in seeds was not changed significantly by irradiation. However, the in vitro protein digestibility was decreased for both cultivars. In addition, the irradiation also caused an increment on the available calcium, phosphorus and iron for both cultivars. Moreover, radiation processing caused an increment on tannin content of the seeds especially at the dose 2 kGy for both cultivars. Regarding these results, irradiation treatment of peanut up to 2 kGy can be used as an effective alternative method to chemical treatments for insect disinfestation and microbial disinfection.

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

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

    2016-01-01

    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 ...

  3. Patatin, the tuber storage protein of potato (Solanum tuberosum L.), exhibits antioxidant activity in vitro.

    Liu, Yen-Wenn; Han, Chuan-Hsiao; Lee, Mei-Hsien; Hsu, Feng-Lin; Hou, Wen-Chi

    2003-07-16

    The potato (Solanum tuberosum L.) tuber storage protein, patatin, was purified to homogeneity with a molecular mass of 45 kDa. The purified patatin showed antioxidant or antiradical activity by a series of in vitro tests, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (half-inhibition concentration, IC(50), was 0.582 mg/mL) scavenging activity assays, anti-human low-density lipoprotein peroxidation tests, and protections against hydroxyl radical-mediated DNA damages and peroxynitrite-mediated dihydrorhodamine 123 oxidations. Using electron paramagnetic resonance (EPR) spectrometry for hydroxyl radical detections, it was found that the intensities of the EPR signal were decreased by the increased amounts of patatin added (IC(50) was 0.775 mg/mL). Through modifications of patatin by iodoacetamide or N-bromosuccinimide, it was found that the antiradical activities of modified patatin against DPPH or hydroxyl radicals were decreased. It was suggested that cysteine and tryptophan residues in patatin might contribute to its antioxidant activities against radicals. PMID:12848515

  4. NOVEL SNAKE VENOM PROTEINS CYTOLYTIC TO CANCER CELLS IN VITRO AND IN VIVO SYSTEMS

    B. V. LIPPS

    1999-01-01

    Full Text Available Cancer cell inhibitors, named Atroporin and Kaotree, having molecular weights of 35 kDa and 6 kDa have been isolated from the venoms of Crotalus atrox and Naja naja kaouthia, respectively, by fractionation on high pressure liquid chromatography. The purified Atroporin and Kaotree showed killing effects on various types of human (breast, colon, liver, ovary, etc. and animal cancer cells in concentrations as low as 0.5µg/ml, and having no effect on normal mouse kidney, liver, spleen, and erythrocytes up to 5.0µg/ml. Both Atroporin and Kaotree prevent the formation of ascitic tumors caused by myeloma cells in Balb/C mice. In addition, both Atroporin and Kaotree showed regression of ascitic tumors formed by myeloma cells. Atroporin and Kaotree complement each other, as in combination they showed elevated anti-cancer activity in vitro and in vivo systems. However, Atroporin and Kaotree are immunologically distinct proteins showing no cross reactivity. Atroporin and Kaotree, individually or in combination, have the potential for cancer biotherapy.

  5. Estramustine-binding protein (EMBP) in renal cell carcinoma immunohistochemistry, immunoscintigraphy and in vitro estramustine effects

    The present report shows that the human renal cell carcinoma (RCC) cell lines, A498 and CAKI-2, express the estramustine-binding protein (EMBP). The RCC cell lines investigated were highly sensitive for estramustine, with cell arrest in atypical metaphase. In vitro experiments using a fluorimetric cytotoxicity assay (FMCA) showed a pronounced cytotoxic effect mediate by estramustine. Immunohistochemical analysis of tumoru specimens from patients with RCC showed positive staining for EMBP in 12/16 cases. Immunoscintigraphy was performed in an experimental system in nude mice, heterotransplanted with the CAKI-2 cell line. A radiolabelled monoclonal anti-EMBP antibody was used. The results show a specific uptake of the antibody in the RCC tumour, expressed as a percentage of the injected dose per gram tissue, which ranged from 4.03 to 6.9. The results obtained from the basis for clinical studies on the feasibility of utilizing estramustine in the management of RCC. Immunoscintigraphy using the monoclonal anti-EMBP antibody is of potential use for in vivo characterization of the malignancy and in the selection patients suitable for treatment with estramustine. (orig.)

  6. Intense pulsed light induces synthesis of dermal extracellular proteins in vitro.

    Cuerda-Galindo, E; Díaz-Gil, G; Palomar-Gallego, M A; Linares-GarcíaValdecasas, R

    2015-09-01

    Intense pulsed light (IPL) devices have been shown to be highly effective for the skin rejuvenation. In our study, we try to elucidate effects of IPL in fibroblast proliferation, in gene expression, and in extracellular matrix protein production. 1BR3G human skin fibroblasts were used to test the effects of an IPL device (MiniSilk FT, Deka®). Fibroblasts were divided into three groups: group 1 was irradiated with filter 800-1200 nm (frequency 10 Hz, 15 s, fluence 60.1 J/cm) twice; group 2 was irradiated with filter 550-1200 nm (double pulse 5 ms + 5 ms, delay 10 ms, fluence 13 J/cm2) twice; and group 3 was irradiated with filter 550-1200 nm (frequency 10 Hz, 15 s, fluence 60.1 J/cm2) twice. To determine changes in gene expression, messenger RNA (mRNA) levels for collagen types I and III and metalloproteinase 1 (MMP-1) were performed 48 h after irradiation. To determine changes in hyaluronic acid, versican, and decorin, mRNA and ELISA tests were performed after 48 h of treatment. In addition to this, a Picro-Sirius red staining for collagen was made. The study showed an increase of mRNA and hyaluronic acid, decorin, and versican production. With RT-PCR assays, an increase mRNA for collagen type I, type III, and MMP-1 was observed. Collagen and hyaluronic synthesis was increased in all groups with no differences among them, while decorin and versican synthesis was higher in those groups irradiated with 550-1200-nm filters with no dependence of type pulse or total energy dose. IPL applied in vitro cultured cells increases fibroblasts activity. Synthesis of extracellular proteins seems to be produced more specifically in determined wavelengths, which could demonstrate a biochemical mechanism light depending. PMID:26188855

  7. Inhibition of hepatitis B virus replication by pokeweed antiviral protein in vitro

    Yong-Wen He; Chun-Xia Guo; Yan-Feng Pan; Cheng Peng; Zhi-Hong Weng

    2008-01-01

    AIM:To explore the inhibitory effects of pokeweed antiviral protein seed(PAP-S)and PAP encoded by a eukaryotic expression plasmid on hepatitis B virus(HBV)replication in vitro.METHODS:HepG2 2.2.15 cells in cultured medium were treated with different concentrations of PAP-S.HBsAg,HBeAg and HBV DNA in supernatants were determined by ELISA and fluorescent quantitative PCR respectively.MTT method was used to assay for cytotoxicity.HepG2 were cotransfected with various amounts of PAP encoded by a eukaryotic expression plasmid and replication competent wild-type HBV 1.3 fold overlength plasmid.On d 3 after transfection,HBsAg and HBeAg were determined by using ELISA.Levels of HBV core-associated DNA and RNA were detected by using Southern and Northern blot,respectively.RESULTS:The inhibitory effects of PAP-S on HBsAg,HBeAg and HBV DNA were gradually enhanced with the increase of PAP concentration.When the concentration of PAP-S was 10 μg/mL,the inhibition rates of HBsAg,HBeAg and HBV DNA were 20.9%,30.2% and 50%,respectively.After transfection of 1.0μg and 2.0μg plasmid pXF3H-PAP,the levels of HBV nucleocapsideassociated DNA were reduced by 38.0% and 74.0% respectively,the levels of HBsAg in the media by 76.8% and 99.7% respectively,and the levels of HBeAg by 72.7% and 99.3% respectively as compared with controls.Transfection with 2μg plasmid pXF3H-PAP reduced the levels of HBV nucleocapside-associated RNA by 69.0%.CONCLUSION:Both PAP-S and PAP encoded by a eukaryotic expression plasmid could effectively inhibit HBV replication and antigen expression in vitro,and the inhibitory effects were dose-dependent.

  8. Qushi Huayu Decoction Inhibits Hepatic Lipid Accumulation by Activating AMP-Activated Protein Kinase In Vivo and In Vitro

    Qin Feng

    2013-01-01

    Full Text Available Qushi Huayu Decoction (QHD, a Chinese herbal formula, has been proven effective on alleviating nonalcoholic fatty liver disease (NAFLD in human and rats. The present study was conducted to investigate whether QHD could inhibit hepatic lipid accumulation by activating AMP-activated protein kinase (AMPK in vivo and in vitro. Nonalcoholic fatty liver (NAFL model was duplicated with high-fat diet in rats and with free fatty acid (FFA in L02 cells. In in vivo experimental condition, QHD significantly decreased the accumulation of fatty droplets in livers, lowered low-density lipoprotein cholesterol (LDL-c, alanine aminotransferase (ALT, and aspartate aminotransferase (AST levels in serum. Moreover, QHD supplementation reversed the HFD-induced decrease in the phosphorylation levels of AMPK and acetyl-CoA carboxylase (ACC and decreased hepatic nuclear protein expression of sterol regulatory element-binding protein-1 (SREBP-1 and carbohydrate-responsive element-binding protein (ChREBP in the liver. In in vitro, QHD-containing serum decreased the cellular TG content and alleviated the accumulation of fatty droplets in L02 cells. QHD supplementation reversed the FFA-induced decrease in the phosphorylation levels of AMPK and ACC and decreased the hepatic nuclear protein expression of SREBP-1 and ChREBP. Overall results suggest that QHD has significant effect on inhibiting hepatic lipid accumulation via AMPK pathway in vivo and in vitro.

  9. Influence of some biologically active substances on amount of MGMT and MARP proteins in human cells in vitro

    Kotsarenko K. V.; Lylo V. V.; Macewicz L. L.; Ruban T. P.; Luchakivska Yu. S.; Kuchuk M. V.; Lukash L. L.

    2014-01-01

    Aim. To investigate an effect of biologically active compounds IFN-α2b, EMAPII, Card medium, fibronectin on the amount of MGMT (O6-methylguanine-DNA methyltransferase) and MARP (anti-Methyltransferase Antibody Recognizable Protein) proteins in human cells in vitro. Methods. The human cells of 4BL, Hep-2 and A102 lines were treated with growth factors and cytokines. Changes in the amount of MGMT and MARP proteins were studied by Western blot analysis with anti-MGMT mAbs. Results. The treatment...

  10. Identification of a novel phosphorylation site in c-jun directly targeted in vitro by protein kinase D

    Protein kinase D (PKD) phosphorylates the c-jun amino-terminal in vitro at site(s) distinct from JNK [C. Hurd, R.T. Waldron, E. Rozengurt, Protein kinase D complexes with c-jun N-terminal kinase via activation loop phosphorylation and phosphorylates the c-jun N-terminus, Oncogene 21 (2002) 2154-2160], but the sites have not been identified. Here, metabolic 32P-labeling of c-jun protein in COS-7 cells indicated that PKD phosphorylates c-jun in vivo at a site(s) between aa 43-93, a region containing important functional elements. On this basis, the PKD-mediated phosphorylation site(s) was further characterized in vitro using GST-c-jun fusion proteins. PKD did not incorporate phosphate into Ser63 and Ser73, the JNK sites in GST-c-jun(1-89). Rather, PKD and JNK could sequentially phosphorylate distinct site(s) simultaneously. By mass spectrometry of tryptic phosphopeptides, Ser58 interposed between the JNK-binding portion of the delta domain and the adjacent TAD1 was identified as a prominent site phosphorylated in vitro by PKD. These data were further supported by kinase reactions using truncations or point-mutations of GST-c-jun. Together, these data suggest that PKD-mediated phosphorylation modulates c-jun at the level of its N-terminal functional domains

  11. The effects of cutting or of stretching skeletal muscle in vitro on the rates of protein synthesis and degradation

    Seider, M. J.; Kapp, R.; Chen, C.-P.; Booth, F. W.

    1980-01-01

    Skeletal muscle preparations using cut muscle fibers have often been used in studies of protein metabolism. The present paper reports an investigation of the effect of muscle cutting or stretching in vitro on the rates of protein synthesis and/or degradation. Protein synthesis and content, and ATP and phosphocreatine levels were monitored in soleus and extensor digitorum longus muscles from the rat with various extents of muscle fiber cuts and following stretching to about 120% the resting length. Rates of protein synthesis are found to be significantly lower and protein degradation higher in the cut muscles than in uncut controls, while ATP and phosphocreatine concentrations decreased. Stretched intact muscles, on the other hand, are observed to have higher concentrations of high-energy phosphates than unstretched muscles, while rates of protein degradation were not affected. Results thus demonstrate that the cutting of skeletal muscle fibers alters many aspects of muscle metabolism, and that moderate decreases in ATP concentration do not alter rates of protein concentration in intact muscles in vitro.

  12. [New regulatory protein isolated from the bovine eye lens and its action on the cataract development in rat in vitro].

    Krasnov, M S; Gurmizov, E P; Iamskova, V P; Gundorova, R A; Iamskov, I A

    2005-01-01

    The regulatory protein was isolated from the eye lens extract by using an early designed scheme including by means of salting-out of proteins by ammonium sulphate, isoelectrofocusing in pH gradient and electrophoresis in PAAG. A high-purity fraction of the regulatory protein was obtained. The localization of the regulatory protein in the rat-eye lens was investigated by means of primary rabbit antibodies obtained within the case study and by FITS-marked secondary antibodies. Cataractogenesis was induced, in vitro, in Wistar rat lenses through adding, to the cultivation medium, hydrogen peroxide (0.5 mM) or calcium chloride (15 mM). The regulatory protein isolated from the bovine eye lens was added alongside with damaging antibodies to the nutrition medium, concentration 10(-12) mg/ml. The lenses were cultivated for as long as 8 days at 37 degrees C. The degree of opacification of lenses was evaluated visually with the help of a lined substrate as well as by spectrophotometry. The studied protein was shown immunohistochemically to be localized in the intercellular space of the lens epithelium in the region of the basic membrane. The cataractogenesis-related research of the regulatory protein was made on rabbit eye lenses, which were cultivated as a whole for as long as 8 days in vitro. Their transparency and morphology were preserved in them in full since they were cultivated in a serum-free nutrition without admixture of any destructive agents. Opacification of lenses was induced in vitro by changing the concentration of calcium ions in the cultivation medium or through adding hydrogen peroxide to the medium. The valuations of the lens opacity degree as observed in different research series and made by visual observation well correlate with the results of spectrophotometry of lenses made after their cultivation. It can be stated that the studied regulatory protein, when added to the cultivation medium, enhances about two-fold the lens transparency versus the lenses

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

    Giugliano Loreny; Lima Renato de; Willer Emerson

    2004-01-01

    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 eff...

  14. In vitro conditions for 14C-leucine incorporation into the protein of cultured ovaries of the silkworm, Bombyx mori

    Vitellogenic ovaries of silkworm pupae were incubated in vitro in different media based on the Wyatt's medium to establish an adequate condition for culture of silkworm ovaries. Incorporation of 14C-leucine into protein fraction was determined to assess the biochemical activity of the ovary. When ovaries were incubated in vitro for a short time by 6 hr, a saturation kinetics of incorporation of the labelled leucine was shown. Sequential substitution of K+ ion to Na+ ion in the medium had no effect on the incorporation of 14C-leucine, but Mg2+ ion appeared to stimulate synthetic activity at more than 10 mM. The activity was not affected at pH range 5.0-7.2. Neither different sugars, nor vitellogenin nor lipoprotein prepared from silkworm haemolymph affected the incorporation of 14C-leucine, when added into the medium. The synthesis of protein depended upon the developmental stages of the cultured ovaries and was most active in 6-day-old ovary. Ovaries developing in pupal body showed comparable changes in synthetic activity. It is concluded that the chemical composition of the medium does not exert a strict effect on synthetic activity of protein in short-term cultures and the ovaries cultured in vitro maintain the activity comparable with those found in in situ condition. (author)

  15. Spermicidal action of a protein isolated from ethanolic root extracts of Achyranthes aspera: an in vitro study.

    Anuja, M M; Nithya, R S; Swathy, S S; Rajamanickam, C; Indira, M

    2011-06-15

    A previous study conducted in our department, showed that 50% ethanolic extract of the roots of Achyranthes aspera possess spermatotoxic effects. Preliminary studies also revealed that the active principle may be a protein. In this study a 58 kDa Achyranthes protein (Ap) was isolated from Achyranthes aspera using standard protocols and their effects on the rat sperm was studied in vitro in comparison with nonoxynol-9 (N-9). The sperm immobilization studies showed that about 150 μg of Ap was able to immobilize sperms completely within seconds at a lower concentration than N-9 (250 μg). The sperm revival test revealed that the spermicidal effect was irreversible. There was also a significant reduction in sperm viability and hypo-osmotic swelling in the Ap-treated and N-9 treated groups in comparison to the control. In the Ap and N-9 treated groups the number of acrosome reacted cells were found to be high and it also caused agglutination of the sperms indicating the loss of intactness of the plasma membrane which was further supported by the significant reduction in the activity of membrane bound 5' nucleotidase and acrosin enzyme. Hence this study showed that the protein isolated from the roots of Achyranthes aspera possess spermicidal activity in vitro and can act as a spermicide similar to that of nonoxynol 9. Ap also possessed spermicidal activity against human sperms in vitro. PMID:21306884

  16. Ascorbate and Apple Phenolics Affect Protein Oxidation in Emulsion-Type Sausages during Storage and in Vitro Digestion.

    Rysman, Tine; Van Hecke, Thomas; De Smet, Stefaan; Van Royen, Geert

    2016-05-25

    The effect of sodium ascorbate and apple phenolics on the oxidative stability of emulsion-type sausages during storage and digestion was investigated. Emulsion-type sausages containing 0.05% sodium ascorbate or 3% freeze-dried apple pomace were subjected to chilled illuminated storage and subsequent in vitro digestion. Lipid oxidation was assessed as TBARS, and protein oxidation was evaluated as thiol oxidation, total carbonyls, and γ-glutamic and α-amino adipic semialdehyde. Proteolysis was measured after digestion to evaluate protein digestibility. The results suggest the presence of protein-ascorbate and protein-phenol interactions, which may decrease protein digestibility and may interfere with spectrophotometric methods for measuring oxidation. PMID:27133801

  17. nusB: a protein factor necessary for transcription antitermination in vitro by phage lambda N gene product.

    Ghosh, B.; Das, A.

    1984-01-01

    We demonstrate that the protein product of the Escherichia coli nusB gene is essential for transcription antitermination in vitro by phage lambda N gene product. We recently have described a convenient biochemical assay for N protein activity in the S30-coupled transcription translation system and demonstrated that N action requires the 69-kDa L factor (nusA), the product of E. coli nusA gene. Using a complementation assay for the restoration of N activity specifically in the nusB mutant extr...

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

    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.

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

    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

  20. In vitro serum protein-binding characteristics of bis-(2-ethylhexyl) phthalate and its principal metabolite, mono-(2-ethylhexyl) phthalate

    The metabolism and toxicity of the ubiquitous plasticizer, bis-(2-ethylhexyl) phthalate (DEHP), and its principal metabolite, mono-(2-ethylhexyl) phthalate (MEHP), have been extensively investigated. In an attempt to understand their disposition in man, the authors studied the in vitro serum protein-binding characteristics of these compounds, using ultracentrifugation and agarose gel electrophoresis. The association of DEHP and lipoproteins was shown to be highly dependent upon, and proportional to, the lipid concentration of the serum. It appears that more than half of the serum DEHP is bound to proteins with density greater than 1.21 g/mL when the concentration of cholesterol is below 300 mg/dL or the cholesterol and triglyceride total concentration is less than 600 mg/dL. As the cholesterol and triglyceride concentrations increase, the percent DEHP bound to VLDL, IDL, and LDL increases. MEHP is bound principally to nonlipoprotein constituents in the serum, and this binding distribution is unaffected by lipid concentration. The percent binding of DEHP and MEHP to individual proteins was also found to be unaffected by their concentrations in serum. These data indicate that the protein-binding characteristics of these compounds, in vitro, is somewhat more complex than previously reported

  1. Effect of Retinoic Acid on Apoptosis and Expression of Fas Proteins in Mouse Blastocysts Cultured In Vitro

    Yan'e XIONG; Duanlian ZHANG

    2008-01-01

    Mouse blastocysts were exposed to doses of 0,1 and 10μmol/L retinoic acid (RA) for 24h and the cytotoxic effect of RA on the mouse blastocysts in vitro was observed. FITC-labeled terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL-FITC) assay was employed to stain apoptotic cells and immunohistochemical S-P staining method was used to detect the expression of Fas protein in mouse blastocysts in vitro. The results showed that RA could induce apoptosis and increase the expression of Fas proteins of trophectoderm (TE) and inner cell mass (ICM) cells in blastocysts. Compared with the findings for the control blastocysts, exposure to RA (10μmol/L) resulted in a more significant apoptosis and higher expression level of Fas proteins (P<0.01). It was concluded that RA could induce apoptosis, which may result in a significant reduction in the average number of total cells and the trophectoderm/inner cell mass in blastocysts and an increased expression of Fas protein, suggesting that RA had a cytotoxic effect on the growth and development of early embryos in mice.

  2. In vitro studies on non-protein nitrogen utilization by rumen microflora. II. Validity of phosphorus-32 in estimating microbial protein synthesis in vitro

    To check the accuracy of the 32P method for estimating microbial growth, the results obtained in two laboratories (Jouy and Ghent) with protein-free substrates are statistically analysed. Linear relationship between phosphorus incorporation (PR) and every other variable is first studied. The best relationship is observed with NH3 utilization (NH)(r=0.94 for the pooled data). Two multivariate analyses - principal components and multiple regression - are applied. The important role of phosphorus incorporation (PR) in characterizing microbial activity intensity is emphasized. PR is the first selected variable for prediction of NH; the multiple regression equations are given. Net NH3 utilization is predicted using these equations with substrates containing proteins likely to be degraded. This method is thought to be more accurate than referring to the N/P ratio in microflora. It is noted that use of these equations should be restricted to the specific experiments described in this report; drastic changes in incubation conditions could alter the ratio total growth/net growth, invalidating the use of the equations. (author)

  3. Potensi Protein Reseptor Fertilisasi Zona Pelusida Kambing Sebagai Kandidat Imunokontrasepsi dengan Fertilisasi in vitro pada Sapi (POTENTIAL OF FERTILIZATION RECEPTOR PROTEIN GOAT ZONA PELLUCIDA AS CANDIDATE OF IMMUNOCONTRACEPTION BY USING IN VITRO FER

    Sri Mulyati

    2013-12-01

    Full Text Available Studies on the role of zona pellucida glycoprotein-3 (ZP3 in immunocontraception have been conductedin many species including goats (gZP3. Our previous study indicated that gZP3 effective in preventingpregnancy in mice. The aim of this study was to prove the evidence of cross reaction in gZP3 to preventfertilization in cow in vitro. Antibody of gZP3 was produced in mice. Immunized mice serum was analyzedusing ELISA technique and dot blot method. Sperm from frozen semen were processed then incubated incapacitation media supplemented with the gZP3, whilst the cow oocyte was incubated in maturationmedia supplemented with antibody of gZP3. Following this, the normal  in vitro fertilization (withoutincubation neither in gZP3 nor in gZP3 antibody was performed, respectively. The results showed thatantibody titer of immunized mice serum was higher (p<0.05 than the control group. Dot blotting analysisshowed that the antibody of immunized mice were able to recognize gZP3 protein. The serum of immunizedmice which was supplemented in the maturation media of cow oocyte were able to decrease the cleavagerate (p<0.05. Protein gZP3 which was supplemented in the capacitation media of the sperm also coulddecrease the cleavage rate (p<0.05. It is concluded that goat-ZP3 protein may produce cross reaction incow.

  4. Hormonal regulation of liver fatty acid-binding protein in vivo and in vitro: effects of growth hormone and insulin.

    Carlsson, L; Nilsson, I; Oscarsson, J

    1998-06-01

    Liver fatty acid-binding protein (LFABP) is an abundant protein in hepatocytes that binds most of the long chain fatty acids present in the cytosol. It is suggested to be of importance for fatty acid uptake and utilization in the hepatocyte. In the present study, the effects of bovine GH (bGH) and other hormones on the expression of LFABP and its messenger RNA (mRNA) were studied in hypophysectomized rats and in vitro using primary cultures of rat hepatocytes. One injection of bGH increased LFABP mRNA levels about 5-fold after 6 h, but there was no effect of this treatment on LFABP levels. However, 7 days of bGH treatment increased both LFABP mRNA and LFABP protein levels 2- to 5-fold. Female rats had higher levels of LFABP than male rats. Hypophysectomy of female rats, but not that of male rats, decreased LFABP levels markedly. Treatment of hypophysectomized rats with bGH for 7 days as two daily injections or as a continuous infusion increased LFABP levels to a similar degree. This finding indicates that the sex difference in the expression of LFABP is not regulated by the sexually dimorphic secretory pattern of GH. Neither insulin nor insulin-like growth factor I treatment of hypophysectomized rats for 6-7 days had any effect on LFABP mRNA or LFABP levels. In vitro, bGH dose-dependently increased the expression of LFABP mRNA, but only in the presence of insulin. Insulin alone had a marked dose-dependent effect on LFABP mRNA levels and was of importance for maintaining the expression of LFABP mRNA during the culture. Incubation with bGH increased LFABP mRNA levels within 3 h. GH had no effect on LFABP mRNA levels in the presence of actinomycin D, indicating a transcriptional effect of GH. Incubation with glucagon in vitro decreased LFABP mRNA levels markedly, indicating that glucagon, in contrast to GH, has an effect opposite that of insulin on LFABP mRNA expression. It is concluded that GH is an important regulator of LFABP in vivo and in vitro. In contrast to

  5. Lessons from in vitro reconstitution analyses of plant microtubule-associated proteins

    Hamada, Takahiro

    2014-01-01

    Plant microtubules, composed of tubulin GTPase, are irreplaceable cellular components that regulate the directions of cell expansion and cell division, chromosome segregation and cell plate formation. To accomplish these functions, plant cells organize microtubule structures by regulating microtubule dynamics. Each microtubule localizes to the proper position with repeated growth and shortening. Although it is possible to reconstitute microtubule dynamics with pure tubulin solution in vitro, ...

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

    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 ...

  7. Influence of some biologically active substances on amount of MGMT and MARP proteins in human cells in vitro

    Kotsarenko K. V.

    2014-05-01

    Full Text Available Aim. To investigate an effect of biologically active compounds IFN-α2b, EMAPII, Card medium, fibronectin on the amount of MGMT (O6-methylguanine-DNA methyltransferase and MARP (anti-Methyltransferase Antibody Recognizable Protein proteins in human cells in vitro. Methods. The human cells of 4BL, Hep-2 and A102 lines were treated with growth factors and cytokines. Changes in the amount of MGMT and MARP proteins were studied by Western blot analysis with anti-MGMT mAbs. Results. The treatment of A102 cells with EMAPII, fibronectin, Laferon and Card medium led to a decreased level of the MGMT protein, whereas the amount of MARP was highly increased in these cells. The treatment with the recombinant protein IFN-α2b increased the amount of MGMT and MARP proteins in Hep-2 cells. The treatment with extracts of transgenic plants,containing human IFN-α2b, caused a significant decrease in the content of both proteins in Hep-2 cells and MARP in 4BL cells. Conclusions. Both MGMT and MARP are highly inducible proteins. Their amount in cells can be changed by some growth factors (Card medium, fibronectin, cytokine (IFN-α2b, cytokine-like (EMAPII or cytokine-containing substances (Laferon and IFN-α2b in plant extracts. This regulation depended not only on the type of biologically active substances but on the cell line used in this study as well.

  8. In vitro assembly into virus-like particles is an intrinsic quality of Pichia pastoris derived HCV core protein

    Different variants of hepatitis C virus core protein (HCcAg) have proved to self-assemble in vitro into virus-like particles (VLPs). However, difficulties in obtaining purified mature HCcAg have limited these studies. In this study, a high degree of monomeric HCcAg purification was accomplished using chromatographic procedures under denaturing conditions. Size exclusion chromatography and sucrose density gradient centrifugation of renatured HCcAg (in the absence of structured RNA) under reducing conditions suggested that it assembled into empty capsids. The electron microscopy analysis of renatured HCcAg showed the presence of spherical VLPs with irregular shapes and an average diameter of 35 nm. Data indicated that HCcAg monomers assembled in vitro into VLPs in the absence of structured RNA, suggesting that recombinant HCcAg used in this work contains all the information necessary for the assembly process. However, they also suggest that some cellular factors might be required for the proper in vitro assembly of capsids

  9. Dose-dependent differential effect of hemin on protein synthesis and cell proliferation in Leishmania donovani promastigotes cultured in vitro

    Jayanta K Pal; Manisha Joshi-Purandare

    2001-06-01

    Leishmania donovani requires an exogenous source of heme for growth and transformation. In in vitro culture of the free-living promastigotes, exogenously added hemin enhances cell proliferation. In this investigation, the question of the function of heme with particular reference to protein synthesis and cell proliferation has been addressed. The results of in vitro cell culture experiments demonstrated that hemin (10 M) alone is suitable for supporting optimum level of protein synthesis, and thereby cell proliferation of promastigotes to an extent that it can replace fetal bovine serum. However, in situ labelling experiments along with Western blots revealed that high concentration of hemin (50 M) reduced the level of protein synthesis in general and of -tubulin in particular with a concomitant induction of hsp90, and induced consequent morphological changes that are observed during in situ transformation of promastigotes in mammalian macrophages. These results therefore suggest that sudden exposure to high concentration of heme in mammalian macrophages may be one of the key factors that trigger promastigote to amastigote transformation in L. donovani. Furthermore, hemin with its dual characteristic could be used as a tool to understand molecular mechanism of cell proliferation and transformation in these parasites.

  10. Recombinant human bone morphogenetic protein-7 expressed from CHO cells possessing the activity of bone-induced in vitro

    LI Xiaoyan; WANG Hao; YANG Yang; TAN Min; XUE Jingya; NI Haidong; GUO Yajun

    2006-01-01

    Objective To express the recombinant human bone morphogenetic protein-7 (rhBMP-7) in Chinese hamster ovary (CHO) cells and to establish the in vitro biological activity assay of rhBMP-7. Methods Human BMP-7 cDNA was subcloned into pcDNA3.1 mammalian expression vector and transfected to CHO cells by using the lipofectin transfection method. BMP-7 expression cell culture supernatants were harvested and purified for target protein. To analyze the bioactivity of the secreted rhBMP-7, a novel in vitro assay was established by measuring its alkaline phosphatase (ALP) stimulating of osteoblast cell line, W-20-17. Results BMP-7 stably expressing cell clone was selected, which secreted mature disulfide-linked homodimer form of hBMP-7 and had an apparent molecular weight of 36kDa. rhBMP-7 with >95% purity was obtained using 3 step chromatography method. Bioactivity assay showed that the purified protein specifically stimulated W-20-17 cell producing ALP, with a 4-fold increase of ALP activity at 100ng/ml or more, and the EC50 of 15.6ng/ml. Conclusion Purified rhBMP-7 from this CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates potential bone regeneration activity.

  11. Functional and anti-nutritional properties, in-vitro protein digestibility and amino acid composition of dehulled afzelia africana seeds

    Analysis of Afzelia africana seed flour showed that the seeds possessed high water absorption capacity (128.31%), good oil absorption capacity (588.49%) and fairly good emulsion property (35.25%). However, it had the Least gelation concentration (6 .00% w/v) and foaming properties ( 8.00%,3 .00%). Anti-nutritional factors were very low, with the highest being phytate (13.59/o) and tannin the least (0.43%). Total amino acid composition was 796.6 mg/g protein. Essentiaal amino acids (48.5%)w ere in high proportion with in-vitro digestibility of 71.5%. (author)

  12. A cell-free approach to accelerate the study of protein–protein interactions in vitro

    Sierecki, E.; Giles, N.; Polinkovsky, M.; Moustaqil, M.; Alexandrov, K.; Gambin, Y.

    2013-01-01

    Protein–protein interactions are highly desirable targets in drug discovery, yet only a fraction of drugs act as binding inhibitors. Here, we review the different technologies used to find and validate protein–protein interactions. We then discuss how the novel combination of cell-free protein expression, AlphaScreen and single-molecule fluorescence spectroscopy can be used to rapidly map protein interaction networks, determine the architecture of protein complexes, and find new targets for d...

  13. Degenerate in vitro genetic selection reveals mutations that diminish alfalfa mosaic virus RNA replication without affecting coat protein binding.

    Rocheleau, Gail; Petrillo, Jessica; Guogas, Laura; Gehrke, Lee

    2004-08-01

    The alfalfa mosaic virus (AMV) RNAs are infectious only in the presence of the viral coat protein; however, the mechanisms describing coat protein's role during replication are disputed. We reasoned that mechanistic details might be revealed by identifying RNA mutations in the 3'-terminal coat protein binding domain that increased or decreased RNA replication without affecting coat protein binding. Degenerate (doped) in vitro genetic selection, based on a pool of randomized 39-mers, was used to select 30 variant RNAs that bound coat protein with high affinity. AUGC sequences that are conserved among AMV and ilarvirus RNAs were among the invariant nucleotides in the selected RNAs. Five representative clones were analyzed in functional assays, revealing diminished viral RNA expression resulting from apparent defects in replication and/or translation. These data identify a set of mutations, including G-U wobble pairs and nucleotide mismatches in the 5' hairpin, which affect viral RNA functions without significant impact on coat protein binding. Because the mutations associated with diminished function were scattered over the 3'-terminal nucleotides, we considered the possibility that RNA conformational changes rather than disruption of a precise motif might limit activity. Native polyacrylamide gel electrophoresis experiments showed that the 3' RNA conformation was indeed altered by nucleotide substitutions. One interpretation of the data is that coat protein binding to the AUGC sequences determines the orientation of the 3' hairpins relative to one another, while local structural features within these hairpins are also critical determinants of functional activity. PMID:15254175

  14. Effects of processing moisture on the physical properties and in vitro digestibility of starch and protein in extruded brown rice and pinto bean composite flours.

    Sumargo, Franklin; Gulati, Paridhi; Weier, Steven A; Clarke, Jennifer; Rose, Devin J

    2016-11-15

    The influence of pinto bean flour and processing moisture on the physical properties and in vitro digestibility of rice-bean extrudates has been investigated. Brown rice: pinto bean flour (0%, 15%, 30%, and 45% bean flour) were extruded under 5 moisture conditions (17.2%, 18.1%, 18.3%, 19.5%, and 20.1%). Physical properties [bulk density, unit density, radial expansion, axial expansion, overall expansion, specific volume, hardness, color, water solubility index, and water absorption index] and in vitro starch and protein digestibilities were determined. Increasing bean flour and processing moisture increased density and hardness while decreasing expansion. Rapidly digestible starch decreased and resistant starch increased as bean substitution and processing moisture increased. In vitro protein digestibility increased with increasing bean flour or with decreasing processing moisture. Incorporating bean flour into extruded snacks can negatively affect physical attributes (hardness, density, and expansion) while positively affecting in vitro starch (decrease) and protein (increase) digestibilities. PMID:27283689

  15. Effects of Industrial Heating Processes of Milk-Based Enteral Formulas on Site-Specific Protein Modifications and Their Relationship to in Vitro and in Vivo Protein Digestibility.

    Wada, Yasuaki; Lönnerdal, Bo

    2015-08-01

    Heat treatments are applied to milk and dairy products to ensure their microbiological safety and shelf lives. Types of heating processes may have different effects on protein modifications, leading to different protein digestibility. In this study, milk-based liquid nutritional formulas (simulating enteral formulas) were subjected to steam injection ultra-high-temperature treatment or in-can sterilization, and the formulas were investigated by proteomic methods and in vitro and in vivo digestion assays. Proteomic analyses revealed that in-can sterilization resulted in higher signals for N(ε)-carboxymethyllysine and dephosphorylation of Ser residues in major milk proteins than in steam-injected formula, reflecting the more severe thermal process of in-can sterilization. In vitro and in vivo digestion assays indicated that steam injection improved protein digestibility, supposedly by denaturation, while the improvement seemed to be overwhelmed by formation of aggregates that showed resistance to digestion in in-can sterilized formula. Adverse effects of heat treatment on protein digestibility are more likely to be manifested in milk-based formulas than in cow's milk. Although the differences might be of limited significance in terms of amino acid bioavailability, these results emphasize the importance of protein quality of raw materials and selection of heating processes. PMID:26161498

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

    Tatiane Almeida Drummond Tetzner; Naiara Zoccal Saraiva; Felipe Perecin; Simone Cristina Méo Niciura; Christina Ramires Ferreira; Clara Slade Oliveira; Joaquim Mansano Garcia

    2011-01-01

    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...

  17. Gamma radiation effects on the proteins of 'in vitro' bovine lenses

    The radiosensitivity of the ocular lens manifested by cataract formation has been of considerable interest in the study on the biological effects of radiations. Cataract can ben produced by different causes and also for the normal process of ageing. The aim of this work was to develop an in vitro system similar to in vivo cataract formation. It was used an aqueous soluton of bovine lenses. The lenses after surgical removal mechanical and ultrasonic disrupted. The suspension was centrifuged and the supernatant was dialyzed and irradiated with different doses of 60Co radiation. The opacification extent was measured in an spectrophotometer. (author)

  18. The chromosomal protein HMGBI inhibits DNA replication in vitro. The role of post-synthetic acetylation

    The effect of HMGB1 protein on the replication of closed circular plasmid DNA in cell free extract have been studied using parental form of the protein, post-synthetically acetylated HMGB1 and HMGB1 lacking its acidic C-terminal tail. We have shown that HMGB1 protein inhibits DNA replication and that this effect is eliminated upon either acetylation of the protein or removal of the acidic C-terminal domain. An explanation of these findings suggests interactions of HMGB1 with a protein(s) of the replication complex resulting in reduction of its functional efficiency. Acetylation of HMGB1 affects these interactions in a way that restores the initial replication capacity of the system. The eventual protein-protein interactions are supposed to proceed via the C-terminal domain of HMGB1. (authors)

  19. In vitro association of mitochondrial ATP-dependent protease with mitochondrial heat-shock proteins

    Specific antibodies against the mitochondrial ATP-dependent protease and heat-shock proteins were used to study the association of these proteins with an abnormal bacterial protein, CRAG. It was shown that the mitochondrial ATP-dependent protease from rat liver and Zajdela hepatoma bind to the CRAG protein and that this binding was mediated through the heat-shock proteins. It was also demonstrated that the protease associated with heat-shock proteins is capable of degrading large proteins as well as small peptides in an ATP-dependent fashion. Zajdela hepatoma mitochondria, with enhanced mitochondrial proteolysis, were shown to contain more ATP-dependent protease associated with heat-shock proteins. (author)

  20. Phospholipid Binding Protein C Inhibitor (PCI Is Present on Microparticles Generated In Vitro and In Vivo.

    Katrin Einfinger

    Full Text Available Protein C inhibitor is a secreted, non-specific serine protease inhibitor with broad protease reactivity. It binds glycosaminoglycans and anionic phospholipids, which can modulate its activity. Anionic phospholipids, such as phosphatidylserine are normally localized to the inner leaflet of the plasma membrane, but are exposed on activated and apoptotic cells and on plasma membrane-derived microparticles. In this report we show by flow cytometry that microparticles derived from cultured cells and activated platelets incorporated protein C inhibitor during membrane blebbing. Moreover, protein C inhibitor is present in/on microparticles circulating in normal human plasma as judged from Western blots, ELISAs, flow cytometry, and mass spectrometry. These plasma microparticles are mainly derived from megakaryocytes. They seem to be saturated with protein C inhibitor, since they do not bind added fluorescence-labeled protein C inhibitor. Heparin partially removed microparticle-bound protein C inhibitor, supporting our assumption that protein C inhibitor is bound via phospholipids. To assess the biological role of microparticle-bound protein C inhibitor we performed protease inhibition assays and co-precipitated putative binding partners on microparticles with anti-protein C inhibitor IgG. As judged from amidolytic assays microparticle-bound protein C inhibitor did not inhibit activated protein C or thrombin, nor did microparticles modulate the activity of exogenous protein C inhibitor. Among the proteins co-precipitating with protein C inhibitor, complement factors, especially complement factor 3, were most striking. Taken together, our data do not support a major role of microparticle-associated protein C inhibitor in coagulation, but rather suggest an interaction with proteins of the complement system present on these phospholipid vesicles.

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

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

    1991-01-01

    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 > 9.3. These new RIP's were shown to be different from dodecandrin with respect to molecular mass....

  2. Apple phenolics as inhibitors of the carbonylation pathway during in vitro metal-catalyzed oxidation of myofibrillar proteins.

    Rysman, Tine; Utrera, Mariana; Morcuende, David; Van Royen, Geert; Van Weyenberg, Stephanie; De Smet, Stefaan; Estévez, Mario

    2016-11-15

    The effect of apple phenolics on the oxidative damage caused to myofibrillar proteins by an in vitro metal-catalyzed oxidation system was investigated. Three pure phenolic compounds (chlorogenic acid, (-)-epicatechin and phloridzin) and an apple peel extract were added to myofibrillar proteins in three concentrations (50, 100 and 200μM), and a blank treatment was included as a control. All suspensions were subjected to Fe(3+)/H2O2 oxidation at 37°C during 10days, and protein oxidation was evaluated as carbonylation (α-amino adipic and γ-glutamic semialdehydes) and Schiff base cross-links. Significant inhibition by apple phenolics was found as compared to the control treatment, with (-)-epicatechin being the most efficient antioxidant and phloridzin showing the weakest antioxidant effect. The higher concentrations of apple extract showed effective antioxidant activity against protein oxidation in myofibrillar proteins, emphasizing the potential of apple by-products as natural inhibitors of protein oxidation in meat products. PMID:27283697

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

    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

  4. Rat sperm immobilisation effects of a protein from Ricinus communis (Linn.): an in vitro comparative study with nonoxynol-9.

    Nithya, R S; Anuja, M M; Rajamanickam, C; Indira, M

    2012-12-01

    Previous study conducted in our department showed that 50% ethanolic extract of the root of Ricinus communis possess reversible antifertility effect and a 62-kDa protein (Rp) from this extract is responsible for the antifertility effects. In this study, we compared the spermicidal effect of this Rp with nonoxynol-9 (N-9) in vitro. The sperm immobilisation studies showed that 100 μg ml(-1) of Rp was able to immobilise the sperms completely within 30 s. Sperm revival test revealed that the spermicidal effect was irreversible. There was also a significant reduction in sperm viability and hypo-osmotic swelling in Rp and N-9 treated groups in comparison with the control. In Rp and N-9 treated groups, the number of acrosome-reacted cells was found to be high and also caused agglutination of the spermatozoa, indicating the loss of intactness of the plasma membrane, which was further supported by the significant reduction in the activity of membrane bound 5'-nucleotidase, acrosomal acrosin. In short, the protein Rp possesses spermicidal activity in vitro and its effects are similar to that of nonoxynol 9. PMID:22486240

  5. The estimation of ruminal protein degradation parameters of various feeds using in vitro modified gas production technique.

    Falahatizow, J; Danesh Mesgaran, M; Vakili, A R; Tahmasbi, A M; Nazari, M R

    2015-01-01

    This study was conducted to determine in vitro crude protein degradation (IVDP) parameters and effective crude protein degradability (EPD) of various feeds using the modified in vitro gas production (GP) technique. Feed samples were alfalfa hay, soybean meal, soybean, rapeseed meal, sunflower meal and fish meal. Rumen fluid was collected before the morning feeding from four rumen fistulated lambs (49.4 ± 3.5 kg, body weight). Approximately 90 ml of buffered rumen fluid (BRF), 400 mg of feed samples and carbohydrates (maltose, xylose and starch) at four concentrations (100, 200, 300, and 400 mg) were added to screw-cap bottles. Gas production (ml) and ammonia nitrogen concentration (mg) in each bottle were measured at 4, 8, 12, 16, 24, and 30 h post incubation and IVDP was calculated via estimated intercept of linear regression between GP (as main variable, X) and ammonia nitrogen (as dependent variable, Y) using the linear regression procedure. Feed, time and feed × time interaction had significant effect on IVDP (P<0.001). Estimated EPD values at the outflow rate of 0.06/h for alfalfa hay, soybean meal, soybean, rapeseed meal, sunflower meal and fish meal were 0.56, 0.77, 0.59, 0.45, 0.50 and 0.38, respectively. PMID:27175150

  6. Effect of salseed-meal tannins on protein synthesis, 35S incorporation and cellulose digestibility by rumen microbes in vitro

    Tannins from seed-meal of sal (Shorea robusta Gaertn. F.) were fractionated after treatments into ethyl acetate (EA) and lead acetate (LA) fractions. In trial 1, incorporation of 35S from (NH4)235SO4 into microbial protein declined due to the effect of both 2% EA and LA fractions as compared to control. Microbial protein synthesis was depressed significantly (P 35S incorporation and cellulolysis at all levels of both the tannin fractions. It may be inferred that both the tannin fractions from salseed-meal showed antimicrobial activity and LA fraction seems to be more deleterious than EA fraction for rumen metabolism. The experiments were conducted in vitro using rumen liquor of a non-pregnant dry cow having permanent rumen fistula. (auth.)

  7. In vitro observation of the stage conversion of transgenic Toxoplasma gondii RH strain expressing dual fluorescent proteins.

    Song, Qijun; Sun, Ximeng; Ji, Yongsheng; Yan, Xinlei; Zou, Jun; Zhao, Shiyun; Suo, Xun; Zhu, Xingquan; Liu, Xianyong

    2016-09-01

    Toxoplasma gondii converts from tachyzoites to bradyzoites after acute infection and thus survives the attack of the host immune responses. In this study, we observed the conversion of tachyzoites to bradyzoites in cell cultures using a transgenic T. gondii RH strain. The transgenic parasites continuously express yellow fluorescent protein (YFP) but only express red fluorescent protein (RFP) at the bradyzoite stage. Red fluorescent bradyzoite-containing cysts were found in transgenic parasite infected cells cultured with atmospheric CO2 supply, indicating the successful induction of the stage conversion. In cell culture with alkalic medium (pH 8.1) and atmospheric CO2 supply, only part of the YFP-expressing parasites in a cyst express RFP marker, suggesting the asynchronous development of T. gondii in vitro. This study provides a possibility for further studies of the gene expression profile during stage conversion and the genes involved. PMID:27447207

  8. Estimated Environmental Exposures for MISSE-7B

    Finckenor, Miria M.; Moore, Chip; Norwood, Joseph K.; Henrie, Ben; DeGroh, Kim

    2012-01-01

    This paper details the 18-month environmental exposure for Materials International Space Station Experiment 7B (MISSE-7B) ram and wake sides. This includes atomic oxygen, ultraviolet radiation, particulate radiation, thermal cycling, meteoroid/space debris impacts, and observed contamination. Atomic oxygen fluence was determined by measured mass and thickness loss of polymers of known reactivity. Diodes sensitive to ultraviolet light actively measured solar radiation incident on the experiment. Comparisons to earlier MISSE flights are discussed.

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

    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

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

    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.

  11. Effects of methamidophos and deltamethrin on in vitro protein phosphorylation in Monochamus alternatus

    Jie Liu; Xi-Wu Gao; Yi-Jun Wu; Wei Li; Qi-Lian Qin; Jiang-Hua Sun

    2008-01-01

    Monochamus alternatus Hope(Coleoptera:Cerambycidae)is not onlY a serious Dest insect to pine trees but alSO the main vector of pine wood nemadote Bursaphelenchus xylophilus,which causes pine wilt disease.To explore the insecticidal mechanism of insecticides to M alternatus,we chose methamidophos and deltamethrin as the representa-tives of two groups of insecticides(organophosphates and pyrethroids),which arc widely used for pest controlin China and investigated their effects on phosphorylation of proteins from the insect.Phosphorylation of proteins from the insect fat body and head was determined by fn vitro 32P-labelling.In the fat body,deltamethtin obviously reduced basal phosphorylation levels of proteins at 111,95,77,and 44 kDa,but enhanced the basal phosphorylation level of a protein at 138 kDa.However,in the presence of calmodulin but not cyclic adenosine monophosphate(cAMP),deitamethrin increased phosphorylation of the protein at 111 kDa.In the head,deltamethrin inhibited basal phosphorylation levels of proteins at 113,98,and 51 kDa,but potentiated phosphorylation of a protein at 167 kDa activated by cAMP.Methamidophos inhibited phosphorylation of a protein at 44 kDa in the fat body.Although methamidophos did not impact basal phosphorylation levels of any proteins in the head,it inhibiled calcium/calmodulin(Ca2+/CAM) stimulated phosphoryla-tion of a protein at 5 1 kDa.Together.our dam indicate that methamidophos and deltamethrin altered phosphorylation levels of various proteins in thc head and fat body of the pine insect and these two kinds of inseeticides acted on the proteins that call be phosphorylated in the tissues respectively,Which is possibly related to their toxicity.

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

    Hansen, Christian; Tarabykina, Svetlana; la Cour, Jonas Marstrand; Lollike, Karsten; Berchtold, Martin W

    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 r...... be a way to regulate and fine tune processes mediated by calcium binding proteins of the penta-EF hand type....

  13. Synthesis, protein kinase inhibitory potencies, and in vitro antiproliferative activities of meridianin derivatives.

    Giraud, Francis; Alves, Georges; Debiton, Eric; Nauton, Lionel; Théry, Vincent; Durieu, Emilie; Ferandin, Yoan; Lozach, Olivier; Meijer, Laurent; Anizon, Fabrice; Pereira, Elisabeth; Moreau, Pascale

    2011-07-14

    The synthesis of new meridianin derivatives is described. The indolic ring system was substituted at the C-4 to C-7 positions either by a bromine atom or by nitro or amino groups. Additionally, an iodine atom or various aryl groups were introduced at the C-5 position of the 2-aminopyrimidine ring. These compounds as well as some of their synthetic intermediates were tested for their kinase inhibitory potencies and for their in vitro antiproliferative activities. We found that this series of compounds is particularly interesting in the development of new inhibitors of DYRK1A and CLK1 kinases. The most effective compounds toward these two kinase families are the 6- and 7-bromo derivatives 30, 33, and 34 that showed more than 45-fold selectivity toward DYRK1A/CLK1 kinases over the other kinases tested. Meridianin derivatives could thus be developed toward potent and selective inhibitors of key RNA splicing regulators and potential therapeutic agents. PMID:21623630

  14. Purification and in vitro activities of the native nitrogen fixation control proteins NifA and NifL.

    Austin, S; Buck, M; Cannon, W; Eydmann, T; Dixon, R

    1994-06-01

    The prokaryotic enhancer-binding protein NifA stimulates transcription at a distance by binding to sequences upstream of nitrogen fixation (nif) promoters and catalyzing the formation of open promoter complexes by RNA polymerase containing the alternative sigma factor, sigma 54. The activity of NifA in vivo is modulated by the negative regulatory protein NifL in response to environmental oxygen and fixed nitrogen. To date, a detailed biochemical analysis of these proteins from the model diazotroph Klebsiella pneumoniae has been hindered by their insolubility. We have now purified NifA and NifL from Azotobacter vinelandii in their native form. NifA is competent in specific DNA binding, transcriptional activation, and response to negative regulation by NifL in vitro. In contrast to the conserved mechanism of phosphotransfer demonstrated by other two-component regulatory systems, our results support a model in which NifL regulates the activity of NifA via a protein-protein steric block interaction rather than a catalytic modification of NifA. PMID:8206822

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

    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.

  16. Regulation and function of the CD3¿ DxxxLL motif: a binding site for adaptor protein-1 and adaptor protein-2 in vitro

    Dietrich, J; Kastrup, J; Nielsen, B L;

    1997-01-01

    /CD3gamma chimeras; and in vitro by binding CD3gamma peptides to clathrin-coated vesicle adaptor proteins (APs). We find that the CD3gamma D127xxxLL131/132 sequence represents one united motif for binding of both AP-1 and AP-2, and that this motif functions as an active sorting motif in monomeric CD4...... and for AP binding in vitro. Furthermore, we provide evidence indicating that phosphorylation of CD3gamma S126 in the context of the complete TCR induces a conformational change that exposes the DxxxLL sequence for AP binding. Exposure of the DxxxLL motif causes an increase in the TCR internalization...

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

    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.

  18. Procalcitonin behaves as a fast responding acute phase protein in vivo and in vitro

    Nijsten, MWN; Olinga, P; The, TH; de Vries, EGE; Groothuis, GMM; Limburg, PC; ten Duis, HJ; Moshage, H; Hoekstra, HJ; Bijzet, J; Zwaveling, JH; Schraffordt Koops, H.

    2000-01-01

    Objectives: Procalcitonin (PCT) is a 13 kD protein of which plasma concentrations are strongly increased in inflammatory states, PCT concentrations are claimed to have a more powerful discriminatory value for bacterial infection than the acute phase proteins serum amyloid A (SAA) or C-reactive prote

  19. Selective binding of proteins on functional nanoparticles via reverse charge parity model: an in vitro study

    Ghosh, Goutam; Panicker, Lata; Barick, K. C.

    2014-03-01

    The conformation of proteins absorbed on nanoparticles surface plays a crucial role in applications of nanoparticles in biomedicine. The surface protein conformation depends on several factors, namely, nature of protein-nanoparticles interaction, chemical composition of the surface of nanoparticles etc. A model of the electrostatic binding of proteins on charged surface nanoparticles has been proposed earlier (Ghosh et al 2013 Colloids Surf. B 103 267). Also, the irreversible denaturation of the protein conformation due to binding of counterions was reported. In this paper, we have used this model, involving reverse charge parity, to show selective binding of proteins on charged surface iron oxide nanoparticles (IONPs). IONPs were surface functionalized with cetylpyridinium chloride (CPC), cetyl(trimethyl)ammonium bromide (CTAB) and cetylpyridinium iodide (CPI). The effect of counterions (Cl-, Br- and I-) on protein conformation has also been investigated. Several proteins such as α-lactalbumin (ALA), β-lactoglobulin (BLG), ovalbumin (OVA), bovin serum albumin (BSA) and HEWL were chosen for this investigation.

  20. Selective binding of proteins on functional nanoparticles via reverse charge parity model: an in vitro study

    The conformation of proteins absorbed on nanoparticles surface plays a crucial role in applications of nanoparticles in biomedicine. The surface protein conformation depends on several factors, namely, nature of protein-nanoparticles interaction, chemical composition of the surface of nanoparticles etc. A model of the electrostatic binding of proteins on charged surface nanoparticles has been proposed earlier (Ghosh et al 2013 Colloids Surf. B 103 267). Also, the irreversible denaturation of the protein conformation due to binding of counterions was reported. In this paper, we have used this model, involving reverse charge parity, to show selective binding of proteins on charged surface iron oxide nanoparticles (IONPs). IONPs were surface functionalized with cetylpyridinium chloride (CPC), cetyl(trimethyl)ammonium bromide (CTAB) and cetylpyridinium iodide (CPI). The effect of counterions (Cl−, Br− and I−) on protein conformation has also been investigated. Several proteins such as α-lactalbumin (ALA), β-lactoglobulin (BLG), ovalbumin (OVA), bovin serum albumin (BSA) and HEWL were chosen for this investigation. (papers)

  1. Relationship between AOPP (Advanced Oxidation Protein Products) and bovine neutrophils "in vitro": AOPP production by neutrophils and AOPP effects on neutrophils ROS production and viability

    Bordignon, Milena

    2013-01-01

    The aim of the present study was to evaluate the relationship between AOPP (Advanced oxidation protein products) and bovine neutrophils 'in vitro'. For this purpose AOPP were produced "in vitro" by oxidizing bovine serum albumin with HOCl (hypochlorous acid) and bovine neutrophils were isolated from whole blood of dairy cattles. AOPP-BSA were incubated with freshly isolated bovine neutrophils, unstimulated and stimulated with PMA a strong activator of the respiratory burst. Neutrophils ROS pr...

  2. Comparison of Plasticity In Vivo and In Vitro in the Developing Visual Cortex of Normal and Protein Kinase A RIβ-Deficient Mice

    Hensch, Takao K.; Gordon, Joshua A.; Brandon, Eugene P.; McKnight, G. Stanley; Idzerda, Rejean L.; Stryker, Michael P.

    1998-01-01

    Developing sensory systems are sculpted by an activity-dependent strengthening and weakening of connections. Long-term potentiation (LTP) and depression (LTD) in vitro have been proposed to model this experience-dependent circuit refinement. We directly compared LTP and LTD induction in vitro with plasticity in vivo in the developing visual cortex of a mouse mutant of protein kinase A (PKA), a key enzyme implicated in the plasticity of a diverse array of systems.

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

    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 flours and the concentrates from both varieties. Thermal properties of both flours as studied by differential scanning calorimetry (DSC) were comparable. However, the endothermic parameters of the two protein concentrates were 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. PMID:23151538

  4. The in vitro antioxidant properties of alcalase hydrolysate prepared from silkie fowl (Gallus gallus) blood protein.

    Cheng, Fu-Yuan; Lai, I-Chun; Lin, Liang-Chuan; Sakata, Ryoichi

    2016-07-01

    Two types of proteins including blood plasma protein and blood cell protein were isolated from silkie fowl (Gallus gallus) blood and hydrolyzed using alcalase for 0, 2, 4 and 6 h. The blood plasma protein hydrolysate (BPH) and blood cell protein hydrolysate (BCH) were analyzed for pH value, peptide content and antioxidative properties. The significantly higher peptide contents were observed in BPH than that of BCH, which showed that blood plasma protein was more suitable to hydrolysis by alcalase than blood cell protein. Both BPH and BCH showed strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity and Fe(2+) chelating ability. BPH at 4 h of hydrolysis (BPH4) demonstrated significantly higher antioxidant capacity than those treated by alcalase in most of the assays. The BPH4 was separated using ultra-filtration and assessment of the fractions and indicated that low molecular weight of peptides (< 3 kDa) possessed greater DPPH scavenging activity, Fe(2+) chelating ability and inhibitory activity of lipid peroxidation. These results show that BPH has the potential to be ingredients in the food industry as a replacement of synthetic antioxidants. PMID:26556592

  5. A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins.

    Diao, Jiajie; Ishitsuka, Yuji; Lee, Hanki; Joo, Chirlmin; Su, Zengliu; Syed, Salman; Shin, Yeon-Kyun; Yoon, Tae-Young; Ha, Taekjip

    2012-05-01

    SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly regulated class of membrane proteins that drive the efficient merger of two distinct lipid bilayers into one interconnected structure. This protocol describes our fluorescence resonance energy transfer (FRET)-based single vesicle-vesicle fusion assays for SNAREs and accessory proteins. Both lipid-mixing (with FRET pairs acting as lipophilic dyes in the membranes) and content-mixing assays (with FRET pairs present on a DNA hairpin that becomes linear via hybridization to a complementary DNA) are described. These assays can be used to detect substages such as docking, hemifusion, and pore expansion and full fusion. The details of flow cell preparation, protein-reconstituted vesicle preparation, data acquisition and analysis are described. These assays can be used to study the roles of various SNARE proteins, accessory proteins and effects of different lipid compositions on specific fusion steps. The total time required to finish one round of this protocol is 3–6 d. PMID:22582418

  6. 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

    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

  7. Purification, renaturation, and reconstituted protein kinase activity of the Sendai virus large (L) protein: L protein phosphorylates the NP and P proteins in vitro.

    Einberger, H; Mertz, R; Hofschneider, P H; Neubert, W J

    1990-01-01

    Sodium dodecyl sulfate-solubilized Sendai virus large (L) protein was highly purified by a one-step procedure, using hydroxylapatite column chromatography. Monoclonal antibodies addressed to the carboxyl-terminal amino acid sequence of the L protein were used for monitoring L protein during purification. By removing sodium dodecyl sulfate from purified L protein, a protein kinase activity was successfully renatured. P and NP proteins served as its substrates. After immunoprecipitation with an...

  8. UV induced DNA-protein cross links in vitro and in vivo

    The review was not intended to cover all the past year's literature in this field; only selective material published in 1974 and 1975 has been surveyed. Covalent linkage of DNA and RNA to proteins induced by UV is considered, but DNA-membrade attachment, amino acids covalently bound to DNA as functions of growth conditions and protein non-covalently bound to DNA involved in cell regulation are excluded. Studies of DNA-protein cross-links upon UV irradiation in chemical model systems, bacteria and tissue culture systems, and an in vivo mammalian system are all surveyed. (U.K.)

  9. Potential bone-inducing activity in vitro of recombinant human bone morphogenetic protein-7 from a CHO expression system

    LI Xiao-yan; SHI Wei-wei; WANG Hao; LI Bo-hua; YANG Yang; TAN Min; XUE Jing-ya; GUO Ya-jun

    2005-01-01

    Objective: To express the recombinant human bone morphogenetic protein-7 (rhBMP-7) in Chinese hamster ovary(CHO) cells, and to establish the in vitro biological activity assay of rhBMP-7.Methods: Human BMP-7 cDNA was subcloned into p114 mammalian expression vector and transfected to CHO cells by using the Lipofectamine2000 transfection method. CHO cell supernatants were harvested and analyzed to identify the molecule mass of secreted rhBMP-7 and examine its biological activity in vitro to stimulate the synthesis of alkaline phophatase(ALP), a characteristic of osteoblast phenotypes. Results:rhBMP-7 was produced stably in CHO cells, as a processed mature disulfide-linked homodimer, with an apparent molecular mass of 36 000. Examination of the rhBMP-7 biological activity showed that rhBMP-7 specifically stimulated the production of ALP(4-fold increase at 100 ng of rhBMP-7/ml). Conclusion: The rhBMP-7 from CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates rhBMP-7 has the potential bone regeneration activity.

  10. In vitro and in vivo evaluation of bone morphogenetic protein-2 (BMP-2) immobilized collagen-coated polyetheretherketone (PEEK)

    Du, Ya-Wei; Zhang, Li-Nan; Ye, Xin; Nie, He-Min; Hou, Zeng-Tao; Zeng, Teng-Hui; Yan, Guo-Ping; Shang, Peng

    2015-03-01

    Polyetheretherketone (PEEK) is regarded as one of the most potential candidates of biomaterials in spinal implant applications. However, as a bioinert material, PEEK plays a limited role in osteoconduction and osseointegration. In this study, recombinant human bone morphogenetic protein-2 (rhBMP-2) was immobilized onto the surface of collagen-coated PEEK in order to prepare a multi-functional material. After adsorbed onto the PEEK surface by hydrophobic interaction, collagen was cross-linked with N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). EDC/NHS system also contributed to the immobilization of rhBMP-2. Water contact angle tests, XPS and SEM clearly demonstrated the surface changes. ELISA tests quantified the amount of rhBMP-2 immobilized and the release over a period of 30 d. In vitro evaluation proved that the osteogenesis differentiation rate was higher when cells were cultured on modified PEEK discs than on regular ones. In vivo tests were conducted and positive changes of major parameters were presented. This report demonstrates that the rhBMP-2 immobilized method for PEEK modification increase bioactivity in vitro and in vivo, suggesting its practicability in orthopedic and spinal clinical applications.

  11. Adrenomedullin promotes differentiation of oligodendrocyte precursor cells into myelin-basic-protein expressing oligodendrocytes under pathological conditions in vitro

    Takakuni Maki

    2015-07-01

    Full Text Available Oligodendrocytes, which are the main cell type in cerebral white matter, are generated from their precursor cells (oligodendrocyte precursor cells: OPCs. However, the differentiation from OPCs to oligodendrocytes is disturbed under stressed conditions. Therefore, drugs that can improve oligodendrocyte regeneration may be effective for white matter-related diseases. Here we show that a vasoactive peptide adrenomedullin (AM promotes the in vitro differentiation of OPCs under pathological conditions. Primary OPCs were prepared from neonatal rat brains, and differentiated into myelin-basic-protein expressing oligodendrocytes over time. This in vitro OPC differentiation was inhibited by prolonged chemical hypoxic stress induced by non-lethal CoCl2 treatment. However, AM promoted the OPC differentiation under the hypoxic stress conditions, and the AM receptor antagonist AM22–52 canceled the AM-induced OPC differentiation. In addition, AM treatment increased the phosphorylation level of Akt in OPC cultures, and correspondingly, the PI3K/Akt inhibitor LY294002 blocked the AM-induced OPC differentiation. Taken together, AM treatment rescued OPC maturation under pathological conditions via an AM-receptor-PI3K/Akt pathway. Oligodendrocytes play critical roles in white matter by forming myelin sheath. Therefore, AM signaling may be a promising therapeutic target to boost oligodendrocyte regeneration in CNS disorders.

  12. Adrenomedullin promotes differentiation of oligodendrocyte precursor cells into myelin-basic-protein expressing oligodendrocytes under pathological conditions in vitro.

    Maki, Takakuni; Takahashi, Yoko; Miyamoto, Nobukazu; Liang, Anna C; Ihara, Masafumi; Lo, Eng H; Arai, Ken

    2015-07-01

    Oligodendrocytes, which are the main cell type in cerebral white matter, are generated from their precursor cells (oligodendrocyte precursor cells: OPCs). However, the differentiation from OPCs to oligodendrocytes is disturbed under stressed conditions. Therefore, drugs that can improve oligodendrocyte regeneration may be effective for white matter-related diseases. Here we show that a vasoactive peptide adrenomedullin (AM) promotes the in vitro differentiation of OPCs under pathological conditions. Primary OPCs were prepared from neonatal rat brains, and differentiated into myelin-basic-protein expressing oligodendrocytes over time. This in vitro OPC differentiation was inhibited by prolonged chemical hypoxic stress induced by non-lethal CoCl(2) treatment. However, AM promoted the OPC differentiation under the hypoxic stress conditions, and the AM receptor antagonist AM(22-52) canceled the AM-induced OPC differentiation. In addition, AM treatment increased the phosphorylation level of Akt in OPC cultures, and correspondingly, the PI3K/Akt inhibitor LY294002 blocked the AM-induced OPC differentiation. Taken together, AM treatment rescued OPC maturation under pathological conditions via an AM-receptor-PI3K/Akt pathway. Oligodendrocytes play critical roles in white matter by forming myelin sheath. Therefore, AM signaling may be a promising therapeutic target to boost oligodendrocyte regeneration in CNS disorders. PMID:26002630

  13. Exposure of surfactant protein A to ozone in vitro and in vivo impairs its interactions with alveolar cells

    Oosting, R.S.; Van Iwaarden, J.F.; Van Bree, L.; Verhoef, J.; Van Golde, L.M.; Haagsman, H.P. (Laboratory of Veterinary Biochemistry, Utrecht University (Netherlands))

    1992-01-01

    This study focused on the question of whether exposure of surfactant protein A (SP-A) to ozone affected properties of this protein that may be involved in regulating alveolar type II cell and alveolar macrophage functions. In vitro exposure of human or canine SP-A to ozone reduced the ability of this protein to inhibit phorbol-ester induced secretion of (3H)phosphatidylcholine by alveolar type II cells in culture. Ozone-exposed human SP-A showed a decreased ability to enhance phagocytosis of herpes simplex virus and to stimulate superoxide anion production by alveolar macrophages. Experiments with elastase showed that ozone-exposed canine SP-A was more susceptible to proteolysis. A conformational change of the protein could underlie this phenomenon. Surfactant isolated from ozone-exposed rats (0.4 ppm ozone for 12 h) was also less able to stimulate superoxide anion production by alveolar macrophages than surfactant from control rats, which suggested that SP-A in vivo was also susceptible to ozone. The results of this study suggest that SP-A-alveolar cell interactions can be inhibited by ozone exposure, which may contribute to the toxicity of ozone in the lungs.

  14. Discordance between in silico & in vitro analyses of ACE inhibitory & antioxidative peptides from mixed milk tryptic whey protein hydrolysate.

    Chatterjee, Alok; Kanawjia, S K; Khetra, Yogesh; Saini, Prerna

    2015-09-01

    ACE inhibitory and antioxidative peptides identified by LCMS/MS, from mixed milk (Bubalus bubalis and Bos taurus) tryptic whey protein hydrolysate, were compared with the in silico predictions. α la and ß lg sequences, both from Bubalus bubalis and Bos taurus, were used for in silico study. SWISS-PROT and BIOPEP protein libraries were accessed for prediction of peptide generation. Study observed gaps in the prediction versus actual results, which remain unaddressed in the literature. Many peptides obtained in vitro, were not reflected in in silico predictions. Differences in identified peptides in separate libraries were observed too. In in silico prediction, peptides with known biological activities were also not reflected. Predictions, towards generation of bioactive peptides, based upon in silico release of proteins and amino acid sequences from different sources and thereupon validation in relation to actual results has often been reported in research literature. Given that computer aided simulation for prediction purposes is an effective research direction, regular updating of protein libraries and an effectual integration, for more precise results, is critical. The gaps addressed between these two techniques of research, have not found any address in literature. Inclusion of more flexibility with the variables, within the tools being used for prediction, and a hierarchy based database with search options for various peptides, will further enhance the scope and strength of research. PMID:26344975

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

    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.

  16. Signal-transduction protein P(II) from Synechococcus elongatus PCC 7942 senses low adenylate energy charge in vitro.

    Fokina, Oleksandra; Herrmann, Christina; Forchhammer, Karl

    2011-11-15

    P(II) proteins belong to a family of highly conserved signal-transduction proteins that occurs widely in bacteria, archaea and plants. They respond to the central metabolites ATP, ADP and 2-OG (2-oxoglutarate), and control enzymes, transcription factors and transport proteins involved in nitrogen metabolism. In the present study, we examined the effect of ADP on in vitro P(II)-signalling properties for the cyanobacterium Synechococcus elongatus, a model for oxygenic phototrophic organisms. Different ADP/ATP ratios strongly affected the properties of P(II) signalling. Increasing ADP antagonized the binding of 2-OG and directly affected the interactions of P(II) with its target proteins. The resulting P(II)-signalling properties indicate that, in mixtures of ADP and ATP, P(II) trimers are occupied by mixtures of adenylate nucleotides. Binding and kinetic activation of NAGK (N-acetyl-L-glutamate kinase), the controlling enzyme of arginine biosynthesis, by P(II) was weakened by ADP, but relief from arginine inhibition remained unaffected. On the other hand, ADP enhanced the binding of P(II) to PipX, a co-activator of the transcription factor NtcA and, furthermore, antagonized the inhibitory effect of 2-OG on P(II)-PipX interaction. These results indicate that S. elongatus P(II) directly senses the adenylate energy charge, resulting in target-dependent differential modification of the P(II)-signalling properties. PMID:21774788

  17. Inhibitory effect of corcin on aggregation of 1N/4R human tau protein in vitro

    Ali Mohammadi Karakani; Gholamhossein Riazi; Seyed Mahmood Ghaffari; Shahin Ahmadian; Farzad Mokhtari; Mahshad Jalili Firuzi; Seyedeh Zahra Bathaie

    2015-01-01

    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 na...

  18. Effects of Osseointegration by Bone Morphogenetic Protein-2 on Titanium Implants In Vitro and In Vivo

    Fu-Yuan Teng; Wen-Cheng Chen; Yin-Lai Wang; Chun-Cheng Hung; Chun-Chieh Tseng

    2016-01-01

    This study designed a biomimetic implant for reducing healing time and achieving early osseointegration to create an active surface. Bone morphogenetic protein-2 (BMP-2) is a strong regulator protein in osteogenic pathways. Due to hardly maintaining BMP-2 biological function and specificity, BMP-2 efficient delivery on implant surfaces is the main challenge for the clinic application. In this study, a novel method for synthesizing functionalized silane film for superior modification with BMP-...

  19. In vitro protein binding of liraglutide in human plasma determined by reiterated stepwise equilibrium dialysis

    Plum, Anne; Jensen, Lisbeth Bjerring; Kristensen, Jesper Bøggild

    2013-01-01

    Liraglutide is a human glucagon-like peptide-1 (GLP-1) analogue approved for the treatment of type 2 diabetes. It is based on human GLP-1 with the addition of a 16-carbon fatty acid, which facilitates binding to plasma proteins, thus prolonging the elimination half-life and allowing once-daily administration. It has not been possible to quantify liraglutide protein binding by ultrafiltration (the usual method of choice), as the lipophilic molecule becomes trapped in the filter membrane. There...

  20. Initiation of bacteriophage lambda DNA replication in vitro with purified lambda replication proteins.

    Wold, M S; Mallory, J B; Roberts, J D; LeBowitz, J H; McMacken, R

    1982-01-01

    We have developed a soluble enzyme system that replicates exogenously added plasmid DNA (lambda dv) bearing the replication origin of the bacteriophage lambda chromosome. The system contains pure phage lambda O and P replication proteins and a partially purified mixture of Escherichia coli replication proteins [the enzyme system of Fuller, R.S., Kaguni, J.M. & Kornberg, A. (1981) Proc. Natl. Acad. Sci. USA 78, 7370-7374). The features of lambda dv replication in this system closely resemble t...

  1. Proteomic analysis of Schistosoma mansoni proteins released during in vitro miracidium-to-sporocyst transformation

    Wu, Xiao-Jun; Sabat, Greg; Brown, James F.; Zhang, Mengzi; Taft, Andrew; Peterson, Nathan; Harms, Amy; YOSHINO, TIMOTHY P.

    2008-01-01

    Free-living miracidia of Schistosoma mansoni, upon penetration of the their snail intermediate host, undergo dramatic morphological and physiological changes as they transform to the parasitic sporocyst stage. During this transformation process, developing larvae release a diverse array of proteins, herein referred to as larval transformation proteins (LTPs), some of which are postulated to serve a parasite protective function. In the present study, nanoLC-tandem MS analysis was performed on ...

  2. In vitro and in vivo biolasing of fluorescent proteins suspended in liquid microdroplet cavities

    Bayraktar, Halil; Kiraz, Alper; Aas, Mehdi; Karadağ, Yasin; Manioğlu, Selen; Jonas, Alexandr; Anand, Suman; McGloin, David

    2014-01-01

    Fluorescent proteins are indispensable for selective, quantitative visualization of localization, dynamics, and interactions of key molecular constituents of live cells. Incorporation of fluorescent proteins into an optical cavity can lead to a significant increase in fluorescence signal levels due to stimulated emission and light amplification in the cavity, forming a laser with biological gain medium. Utilization of lasing emission from fluorescent biological molecules can then greatly enha...

  3. Deoxynivalenol affects in vitro intestinal epithelial cell barrier integrity through inhibition of protein synthesis

    Deoxynivalenol (DON), one of the most common mycotoxin contaminants of raw and processed cereal food, adversely affects the gastrointestinal tract. Since DON acts as a protein synthesis inhibitor, the constantly renewing intestinal epithelium could be particularly sensitive to DON. We analyzed the toxicological effects of DON on intestinal epithelial protein synthesis and barrier integrity. Differentiated Caco-2 cells, as a widely used model of the human intestinal barrier, were exposed to realistic intestinal concentrations of DON (50, 500 and 5000 ng/ml) during 24 h. DON caused a concentration-dependent decrease in total protein content associated with a reduction in the incorporation of [3H]-leucine, demonstrating its inhibitory effect on protein synthesis. DON simultaneously increased the paracellular permeability of the monolayer as reflected through a decreased transepithelial electrical resistance associated with an increased paracellular flux of the tracer [3H]-mannitol. A concentration-dependent reduction in the expression level of the tight junction constituent claudin-4 was demonstrated by Western blot, which was not due to diminished transcription, increased degradation, or NF-κB, ERK or JNK activation, and was also observed for a tight junction independent protein, i.e. intestinal alkaline phosphatase. These results demonstrate a dual toxicological effect of DON on differentiated Caco-2 cells consisting in an inhibition of protein synthesis as well as an increase in monolayer permeability, and moreover suggest a possible link between them through diminished synthesis of the tight junction constituent claudin-4.

  4. In vitro bioactive properties of intact and enzymatically hydrolysed whey protein: targeting the enteroinsular axis.

    Power-Grant, O; Bruen, C; Brennan, L; Giblin, L; Jakeman, P; FitzGerald, R J

    2015-03-01

    Enzymatically hydrolysed milk proteins have a variety of biofunctional effects some of which may be beneficial in the management of type 2 diabetes mellitus. The purpose of this study was to evaluate the effect of commercially available intact and hydrolysed whey protein ingredients (DH 32, DH 45) on markers of the enteroinsular axis (glucagon like peptide-1 secretion, dipeptidyl peptidase IV inhibition, insulin secretion and antioxidant activity) before and after simulated gastrointestinal digestion (SGID). A whey protein hydrolysate, DH32, significantly enhanced (P whey protein hydrolysates inhibited dipeptidyl peptidase IV activity, yielding half maximal inhibitory concentration values (IC50) of 1.5 ± 0.1 and 1.1 ± 0.1 mg mL(-1) for the DH 32 and DH 45, samples respectively, and were significantly more potent than the intact whey (P whey protein significantly enhanced (P antioxidant activity compared to intact whey, as measured by the oxygen radical absorbance capacity assay (ORAC). This antioxidant activity was maintained (DH 32, P > 0.05) or enhanced (DH 45, P whey stimulated GLP-1 secretion from enteroendocrine cells compared to vehicle control (P whey proteins and peptides can act through multiple targets within the enteroinsular axis and as such may have glucoregulatory potential. PMID:25666373

  5. Alfalfa mosaic virus coat protein bridges RNA and RNA-dependent RNA polymerase in vitro.

    Reichert, Vienna L; Choi, Mehee; Petrillo, Jessica E; Gehrke, Lee

    2007-07-20

    Alfalfa mosaic virus (AMV) RNA replication requires the viral coat protein (CP). AMV CP is an integral component of the viral replicase; moreover, it binds to the viral RNA 3'-termini and induces the formation of multiple new base pairs that organize the RNA conformation. The results described here suggest that AMV coat protein binding defines template selection by organizing the 3'-terminal RNA conformation and by positioning the RNA-dependent RNA polymerase (RdRp) at the initiation site for minus strand synthesis. RNA-protein interactions were analyzed by using a modified Northwestern blotting protocol that included both viral coat protein and labeled RNA in the probe solution ("far-Northwestern blotting"). We observed that labeled RNA alone bound the replicase proteins poorly; however, complex formation was enhanced significantly in the presence of AMV CP. The RNA-replicase bridging function of the AMV CP may represent a mechanism for accurate de novo initiation in the absence of canonical 3' transfer RNA signals. PMID:17400272

  6. Sri Lankan black tea (Camellia sinensis L.) inhibits the methylglyoxal mediated protein glycation and potentiates its reversing activity in vitro

    Wanigasekara Daya Ratnasooriya

    2016-01-01

    Objective: To evaluate inhibitory activity of methylglyoxal (MGO) mediated protein glycation and ability to potentiate its reversing activity and range of antioxidant properties of Sri Lankan low grown orthodox orange pekoe grade black tea. Methods: Freeze dried black tea brew (BTB) was used as the sample in this study. Anti-glycation and glycation reversing activity was studied in bovine serum albumin (BSA)-MGO model. Antioxidant properties were studied using total polyphenolic content, total flavonoid content, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid, 1,1-diphenyl-2-picrylhydrazine and ferric reducing antioxidant power in vitro antioxidant assays. Results: The results demonstrated significant (P Conclusions: The novel properties observed for Sri Lankan orange pekoe grade black tea indicate its usefulness as a supplementary beverage in managing MGO and advanced glycation end products related diseases and ailments.

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

    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. PMID:26465145

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

    Aihua Zheng

    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.

  9. In vitro biosynthesis of globular proteins by murine splenic lymphocytes: effect of serum components as supplement

    Studies on replacement of foetal calf serum (FCS) with precipitable protein, non precipitable protein, dialysable and non dialysable components of the FCS in media for the growth and proliferation of murine splenic rat lymphocytes have revealed that the whole serum could be completely replaced by either of the components without any appreciable deleterious effect on the mitogenic response but none of these components could offer optimum immune response. These findings establish a covalent association of whole FCS for synthesis and secretion of immunologically important pulsed proteins in terms of turnover rate and quantification by FPLC and suggest an important and yet undefined role of FCS in the process of immunoglobulin synthesis. (author). 10 refs., 2 figs., 1 tab

  10. Inhibitory effect of corcin on aggregation of 1N/4R human tau protein in vitro

    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.

  11. Application of Fluorescent Protein Expressing Strains to Evaluation of Anti-Tuberculosis Therapeutic Efficacy In Vitro and In Vivo.

    Kong, Ying; Yang, Dong; Cirillo, Suat L G; Li, Shaoji; Akin, Ali; Francis, Kevin P; Maloney, Taylor; Cirillo, Jeffrey D

    2016-01-01

    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. PMID:26934495

  12. Application of Fluorescent Protein Expressing Strains to Evaluation of Anti-Tuberculosis Therapeutic Efficacy In Vitro and In Vivo.

    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.

  13. Anabolic Properties of High Mobility Group Box Protein-1 in Human Periodontal Ligament Cells In Vitro

    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.

  14. In-vitro antioxidant and antibacterial properties of fermentatively and enzymatically prepared chicken liver protein hydrolysates.

    Chakka, Ashok Kumar; Elias, Mercy; Jini, R; Sakhare, P Z; Bhaskar, N

    2015-12-01

    Protein hydrolysates were prepared from chicken liver using fermentation and enzymatic hydrolysis. The lactic acid bacteria Pediococcus acidilactici NCIM5368 was employed in the fermentation process and a commercial protease (Alcalase® 2.5) was used in enzymatic hydrolysis. Chicken liver hydrolysates prepared by fermentation (FCLH) and enzymatic hydrolysis (ECLH) revealed appreciable amounts of protein [55.85 and 61.34 %; on dry weight basis, respectively]. Fermentation and enzymatic hydrolysis resulted in 14.3 and 26.12 % of degree of hydrolysis. Total antioxidant activity, reducing power, scavenging of superoxide, 2- diphenyl-1-picrylhydrazyl (DPPH) and 2, 2-azino-bis-3-ethyl-benzthiazoline-6-sulphonic acid (ABTS) radicals were determined for both FCLH & ECLH. FCLH & ECLH showed total antioxidant activity of 0.99 and 1.13 μg AAE mg(-1) proteins, respectively; while, they scavenged 96.14 and 92.76 % of DPPH radicals respectively. FCLH showed higher ABTS radical scavenging activity (32.16 %) than ECLH (19.29 %). Superoxide anion scavenging activity of FCLH & ECLH were found to be 95.02 & 88.94 %, respectively. Residues obtained after both treatments also exhibited antioxidant activities. FCLH reported highest antagonistic activity against Listeria monocytogenes (30 mm); while, ECLH showed antibacterial activity only against Micrococcus luteus (12 mm). Both hydrolysates have the potential to be a protein rich ingredient for use in formulated foods and possible help in reduction of oxidative stress. PMID:26604378

  15. Protein Hydrolysates from Beta-Conglycinin Enriched Soybean Genotypes Inhibit Lipid Accumulation and Inflammation in Vitro

    Obesity is a worldwide health concern and a well recognized predictor of premature mortality associated with a state of chronic inflammation. The objective was to evaluate the effect of soy protein hydrolysates (SPH) produced from different soybean genotypes by alcalase (SAH) or simulated gastroint...

  16. Variation in crude protein and in vitro dry matter digestion of wheat forage

    Pasture bloat can be a serious problem for ruminant livestock grazing winter wheat (Triticum aestivum L.). Decreased gains with nonlethal bloat episodes, yearly herd deaths up to 2%, and the cost and risk of active intervention reduce profi ts. Wheat has high digestibility, crude protein (CP), and s...

  17. Two distinct domains of protein 4.1 critical for assembly offunctional nuclei in Vitro

    Krauss, Sharon Wald; Heald, Rebecca; Lee, Gloria; Nunomura, Wataru; Gimm,J. Aura; Mohandas, Narla; Chasis, Joel AnneJ. Aura; Mohandas, Narla; Chasis, Joel Anne

    2002-11-15

    Protein 4.1R, a multifunctional structural protein, acts asan adaptor in mature red cell membrane skeletons linking spectrin-actincomplexes to plasma membrane-associated proteins. In nucleated cellsprotein 4.1 is not associated exclusively with plasma membrane but isalso detected at several important subcellular locations crucial for celldivision. To identify 4.1 domains having critical functions in nuclearassembly, 4.1 domain peptides were added to Xenopus egg extract nuclearreconstitution reactions. Morphologically disorganized, replicationdeficient nuclei assembled when spectrin-actin binding domain orNuMA-binding C-terminal domain peptides were present. However, controlvariant spectrin-actin binding domain peptides incapable of bindingactin, or mutant C-terminal domain peptides with reduced NuMA binding,had no deleterious effects on nuclear reconstitution. To test if 4.1 isrequired for proper nuclear assembly, 4.1 isoforms were depleted withspectrin-actin binding or C-terminal domain-specific antibodies. Nucleiassembled in depleted extracts ha d deranged phenotypes. However, nuclearassembly could be rescued by addition of recombinant 4.1R. Our dataestablishes that protein 4.1 is essential for nuclear assembly andidentifies two distinct 4.1 domains, initially characterized incytoskeletal interactions, that have crucial and versatile functions innuclear assembly.

  18. In vitro digestion of Pickering emulsions stabilized by soft whey protein microgel particles: influence of thermal treatment.

    Sarkar, Anwesha; Murray, Brent; Holmes, Melvin; Ettelaie, Rammile; Abdalla, Azad; Yang, Xinyi

    2016-04-13

    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. PMID:26959339

  19. LegC3, an effector protein from Legionella pneumophila, inhibits homotypic yeast vacuole fusion in vivo and in vitro.

    Terry L Bennett

    Full Text Available 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.

  20. Impact of dehulling on the physico-chemical properties and in vitro protein digestion of common beans (Phaseolus vulgaris L.).

    Romano, A; Giosafatto, C V L; Masi, P; Mariniello, L

    2015-04-01

    The objective of this study was to understand the effect of dehulling on the microstructural, physico-chemical characteristics, and in vitro protein digestion of common bean flours with particular regard to differences between adults and infants. The microstructure of flour samples from undehulled (WB) and manually dehulled (SB) beans, observed through scanning electron microscopy, showed that WB starch granules appeared to be surrounded by an integral matrix, while the SB starch granule structure was still visible although covered by protein clusters. The starch granules were oval and spherical, with heterogeneous sizes ranging from 19 to 30 μm in diameter. Particle size analysis determined with a laser diffraction particle size analyzer showed similar bimodal particle size distributions of small (1-25 μm) and large (>100 μm) granules, though the particle size of WB was obviously higher than SB. Color and other physico-chemical analyses showed that dehulling had significant (P flour protein digestion which could be utilized in various food applications. PMID:25778680

  1. In vitro biofilm development of Streptococcus pneumoniae and formation of choline-binding protein-DNA complexes.

    Domenech, Mirian; Ruiz, Susana; Moscoso, Miriam; García, Ernesto

    2015-10-01

    Extracellular deoxyribonucleic acid (eDNA) is an essential component of bacterial biofilm matrices, and is required in their formation and maintenance. Extracellular DNA binds to exopolysaccharides or extracellular proteins, affording biofilms greater structural integrity. Recently, we reported evidence of intercellular eDNA-LytC complexes in pneumococcal biofilms. The LytC lysozyme is a member of the choline-binding family of proteins (CBPs) located on the pneumococcal surface. The present work shows that other CBPs, i.e. LytA, LytB, Pce, PspC and CbpF, which have a pI between 5 and 6, can bind DNA in vitro. This process requires the presence of divalent cations other than Mg(2+). This DNA binding capacity of CBPs appears to be independent of their enzymatic activity and, at least in the case of LytA, does not require the choline-binding domain characteristic of CBPs. Positively charged, surface-exposed, 25 amino acid-long peptides derived from the catalytic domain of LytB, were also found capable of DNA binding through electrostatic interactions. Confocal laser scanning microcopy revealed the existence of cell-associated LytB-eDNA complexes in Streptococcus pneumoniae biofilms. These and other findings suggest that these surface-located proteins of S. pneumoniae could play roles of varying importance in the colonization and/or invasion of human host where different environmental conditions exist. PMID:25950767

  2. Effect of protein corona magnetite nanoparticles derived from bread in vitro digestion on Caco-2 cells morphology and uptake.

    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. PMID:26520468

  3. The inhibitory effect of selenium nanoparticles on protein glycation in vitro

    Selenium nanoparticles (Se NPs) possess well-known excellent biological activities and low toxicity, and have been employed for numerous applications except as inhibitors to protein glycation. Herein, the present study is carried out to investigate the inhibitory effect of Se NPs on protein glycation in a bovine serum albumin (BSA)/glucose system. By measuring the amount of glucose covalently bound onto BSA, the formation of fructosamine and fluorescent products, it is found that Se NPs can hinder the development of protein glycation in a dose-dependent but time-independent manner under the selected reaction conditions (55 °C, 40 h). And after comparing the increase of inhibitory rate in different stages, it is observed that Se NPs show the greatest inhibitory effect in the early stage, then in the advanced stage, but no effect in the intermediate stage. Fourier transform infrared spectroscopy characterization of Se NPs collected after glycation and determination of ·OH influence and glyoxal formation show that the mechanism for the inhibitory efficacy of Se NPs is related to their strong competitive activity against available amino groups in proteins, their great scavenging activity on reactive oxygen species and their inhibitory effect on α-dicarbonyl compounds’ formation. In addition, it is proved that Se NPs protect proteins from structural modifications in the system and they do not exhibit significant cytotoxicity towards BV-2 and BRL-3A cells at low concentrations (10 and 50 μg mL−1). Consequently, Se NPs may be suitable for further in vivo studies as novel anti-glycation agents. (paper)

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

    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.

  5. RepA and DnaA proteins are required for initiation of R1 plasmid replication in vitro and interact with the oriR sequence.

    Masai, H.; Arai, K.

    1987-01-01

    RepA, an initiation protein of R1 plasmid replication, was purified from an Escherichia coli strain overproducing the protein. The purified RepA protein specifically initiated replication in vitro of plasmid DNA bearing the replication origin of R1 plasmid (oriR). The replication, strictly dependent on added RepA protein, was independent of host RNA polymerase but required other host replication functions (DnaB and DnaC proteins, the single-stranded-DNA-binding protein SSB, and DNA gyrase). T...

  6. Recombinant Neural Protein PrP Can Bind with Both Recombinant and Native Apolipoprotein E In Vitro

    Chen GAO; Wei ZHOU; Xiao-Ping DONG; Yan-Jun LEI; Jun HAN; Qi SHI; Lan CHEN; Yan GUO; Yong-Jun GAO; Jian-Ming CHEN; Hui-Ying JIANG

    2006-01-01

    The most essential and crucial step during the pathogenesis of transmissible spongiform encephalopathy is the conformational change of cellular prion protein (PrPC) to pathologic isoform (prpSc). A lot of data revealed that caveolae-like domains (CLDs) in the cell surface were the probable place where the conversion of PrP proteins happened. Apolipoprotein E (ApoE) is an apolipoprotein which is considered to play an important role in the development of Alzheimer's disease and other neurodegenerative diseases by forming protein complex through binding to the receptor located in the clathrin-coated pits of the cell surface.In this study, a 914-bp cDNA sequence encoding human ApoE3 was amplified from neuroblastoma cell line SH-SY5Y. Three human ApoE isomers were expressed and purified from Escherichia coli. ApoE-specific antiserum was prepared by immunizing rabbits with the purified ApoE3. GST/His pull-down assay,immunoprecipitation and ELISA revealed that three full-length ApoE isomers interact with the recombinant full-length PrP protein in vitro. The regions corresponding to protein binding were mapped in the N-terminal segment of ApoE (amino acid 1-194) and the N-terminal of PrP (amino acid 23-90). Moreover, the recombinant PrP showed the ability to form a complex with the native ApoE from liver tissues. Our data provided direct evidence of molecular interaction between ApoE and PrP. It also supplied scientific clues for assessing the significance of CLDs on the surface of cellular membrane in the process of conformational conversion from PrPC to PrPSc and probing into the pathogenesis of transmissible spongiform encephalopathy.

  7. The Key Factors Affecting Tuber Development of Potato in vitro and the Relation with Protein Fractions

    WANG Da-yong; LIAN Yong; ZHU De-wei

    2002-01-01

    According to previous analysis, some properties bounding up with tuber yield were investigated.The results showed that tuber average weight, plastid Mg2+ -ATPase activity, plastid Ca2+ -ATPase activity,mitochondria Mg2+-ATPase activity, total soluble protein content, tuber average diameter, and Q-enzyme activity were important factors determining the tuber yield. The linear regression equation was:Y = 0.5211 +0.0595X(1) + 0.8389X(2) + 0.0882X(3) - 0. 0073X(4) + 0. 1449X(5) + 0. 3510X(6) + 0. 0031X(7) -0.00003X(8) + 0.3412X(9)+ 0.0127X(10) + 0.2904X(11) + 0.0570X(12) + 0.0159X(13) + 0.3585X(14)+ 0.0134X(15) -0.1012X(16). At the same time, the relation between several important properties and soluble protein fractions were analyzed.

  8. Hypoxia-inducible factor regulates expression of surfactant protein in alveolar type II cells in vitro.

    Ito, Yoko; Ahmad, Aftab; Kewley, Emily; Mason, Robert J

    2011-11-01

    Alveolar type II (ATII) cells cultured at an air-liquid (A/L) interface maintain differentiation, but they lose these properties when they are submerged. Others showed that an oxygen tension gradient develops in the culture medium as ATII cells consume oxygen. Therefore, we wondered whether hypoxia inducible factor (HIF) signaling could explain differences in the phenotypes of ATII cells cultured under A/L interface or submerged conditions. ATII cells were isolated from male Sprague-Dawley rats and cultured on inserts coated with a mixture of rat-tail collagen and Matrigel, in medium including 5% rat serum and 10 ng/ml keratinocyte growth factor, with their apical surfaces either exposed to air or submerged. The A/L interface condition maintained the expression of surfactant proteins, whereas that expression was down-regulated under the submerged condition, and the effect was rapid and reversible. Under submerged conditions, there was an increase in HIF1α and HIF2α in nuclear extracts, mRNA levels of HIF inducible genes, vascular endothelial growth factor, glucose transporter-1 (GLUT1), and the protein level of pyruvate dehydrogenase kinase isozyme-1. The expression of surfactant proteins was suppressed and GLUT1 mRNA levels were induced when cells were cultured with 1 mM dimethyloxalyl glycine. The expression of surfactant proteins was restored under submerged conditions with supplemented 60% oxygen. HIF signaling and oxygen tension at the surface of cells appears to be important in regulating the phenotype of rat ATII cells. PMID:21454802

  9. Imaging of Protein Synthesis: In Vitro and In Vivo Evaluation of Sc-44-DOTA-Puromycin

    Eigner, Sebastian; Beckford, Denis R.; Fellner, M.; Loktionova, N.; Piel, M.; Lebeda, Ondřej; Rosch, F.; Ross, T. L.; Eigner-Henke, Kateřina

    2013-01-01

    Roč. 15, č. 1 (2013), s. 79-86. ISSN 1536-1632 R&D Projects: GA MŠk 2B06165 Institutional support: RVO:61389005 Keywords : protein synthesis * Scandium-44 * DOTA-Pur * therapy control * mu PET * preclinical imaging Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 2.869, year: 2013 http://link.springer.com/content/pdf/10.1007%2Fs11307-012-0561-3

  10. In vitro and in vivo screening for novel essential cell-envelope proteins in Pseudomonas aeruginosa

    Regina Fernández-Piñar; Alessandra Lo Sciuto; Alice Rossi; Serena Ranucci; Alessandra Bragonzi; Francesco Imperi

    2015-01-01

    The Gram-negative bacterium Pseudomonas aeruginosa represents a prototype of multi-drug resistant opportunistic pathogens for which novel therapeutic options are urgently required. In order to identify new candidates as potential drug targets, we combined large-scale transposon mutagenesis data analysis and bioinformatics predictions to retrieve a set of putative essential genes which are conserved in P. aeruginosa and predicted to encode cell envelope or secreted proteins. By generating unma...

  11. Modulation of Human Neutrophil Functions In Vitro by Treponema denticola Major Outer Sheath Protein

    Puthengady Thomas, Bina; Sun, Chun Xiang; Bajenova, Elena; Ellen, Richard P.; Glogauer, Michael

    2006-01-01

    In this study of human polymorphonuclear leukocytes (PMNs), pretreatment with Treponema denticola major outer sheath protein (Msp) inhibited formyl-methionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis, phagocytosis of immunoglobulin G-coated microspheres, fMLP-stimulated calcium transients, and actin assembly. Msp neither altered oxidative responses to phorbol myristate or fMLP nor induced apoptosis. Msp selectively impairs chemotaxis and phagocytosis by impacting the PMN cytoskeleton.

  12. Functional characterization of "Bartonella" effector protein - BepE during "in vivo" and "in vitro" infection

    Okujava, Rusudan

    2013-01-01

    The bartonellae is a family of gram-negative, fastidious, facultative intracellular, zoonotic bacteria. Most of the Bartonella species are highly adapted to establish asymptomatic bacteremia of their reservoir host within which the bacteria colonize erythrocytes as privileged host niche and develop long-lasting persistent infections. Bartonella uses a VirB type IV secretion system (T4SS) to translocate Bartonella effector proteins (Beps) into the infected cells. By using such a tool box it su...

  13. In Vitro Protein Digestibility and Physical Properties of Instant Teh Talua Dried by Spray Dryer

    Rina Yenrina; Deivy Andhika Permata; Dini Rasjmida; Rahmal Tayandi

    2016-01-01

    Abstract—This study aims to learn the effect of the addition of different concentrations of tea on protein digestibility and physical properties of the  product. This study has been completed from February to July 2014. This study begins with the process of making instant teh talua, then continue with prodct analysis. This study used a completely randomized design (CRD) with 5 treatments and 3 replications. Data were analyzed statistically by F test and if significantly different, followed by...

  14. Immunostimulatory Activity of Protein Hydrolysate from Oviductus Ranae on Macrophage In Vitro

    2014-01-01

    Oviductus Ranae is the dry oviduct of Rana chensinensis, which is also called R. chensinensis oil. Oviductus Ranae is a valuable Chinese crude drug and is recorded in the Pharmacopoeia of the People's Republic of China. The aim of this study was to investigate the immunostimulatory activity of protein hydrolysate of Oviductus Ranae (ORPH) and to assess its possible mechanism. Immunomodulatory activity of ORPH was examined in murine macrophage RAW 264.7 cells. The effect of ORPH on the phagocy...

  15. Identification of bone morphogenetic protein 9 (BMP9) as a novel profibrotic factor in vitro.

    Muñoz-Félix, José M; Cuesta, Cristina; Perretta-Tejedor, Nuria; Subileau, Mariela; López-Hernández, Francisco J; López-Novoa, José M; Martínez-Salgado, Carlos

    2016-09-01

    Upregulated synthesis of extracellular matrix (ECM) proteins by myofibroblasts is a common phenomenon in the development of fibrosis. Although the role of TGF-β in fibrosis development has been extensively studied, the involvement of other members of this superfamily of cytokines, the bone morphogenetic proteins (BMPs) in organ fibrosis has given contradictory results. BMP9 is the main ligand for activin receptor-like kinase-1 (ALK1) TGF-β1 type I receptor and its effect on fibrosis development is unknown. Our purpose was to study the effect of BMP9 in ECM protein synthesis in fibroblasts, as well as the involved receptors and signaling pathways. In cultured mice fibroblasts, BMP9 induces an increase in collagen, fibronectin and connective tissue growth factor expression, associated with Smad1/5/8, Smad2/3 and Erk1/2 activation. ALK5 inhibition with SB431542 or ALK1/2/3/6 with dorsomorphin-1, inhibition of Smad3 activation with SIS3, and inhibition of the MAPK/Erk1/2 with U0126, demonstrates the involvement of these pathways in BMP9-induced ECM synthesis in MEFs. Whereas BMP9 induced Smad1/5/8 phosphorylation through ALK1, it also induces Smad2/3 phosphorylation through ALK5 but only in the presence of ALK1. Summarizing, this is the first study that accurately identifies BMP9 as a profibrotic factor in fibroblasts that promotes ECM protein expression through ALK1 and ALK5 receptors. PMID:27208502

  16. In Vitro Protein Digestibility and Physical Properties of Instant Teh Talua Dried by Spray Dryer

    Rina Yenrina

    2016-02-01

    Full Text Available Abstract—This study aims to learn the effect of the addition of different concentrations of tea on protein digestibility and physical properties of the  product. This study has been completed from February to July 2014. This study begins with the process of making instant teh talua, then continue with prodct analysis. This study used a completely randomized design (CRD with 5 treatments and 3 replications. Data were analyzed statistically by F test and if significantly different, followed by Duncan's test New Multiple Range Test (DNMRT at 5% level. The treatment in this study include A (Without Tea Extract, B (5 g of Tea Extract in 100 ml of water, C (10 g of Tea Extract in 100 ml of water, D (15 g of Tea Extract in 100 ml of water, and E (20 g of Tea Extract in 100 ml of water. The results of this study showed that the addition of treatment between different tea extract gives significant effect on protein content, water-soluble portion, protein digestibility, and no significant effect on moisture content and water activities.

  17. SLUDGE BATCH 7B GLASS VARIABILITY STUDY

    Johnson, F.; Edwards, T.

    2011-10-25

    The Defense Waste Processing Facility (DWPF) is preparing to initiate processing Sludge Batch 7b (SB7b). In support of the upcoming processing, the Savannah River National Laboratory (SRNL) provided a recommendation to utilize Frits 418 with a 6% Na{sub 2}O addition (26 wt% Na{sub 2}O in sludge) and 702 with a 4% Na{sub 2}O addition (24 wt% Na{sub 2}O in sludge) to process SB7b. This recommendation was based on assessments of the compositional projections for SB7b available at the time from the Savannah River Remediation (SRR). To support qualification of SB7b, SRNL executed a variability study to assess the applicability of the current durability models for SB7b. The durability models were assessed over the expected composition range of SB7b, including potential caustic additions, combined with Frits 702 and 418 over a 32-40% waste loading (WL) range. Thirty four glasses were selected based on Frits 418 and 702 coupled with the sludge projections with an additional 4-6% Na{sub 2}O to reflect the potential caustic addition. Six of these glasses, based on average nominal sludge compositions including the appropriate caustic addition, were developed for both Frit 418 and Frit 702 at 32, 36 and 40% WL to provide coverage in the center of the anticipated SB7b glass region. All glasses were fabricated and characterized using chemical composition analysis, X-ray diffraction (XRD) and the Product Consistency Test (PCT). To comply with the DWPF Glass Product Control Program, a total of thirty four glasses were fabricated to assess the applicability of the current DWPF PCCS durability models. Based on the measured PCT response, all of the glasses were acceptable with respect to the Environmental Assessment (EA) benchmark glass regardless of thermal history. The NL[B] values of the SB7b variability study glasses were less than 1.99 g/L as compared to 16.695 g/L for EA. A small number of the D-optimally selected 'outer layer' extreme vertices (EV) glasses were not

  18. The human metapneumovirus matrix protein stimulates the inflammatory immune response in vitro.

    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.

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

    Mundy, John; Hejgaard, Jørn; Hansen, Annette;

    1986-01-01

    . Thus, 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....... In contrast, synthesis of alpha-amylase (included as control) and of ASI showed antagonistic hormonal control: while GA promotes and ABA reduces accumulation of mRNA for alpha-amylase, these hormones have the opposite effect on ASI mRNA levels....

  20. Combined Beta-Agonists and Corticosteroids Do Not Inhibit Extracellular Matrix Protein Production In Vitro

    Qi Ge; Poniris, Maree H; Moir, Lyn M.; Black, Judith L; Burgess, Janette K.

    2012-01-01

    Background. Persistent asthma is characterized by airway remodeling. Whereas we have previously shown that neither β 2-agonists nor corticosteroids inhibit extracellular matrix (ECM) protein release from airway smooth muscle (ASM) cells, the effect of their combination is unknown and this forms the rationale for the present study. Methods. ASM cells from people with and without asthma were stimulated with TGFβ1 (1 ng/ml) with or without budesonide (10−8 M) and formoterol (10−10 and 10−8 M), a...

  1. Combined Beta-agonists and corticosteroids do not inhibit extracellular matrix protein production in vitro

    Ge, Qi; Poniris, Maree H; Moir, Lyn M.; Black, Judith L; Burgess, Janette K.

    2012-01-01

    Background. Persistent asthma is characterized by airway remodeling. Whereas we have previously shown that neither β(2)-agonists nor corticosteroids inhibit extracellular matrix (ECM) protein release from airway smooth muscle (ASM) cells, the effect of their combination is unknown and this forms the rationale for the present study. Methods. ASM cells from people with and without asthma were stimulated with TGFβ1 (1 ng/ml) with or without budesonide (10(-8) M) and formoterol (10(-10) and 10(-8...

  2. In vitro characterization of the Bacillus subtilis protein tyrosine phosphatase YwqE

    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....

  3. Construction and in vitro Expression of Streptococcus Mutans Surface Protein Encoding DNA Vaccine

    PENG; Zhixiang(

    2001-01-01

    [1]樊明文主编.口腔生物学.北京:人民卫生出版社 1996.132[2]Senpuku H Iizima T Yamaguchi Y et al.Immunogenicity of peptides coupled with multiple T-cell epitopes of a surface protein antigen of Streptococcus mutans.Immunology 1996 88:2275[3]Kato H Takeuchi H Oishi Y et al.The immunogenicity of various peptide antigens inducing cross-reacting antibodies to a cell surface protein antigen of Streptococcus mutans.Oral Microbiol Immunol 1999 14:213[4]Okahashi N Sasakawa C Yoshikawa M et al.Molecular characterization of a surface protein antigen gene from serotype c Streptococcus mutans implicated in dental caries.Mol Microbiol 1989 3:673[5]Okahashi N Takahashi I Nakai M et al.Identification of antigenic epitopes in an alanine-rich repeating region of a surface protein antigen of Streptococcus mutans.Infeet Immun 1993 61(4):1301[6]Brady L J Cvitkovitch D G Geric C M et al.Deletion of the central proline-rich repeat domain results in altered antigenicity and lack of surface expression of the Streptococcus mutans P1 adhesin molecule.Infect Immun 1998 66(9):4274[7]彭志翔 樊明文 边专.变形链球菌表面蛋白PAc结构基因克隆工程数据分析.口腔医学纵横杂志 2000 16(2):90[8]彭志翔 钟燕 樊明文等.含变链菌PAc蛋白编码基因保守区重组质粒pCIA-P的亚克隆构建.中华口腔医学杂志 2000 35(5):339[9]Peng Z X Zhong Y Fan M W et al.Design and preparation of cloned DNA fragment from pac gene of Streptococcus mutans.J Comprehensive Stomatology 2000 16(1):54

  4. Effects of manipulation of the caspase system on myofibrillar protein degradation in vitro.

    Kemp, C M; Wheeler, T L

    2011-10-01

    Apoptosis via the intrinsic caspase 9 pathway can be induced by oxidative stressors hydrogen peroxide (H₂O₂) and N-(4 hydroxyphenol) rentinamide (fenretinide), a synthetic retinoid. Accelerated muscle atrophy and proteolysis in muscle-wasting conditions have been linked to oxidative stress and activated protease systems. Therefore, the hypothesis of this study was that proteolysis of myofibrillar proteins could be manipulated through the induction or inhibition of the caspase system. After slaughter, LM and supraspinatus muscles from callipyge (n = 5) and normal (n = 3) lambs were excised, finely diced, and incubated with treatment buffers containing oxidative stressors fenretinide or H₂O₂, recombinant caspase 3, caspase-specific inhibitor N-acetyl-Asp-Glu-Val-Asp-CHO (DEVD), or control solution. Muscle samples were incubated for 1, 2, 7, and 21 d at 4°C. Activation of the initiator caspase, caspase 9, and myofibrillar protein degradation was determined by SDS-PAGE and Western blotting. Results showed that fenretinide, H₂O₂, and recombinant caspase 3 increased (P tenderization. However, these stimulated changes were not sufficient to overcome the lack of proteolysis that is characteristic of muscle from callipyge lambs. PMID:21622882

  5. Biophysical study on the interaction of etomidate and the carrier protein in vitro.

    Liu, Wei; Liu, Aijie; Liu, Guoqiang; Wang, Dewei; Chen, Kui; Wang, Hongying

    2016-08-01

    Etomidate is a unique drug used for induction of general anesthesia and sedation, and is usually used through intravenous injection clinically. Before targeting to the receptor, etomidate binds proteins in blood when it comes into veins. Thus to study the interaction of etomidate and serum albumin would be of great toxicological and pharmacological importance. In this study, the interaction between etomidate and human serum albumin (HSA) was studied using fluorescence spectroscopy, UV-vis absorption spectroscopy, Fourier transform infrared spectroscopy (FT-IR), circular dichroism (CD) spectroscopy, site maker displacement and molecular modeling methods. Investigations of the binding constant (K = 3.55 × 10(5 )M(-1), 295 K), the number of binding sites (n = 1.16), thermodynamic parameters (ΔG = 3.13 × 10(4 )J·mol(-1), ΔS = 364 J·mol(-1)·K(-1) and ΔH = -6.85 × 10(5 )J·mol(-1)) for the reaction and changes to the binding sites and conformation in HSA in response to etomidate were presented. Results show that etomidate can bind HSA tightly through electrostatic forces, and the protein skeleton conformation and secondary structure changes thereby. This is the first spectroscopic report for etomidate-HSA interactions which illustrates the complex nature of this subject. PMID:25757642

  6. A Histone-Like Protein Induces Plasmid DNA to Form Liquid Crystals in Vitro and Gene Compaction in Vivo

    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.

  7. In vitro unfolding, refolding, and polymerization of human gammaD crystallin, a protein involved in cataract formation.

    Kosinski-Collins, Melissa S; King, Jonathan

    2003-03-01

    Human gammaD crystallin (HgammaD-Crys), a major protein of the human eye lens, is a primary component of cataracts. This 174-residue primarily beta-sheet protein is made up of four Greek keys separated into two domains. Mutations in the human gene sequence encoding HgammaD-Crys are implicated in early-onset cataracts in children, and the mutant protein expressed in Escherichia coli exhibits properties that reflect the in vivo pathology. We have characterized the unfolding, refolding, and competing aggregation of human wild-type HgammaD-Crys as a function of guanidinium hydrochloride (GuHCl) concentration at neutral pH and 37 degrees C, using intrinsic tryptophan fluorescence to monitor in vitro folding. Wild-type HgammaD-Crys exhibited reversible refolding above 1.0 M GuHCl. The GuHCl unfolded protein was more fluorescent than its native counterpart despite the absence of metal or ion-tryptophan interactions. Aggregation of refolding intermediates of HgammaD-Crys was observed in both equilibrium and kinetic refolding processes. The aggregation pathway competed with productive refolding at denaturant concentrations below 1.0 M GuHCl, beyond the major conformational transition region. Atomic force microscopy of samples under aggregating conditions revealed the sequential appearance of small nuclei, thin protofibrils, and fiber bundles. The HgammaD-Crys fibrous aggregate species bound bisANS appreciably, indicating the presence of exposed hydrophobic pockets. The mechanism of HgammaD-Crys aggregation may provide clues to understanding age-onset cataract formation in vivo. PMID:12592018

  8. Relationship of LRP-human major vault protein to in vitro and clinical resistance to anticancer drugs.

    Izquierdo, M A; Scheffer, G L; Flens, M J; Shoemaker, R H; Rome, L H; Scheper, R J

    1996-01-01

    Multidrug resistance (MDR) has been related to two members of the ABC-superfamily of transporters, P-glycoprotein (Pgp) and Multidrug Resistance-associated Protein (MRP). We have described a 110 kD protein termed the Lung Resistance-related Protein (LRP) that is overexpressed in several non-Pgp MDR cells lines of different histogenetic origin. Reversal of MDR parallels a decrease in LRP expression. In a panel of 61 cancer cell lines which have not been subjected to laboratory drug selection, LRP was a superior predictor for in vitro resistance to MDR-related drugs when compared to Pgp and MRP, and LRP's predictive value extended to MDR unrelated drugs, such as platinum compounds. LRP is widely distributed in clinical cancer specimens, but the frequency of LRP expression inversely correlates with the known chemosensitivity of different tumour types. Furthermore, LRP expression at diagnosis has been shown to be a strong and independent prognostic factor for response to chemotherapy and outcome in acute myeloid leukemia and ovarian carcinoma (platinum-based treatment) patients. Recently, LRP has been identified as the human major protein. Vaults are novel cellular organelles broadly distributed and highly conserved among diverse eukaryotic cells, suggesting that they play a role in fundamental cell processes. Vaults localise to nuclear pore complexes and may be the central plug of the nuclear pore complexes. Vaults structure and localisation support a transport function for this particle which could involve a variety of substrates. Vaults may therefore play a role in drug resistance by regulating the nucleocytoplasmic transport of drugs. PMID:8862006

  9. VIP21-caveolin, a membrane protein constituent of the caveolar coat, oligomerizes in vivo and in vitro.

    Monier, S; Parton, R G; Vogel, F; Behlke, J; Henske, A; Kurzchalia, T V

    1995-01-01

    VIP21-caveolin is a membrane protein, proposed to be a component of the striated coat covering the cytoplasmic surface of caveolae. To investigate the biochemical composition of the caveolar coat, we used our previous observation that VIP21-caveolin is present in large complexes and insoluble in the detergents CHAPS or Triton X-114. The mild treatment of these insoluble structures with sodium dodecyl sulfate leads to the detection of high molecular mass complexes of approximately 200, 400, and 600 kDa. The 400-kDa complex purified to homogeneity from dog lung is shown to consist exclusive of the two isoforms of VIP21-caveolin. Pulse-chase experiments indicate that the oligomers form early after the protein is synthesized in the endoplasmic reticulum (ER). VIP21-caveolin does indeed insert into the ER membrane through the classical translocation machinery. Its hydrophobic domain adopts an unusual loop configuration exposing the N- and C-flanking regions to the cytoplasm. Similar high molecular mass complexes can be produced from the in vitro-synthesized VIP21-caveolin. The complex formation occurs only if VIP21-caveolin isoforms are properly inserted into the membrane; formation is cytosol-dependent and does not involve a vesicle fusion step. We propose that high molecular mass oligomers of VIP21-caveolin represent the basic units forming the caveolar coat. They are formed in the ER and later, between the ER and the plasma membrane, these oligomers could associate into larger detergent-insoluble structures. Images PMID:7579702

  10. Ultraviolet-induced photodegradation of cucumber (Cucumis sativus L.) microsomal and soluble protein tryptophanyl residues in vitro

    The in vitro effects of ultraviolet B (280--320 nm) radiation on microsomal membrane proteins and partially purified ribulose bisphosphate carboxylase (Rubisco) from cucumber (Cucumis sativus L.) was investigated by measuring the direct photolytic reduction of tryptophan fluorescence and the formation of fluorescent photooxidation products. Exposure of microsomes and Rubisco to monochromatic 300-nm radiation resulted in the loss of intrinsic tryptophan fluorescence and the production of blue-emitting fluorophores. The major product of tryptophan photolysis was tentatively identified as N-formylkynurenine (N-FK). Even though the rates of tryptophan photodegradation and N-FK formation were similar, the amount of blue fluorescence produced was significantly higher in the microsomes relative to Rubisco. Studies with various free radical scavengers and other modifiers indicated that tryptophan photodegradation requires oxygen species. The optimum wavelengths for loss of tryptophan fluorescence were 290 nm for the microsomes and 280 nm for Rubisco. The temperature dependence of tryptophan fluorescence and rate of tryptophan photodegradation indicated an alteration in the cucumber microsomal membranes at about 24 degrees C, which influenced protein structure and tryptophan photosensitivity. 29 refs., 6 figs., 1 tab

  11. Synthesis and in vitro antioxidant functions of protein hydrolysate from backbones of Rastrelliger kanagurta by proteolytic enzymes.

    Sheriff, Sheik Abdulazeez; Sundaram, Balasubramanian; Ramamoorthy, Baranitharan; Ponnusamy, Ponmurugan

    2014-01-01

    Every year, a huge quantity of fishery wastes and by-products are generated by fish processing industries. These wastes are either underutilized to produce low market value products or dumped leading to environmental issues. Complete utilization of fishery wastes for recovering value added products would be beneficial to the society and individual. The fish protein hydrolysates and derived peptides of fishery resources are widely used as nutritional supplements, functional ingredients, and flavor enhancers in food, beverage and pharmaceutical industries. Antioxidants from fishery resources have attracted the attention of researchers as they are cheaper in cost, easy to derive, and do not have side effects. Thus the present investigation was designed to produce protein hydrolysate by pepsin and papain digestion from the backbones of Rastrelliger kanagurta (Indian mackerel) and evaluate its antioxidant properties through various in vitro assays. The results reveal that both hydrolysates are potent antioxidants, capable of scavenging 46% and 36% of DPPH (1,1-diphenyl-2 picrylhydrazyl) and 58.5% and 37.54% of superoxide radicals respectively. The hydrolysates exhibit significant (p hydrolysates produced, pepsin derived fraction is superior than papain derived fraction in terms of yield, DH (Degree of hydrolysis), and antioxidant activity. PMID:24596496

  12. In vitro Expression in Eukaryotic Cells of a Prion Protein Gene Cloned from Scrapie-Infected Mouse Brain

    Caughey, Byron; Race, Richard E.; Vogel, Mari; Buchmeier, Michael J.; Chesebro, Bruce

    1988-07-01

    It has been proposed that the causative agent of scrapie represents a class of infectious particle that is devoid of nucleic acid and that an altered form of the endogenous prion protein (PrP) is the agent. However, it has been difficult to exclude the possibility that PrP purified from scrapie tissues might be contaminated with a more conventional viral agent. To obtain PrP uncontaminated by scrapie-infected tissues, PrP cDNA cloned from a scrapie-infected mouse brain was expressed in mouse C127 cells in vitro. mRNA and protein encoded by the cloned PrP gene were identified. The expressed PrP polypeptides appeared to be glycosylated and were released from the cell surface into the medium. Homogenates of the cells expressing the cloned PrP gene were inoculated into susceptible mice but failed to induce clinical signs of scrapie. Thus, either PrP is not the transmissible agent of scrapie or the expressed PrP requires additional modification to be infectious.

  13. A novel protein fraction from Sesbania grandiflora shows potential anticancer and chemopreventive efficacy, in vitro and in vivo.

    Laladhas, Krishna P; Cheriyan, Vino T; Puliappadamba, Vineshkumar T; Bava, Smitha V; Unnithan, Rajesh G; Vijayammal, Parvathy L; Anto, Ruby John

    2010-03-01

    We report mechanism-based evidence for the anticancer efficacy of a protein fraction, SF2 (Sesbania fraction 2) isolated from the flower of the medicinal plant, Sesbania grandiflora (S. grandiflora). The fraction was evaluated in two murine ascites tumour cell lines and human cancer cell lines of different origin for its anticancer effect. SF2 inhibited cell proliferation and induced apoptosis as demonstrated by DNA fragmentation and externalization of phosphatidyl serine in Daltons lymphoma ascites (DLA) and colon cancer cells (SW-480). Sensitivity to SF2 in these cells was associated with activation of caspases 3, 8 and 9, poly (ADP-ribose) polymerase cleavage and cytochrome C release which attests apoptosis induced cell death. Mechanistically, SF2 down-regulated phorbol myristate acetate (PMA) induced NF-kappaB, a transcription factor which controls the expression of genes encoding proteins involved in cell regulation and growth control. Additionally, SF2 also down-regulated anti-apoptotic factors such as Bcl-2, p-Akt and cyclooxygenase-2 induced by the tumour promoter PMA suggestive of a possible explanation for its anticancer effect. In vivo studies using ascites and solid tumour models strongly support in vitro findings as SF2 administration increased the life span and decreased the tumour volume in mice bearing tumour. In vivo toxicological evaluation revealed the pharmacological safety of SF2 and may serve as a potential anticancer drug candidate. PMID:19183244

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

    Wu, Y.; Liu, Y; Lee, L.(Department of Physics, Yale University, New Haven, CT, USA); 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...

  15. Copper transportion of WD protein in hepatocytes from Wilson disease patients in vitro

    Guo-Qing Hou; Xiu-Ling Liang; Rong Chen; Li-Wen Tang; Ying Wang; Ping-Yi Xu; Ying-Ru Zhang; Cui-Hua Ou

    2001-01-01

    AIM: To study the effect of copper transporting P-type ATPase in copper metabolism of hepatocyte and pathogenesis of Wilson disease (WD). METHODS: WD copper transporting properties in some organelles of the cultured hepatocytes were studied from WD patients and normal controls. These cultured hepatocytes were incubated in the media of copper 15mg.L-1 only, copper 15 mg. L-1 with vincristine (agonist of P-type ArPase) 0.5mg. L-1, or copper 15 mg. L-1 withvanadate (antagonist of P-type ATPase) 18.39 mg. L-1separately. Microsome (endoplasmic reticulum and Golgi apparatus), lysosome, mitochondria, and cytosol were isolated by differential centrifugation. Copper contents in these organelles were measured with atomic absorption spectrophotometer, and the influence in copper transportion of these organelles by vanadate and vincristine were comparatively analyzed between WD patients and controls.WD copper transporting P-type ATPase was detected by SDS-PAGE in conjunction with Western blot in liver samples of WD patients and controls. RESULTS: The specific WD proteins (Mr155 000 lanes) were expressed in human hepatocytes, including the control and WD patients. After incubation with medium containing copper for 2 h or 24 h, the microsome copper concentration in WD patients was obviously lower than that of controls,and the addtion of vanadate or vincristine would change the copper transporting of microsomes obviously. When incubated with vincristine, levels of copper in microsome were significantly increased, while incubated with vanadate,the copper concentrations in microsome were obviously decreased. The results indicated that there were Wdproteins, the copper transportion P-type ATPase in the microsome of hepatocytes. WD patients possessed abnormal copper transporting function of WD protein in the microsome, and the agonist might correct the defect of copper transportion by promoting the activity of copper transportion P-type ATPase. CONCLUSION: Copper transportion P

  16. Protein kinase D2 regulates migration and invasion of U87MG glioblastoma cells in vitro

    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-JunS73 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

  17. Protein kinase D2 regulates migration and invasion of U87MG glioblastoma cells in vitro

    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

  18. FLEXITau: Quantifying Post-translational Modifications of Tau Protein in Vitro and in Human Disease.

    Mair, Waltraud; Muntel, Jan; Tepper, Katharina; Tang, Shaojun; Biernat, Jacek; Seeley, William W; Kosik, Kenneth S; Mandelkow, Eckhard; Steen, Hanno; Steen, Judith A

    2016-04-01

    Tauopathies, including Alzheimer's disease (AD), are associated with the aggregation of modified microtubule associated protein tau. This pathological state of tau is often referred to as "hyperphosphorylated". Due to limitations in technology, an accurate quantitative description of this state is lacking. Here, a mass spectrometry-based assay, FLEXITau, is presented to measure phosphorylation stoichiometry and provide an unbiased quantitative view of the tau post-translational modification (PTM) landscape. The power of this assay is demonstrated by measuring the state of hyperphosphorylation from tau in a cellular model for AD pathology, mapping, and calculating site occupancies for over 20 phosphorylations. We further employ FLEXITau to define the tau PTM landscape present in AD post-mortem brain. As shown in this study, the application of this assay provides mechanistic understanding of tau pathology that could lead to novel therapeutics, and we envision its further use in prognostic and diagnostic approaches for tauopathies. PMID:26877193

  19. Dynamin Binding Protein (Tuba) Deficiency Inhibits Ciliogenesis and Nephrogenesis in Vitro and in Vivo.

    Baek, Jeong-In; Kwon, Sang-Ho; Zuo, Xiaofeng; Choi, Soo Young; Kim, Seok-Hyung; Lipschutz, Joshua H

    2016-04-15

    Dysfunction of renal primary cilia leads to polycystic kidney disease. We previously showed that the exocyst, a protein trafficking complex, is essential for ciliogenesis and regulated by multiple Rho and Rab family GTPases, such as Cdc42. Cdc42 deficiency resulted in a disruption of renal ciliogenesis and a polycystic kidney disease phenotype in zebrafish and mice. Here we investigate the role of Dynamin binding protein (also known as Tuba), a Cdc42-specific guanine nucleotide exchange factor, in ciliogenesis and nephrogenesis using Tuba knockdown Madin-Darby canine kidney cells and tuba knockdown in zebrafish. Tuba depletion resulted in an absence of cilia, with impaired apical polarization and inhibition of hepatocyte growth factor-induced tubulogenesis in Tuba knockdown Madin-Darby canine kidney cell cysts cultured in a collagen gel. In zebrafish, tuba was expressed in multiple ciliated organs, and, accordingly, tuba start and splice site morphants showed various ciliary mutant phenotypes in these organs. Co-injection of tuba and cdc42 morpholinos at low doses, which alone had no effect, resulted in genetic synergy and led to abnormal kidney development with highly disorganized pronephric duct cilia. Morpholinos targeting two other guanine nucleotide exchange factors not known to be in the Cdc42/ciliogenesis pathway and a scrambled control morpholino showed no phenotypic effect. Given the molecular nature of Cdc42 and Tuba, our data strongly suggest that tuba and cdc42 act in the same ciliogenesis pathway. Our study demonstrates that Tuba deficiency causes an abnormal renal ciliary and morphogenetic phenotype. Tuba most likely plays a critical role in ciliogenesis and nephrogenesis by regulating Cdc42 activity. PMID:26895965

  20. Expression of ATP7B in human gastric cardiac carcinomas in comparison with distal gastric carcinomas

    Da-Long Wu; Hui-Xing Yi; Feng-Ying Sui; Xiao-Hong Jiang; Xiao-Ming Jiang; Ying-Ying Zhao

    2006-01-01

    AIM: To analyze expression of ATP7B in gastric cardiac adenocarcinomas, its clinicopathologic significance, in comparison with distal gastric adenocarcinomas.METHODS: Immunohistochemical avidin-biotin peroxidase complex method was applied to detect the expression of ATP7B in 49 cases of cardiac carcinomas,the corresponding adjacent non-neoplastic epithelium and 55 cases of distal gastric carcinomas.RESULTS: The proportion of ATP7B positive samples in gastric cardiac carcinomas (51.0%, 25 of 49) was significantly higher than that in the corresponding adjacent non-neoplastic epithelium (22.4%, 11 of 49)(P = 0.003). ATP7B expression in poorly differentiated gastric cardiac carcinomas was significantly higher than that in well/moderately differentiated gastric cardiac carcinomas (P = 0.030). ATP7B expression in gastric cardiac carcinomas was independent of age, tumor size, nodal stage and metastasis status. ATP7B protein was detected in 30.9% (17/55 cases) of distal gastric carcinomas, markedly lower than that in gastric cardiac carcinomas (P = 0.037).CONCLUSION: ATP7B protein is frequently overexpressed in gastric cardiac carcinomas, and correlated with the differentiation of cardiac carcinoma. ATP7B expression in gastric cardiac carcinomas is significantly higher than that in distal gastric carcinomas, which might partially explain the difference of chemotherapy response and prognosis between these two gastric carcinomas.

  1. In vivo and in vitro effects of growth hormone on the incorporation of [14C]leucine into protein of liver and muscle of the eel

    In vivo administration of ovine GH (2 micrograms/g body wt) increased [14C]leucine incorporation into protein of the liver, skeletal muscle, and opercular muscle of hypophysectomized eels. Addition of ovine GH into the medium slightly increased [14C]leucine incorporation into protein of liver slices during 5 hr in vitro incubation, but did not affect protein synthesis from [14C]leucine in opercular muscle. In vivo pretreatment with ovine GH 48 hr prior to tissue preparation clearly increased [14C]leucine incorporation into protein of liver slices in vitro. However, no statistically significant change was observed for in vitro incorporation of [14C]leucine into protein of opercular muscle of hypophysectomized eels which had been previously treated with ovine GH. These results indicate that ovine GH has a protein anabolic action in the liver and muscle of the eel and that compared to mammals a rather long lag period is needed to elicit such protein anabolic actions of GH in these animals

  2. Spectroscopic investigation on the sonodynamic damage to proteins in the presence of berberine in vitro

    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.

  3. Inhibitory effects of rutin on the endothelial protein C receptor shedding in vitro and in vivo.

    Ku, Sae-Kwang; Lee, In-Chul; Han, Min-Su; Bae, Jong-Sup

    2014-10-01

    Endothelial cell protein C receptor (EPCR) has important functions in regulation of coagulation and inflammation. EPCR shedding from the cell surface is mediated by tumor necrosis factor-α converting enzyme (TACE). Rutin is one of the major flavonoids from the buckwheat plant Fagopyrum tataricum. In this study, we investigated the effects of rutin on phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and on cecal ligation and puncture (CLP)-mediated EPCR shedding. We used a CLP model because this model more closely resembles human sepsis. Data showed rutin was a potent inhibitor of PMA, TNF-α, IL-1β, and CLP-induced EPCR shedding by suppression of TACE expression. Treatment with rutin resulted in a decrease of PMA-stimulated phosphorylation of p38, extracellular regulated kinases 1/2, and c-Jun N-terminal kinase. These results suggest the potential application of rutin for treatment of PMA and CLP-mediated EPCR shedding. PMID:24622777

  4. Modulation of enamel matrix proteins on the formation and nano-assembly of hydroxyapatite in vitro

    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: ► An aligned hydroxyapatite crystal bundled by rod-like nanosize crystals was obtained. ► An organized Amel/ACP would be the precursor of the bundled hydroxyapatite crystal prism. ► EMPs inhibit the growth of octacalcium phosphate in a defined plane.

  5. Modulation of enamel matrix proteins on the formation and nano-assembly of hydroxyapatite in vitro

    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.

  6. Interaction of plasminogen-related protein B with endothelial and smooth muscle cells in vitro.

    Morioka, Hideo; Morii, Takeshi; Vogel, Tikva; Hornicek, Francis J; Weissbach, Lawrence

    2003-07-01

    Plasminogen-related protein B (PRP-B) closely resembles the N-terminal plasminogen activation peptide, which is released from plasminogen during conversion to plasmin. We have previously demonstrated that the steady-state level of mRNA encoding PRP-B is increased within tumor tissues, and that recombinant PRP-B antagonizes neoplastic growth when administered systemically to mice harboring tumors, but no insights into the cell targets of PRP-B have been presented. Employing serum-free medium optimized for culturing human endothelial or smooth muscle cells, we show that recombinant PRP-B inhibits basic fibroblast growth factor-dependent cell migration for both cell types, as well as tube formation of endothelial cells. Comparison with the angiogenesis inhibitors angiostatin and endostatin revealed similar results. Recombinant PRP-B is effective in promoting cell attachment of endothelial and smooth muscle cells, and antibody interference experiments reveal that the interaction of recombinant PRP-B with endothelial cells is mediated at least in part by alpha(v)-containing integrins. Inhibition of angiogenesis in vivo by PRP-B was demonstrated in the chicken chorioallantoic membrane assay. PRP-B and other antiangiogenic molecules may elicit metabolic perturbations in endothelial cells as well as perivascular mesenchymal cells such as smooth muscle cells and pericytes. PMID:12799192

  7. The Ability to Associate with Activation Domains in vitro is not Required for the TATA Box-Binding Protein to Support Activated Transcription in vivo

    Tansey, William P.; Herr, Winship

    1995-11-01

    The TATA box-binding protein (TBP) interacts in vitro with the activation domains of many viral and cellular transcription factors and has been proposed to be a direct target for transcriptional activators. We have examined the functional relevance of activator-TBP association in vitro to transcriptional activation in vivo. We show that alanine substitution mutations in a single loop of TBP can disrupt its association in vitro with the activation domains of the herpes simplex virus activator VP16 and of the human tumor suppressor protein p53; these mutations do not, however, disrupt the transcriptional response of TBP to either activation domain in vivo. Moreover, we show that a region of VP16 distinct from its activation domain can also tightly associate with TBP in vitro, but fails to activate transcription in vivo. These data suggest that the ability of TBP to interact with activation domains in vitro is not directly relevant to its ability to support activated transcription in vivo.

  8. Casein kinase Ⅱ interacts with prion protein in vitro and forms complex with na-tive prion protein in vivo

    2008-01-01

    The most essential and crucial step during the pathogenesis of transmissible spongiform encephalopathy is the conformational change of cellular prion protein to pathologic isoform. Casein kinase Ⅱ (CK2) is a ubiquitously expressed and evolutiouarily conserved pleiotropic protein kinase that is essential for viability. To explore the possible molecular interaction between CK2 and prion protein (PrP), the full-length sequences of human CK2α and CK2β complementary DNA were amplified with reverse transcription-polymerase chain reaction using the total messenger RNA from cell line SH-SY5Y as the template; then, the fusion proteins histidine-CK2α and glutathione S-transferase-histidine-CK2β were expressed in Escherichia coll. The interaction between CK2 and PrP was evaluated with co-immunoprecipi-tation and pull-down assays. The results demonstrated that recombinant PrP bound specifically with CK2α, but not with CK2β. The native CK2 and PrP in hamster brains interacted with each other, forming protein complexes. Three different glycosylated forms of PrP (diglycosylated, monoglycosylated and unglycosylated PrP) from normal brains interacted with the CK2α subunit, though the unglycosylated PrP seemed to have a stronger binding ability with CK2α subunit. The domain responsible for interacting with CK2α was located at the C-terminal segment of PrP (residues 91-231). This study proposed reliable experimental data for the molecular interaction between PrP and CK2α (both in recombinant and native categories), scientific clues for further assessing the potential biological significance of the PrP-CK2 interaction, and the possible role of CK2 in the pathogenesis of prion diseases.

  9. Deguelin regulates nuclear pore complex proteins Nup98 and Nup88 in U937 cells in vitro

    Hong-li LIU; Yan CHEN; Guo-hui CUI; Qiu-ling WU; Jing HE; Wei-hua CHEN; Jian-feng ZHOU

    2005-01-01

    Aim: To investigate the anticancer effects and the molecular mechanisms of deguelin on human U937 leukemia cells, and to explore the underlying mechanism regulating nucleoporin 98 (Nup98) and nucleoporin 88 (Nup88) in vitro. Methods: The effects of deguelin on the growth of U937 cells were studied by MTT assay.The effect of deguelin on the cell cycle of U937 cells was studied by using a propidium iodide method. The localization of the nuclear pore complex proteins Nup98 and Nup88 was investigated by using immunofluorescence and immunoelectron microscopy. The expression of Nup98 and Nup88 in U937 cells was investigated by using flow cytometry and Western blot. Results: The proliferation of U937 cells was inhibited in the deguelin-treated group, with a 24-h IC50 value of 21.61 nmol/L and a 36-h IC50 value of 17.07 nmol/L. U937 cells treated with deguelin had reduced percentages of cells in the G0/G1 phase, whereas cells accumulated in the S and G2/M phases. Nup88 and Nup98 were found on both the nuclear and cytoplasmic sides of the U937 cells by using immunofluorescence and immunoelectron microscopy. The expression of Nup98 was upregulated and that of the Nup88 protein was downregulated in U937 cells treated with deguelin.Conclusion: Deguelin is able to inhibit the proliferation of U937 cells by regulating the cell cycle such that cells are arrested at the S and G2/M phases, so that the proportion of cells in the G0/G1 phase decreases. The antitumor effects of deguelin are related to upregulating the expression of Nup98 and downregulating the expression of Nup88 protein in U937 cells.

  10. In vitro haematic proteins adsorption and cytocompatibility study on acrylic copolymer to realise coatings for drug-eluting stents

    In the present paper, a preliminary in vitro analysis of biocompatibility of newly-synthesised acrylic copolymers is reported. In particular, with the aim to obtain coatings for drug-eluting stents, blood protein absorption and cytocompatibility were studied. For protein absorption tests, bovine serum albumin and bovine plasma fibrinogen were considered. Cytocompatibility was tested using C2C12 cell line as model, analysing the behaviour of polymeric matrices and of drug-eluting systems, obtained loading polymeric matrices with paclitaxel, an anti-mitotic drug, in order to evaluate the efficacy of a pharmacological treatment locally administered from these materials. Results showed that the amount of albumin absorbed was greater than the amount of fibrinogen (comprised in the range of 70%–85% and 10%–22% respectively) and it is a good behaviour in terms of haemocompatibility. Cell culture tests showed good adhesion properties and a relative poor proliferation. In addition, a strong effect related to drug elution and a correlation with the macromolecular composition were detected. In this preliminary analysis, tested materials showed good characteristics and can be considered possible candidates to obtain coatings for drug-eluting stents. Highlights: ► Preliminary evaluation of haemo- and cytocompatibility of newly-synthesised acrylic copolymers ► Materials adsorb higher amounts of albumin and with a faster rate than fibrinogen. ► Protein adsorption depended on the macromolecular composition and surface properties. ► Cell viability on pure samples and efficacy of paclitaxel release were verified in C2C12 cultures.

  11. Production of Fibronectin Binding Protein A at the surface of Lactococcus lactis increases plasmid transfer in vitro and in vivo.

    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.

  12. In vitro haematic proteins adsorption and cytocompatibility study on acrylic copolymer to realise coatings for drug-eluting stents

    Gagliardi, Mariacristina, E-mail: mariacristina.gagliardi@iit.it

    2012-12-01

    In the present paper, a preliminary in vitro analysis of biocompatibility of newly-synthesised acrylic copolymers is reported. In particular, with the aim to obtain coatings for drug-eluting stents, blood protein absorption and cytocompatibility were studied. For protein absorption tests, bovine serum albumin and bovine plasma fibrinogen were considered. Cytocompatibility was tested using C2C12 cell line as model, analysing the behaviour of polymeric matrices and of drug-eluting systems, obtained loading polymeric matrices with paclitaxel, an anti-mitotic drug, in order to evaluate the efficacy of a pharmacological treatment locally administered from these materials. Results showed that the amount of albumin absorbed was greater than the amount of fibrinogen (comprised in the range of 70%-85% and 10%-22% respectively) and it is a good behaviour in terms of haemocompatibility. Cell culture tests showed good adhesion properties and a relative poor proliferation. In addition, a strong effect related to drug elution and a correlation with the macromolecular composition were detected. In this preliminary analysis, tested materials showed good characteristics and can be considered possible candidates to obtain coatings for drug-eluting stents. Highlights: Black-Right-Pointing-Pointer Preliminary evaluation of haemo- and cytocompatibility of newly-synthesised acrylic copolymers Black-Right-Pointing-Pointer Materials adsorb higher amounts of albumin and with a faster rate than fibrinogen. Black-Right-Pointing-Pointer Protein adsorption depended on the macromolecular composition and surface properties. Black-Right-Pointing-Pointer Cell viability on pure samples and efficacy of paclitaxel release were verified in C2C12 cultures.

  13. Liver fatty acid-binding protein binds monoacylglycerol in vitro and in mouse liver cytosol.

    Lagakos, William S; Guan, Xudong; Ho, Shiu-Ying; Sawicki, Luciana Rodriguez; Corsico, Betina; Kodukula, Sarala; Murota, Kaeko; Stark, Ruth E; Storch, Judith

    2013-07-01

    Liver fatty acid-binding protein (LFABP; FABP1) is expressed both in liver and intestinal mucosa. Mice null for LFABP were recently shown to have altered metabolism of not only fatty acids but also monoacylglycerol, the two major products of dietary triacylglycerol hydrolysis (Lagakos, W. S., Gajda, A. M., Agellon, L., Binas, B., Choi, V., Mandap, B., Russnak, T., Zhou, Y. X., and Storch, J. (2011) Am. J. Physiol. Gastrointest. Liver Physiol. 300, G803-G814). Nevertheless, the binding and transport of monoacylglycerol (MG) by LFABP are uncertain, with conflicting reports in the literature as to whether this single chain amphiphile is in fact bound by LFABP. In the present studies, gel filtration chromatography of liver cytosol from LFABP(-/-) mice shows the absence of the low molecular weight peak of radiolabeled monoolein present in the fractions that contain LFABP in cytosol from wild type mice, indicating that LFABP binds sn-2 MG in vivo. Furthermore, solution-state NMR spectroscopy demonstrates two molecules of sn-2 monoolein bound in the LFABP binding pocket in positions similar to those found for oleate binding. Equilibrium binding affinities are ∼2-fold lower for MG compared with fatty acid. Finally, kinetic studies examining the transfer of a fluorescent MG analog show that the rate of transfer of MG is 7-fold faster from LFABP to phospholipid membranes than from membranes to membranes and occurs by an aqueous diffusion mechanism. These results provide strong support for monoacylglycerol as a physiological ligand for LFABP and further suggest that LFABP functions in the efficient intracellular transport of MG. PMID:23658011

  14. Liver Fatty Acid-binding Protein Binds Monoacylglycerol in Vitro and in Mouse Liver Cytosol*

    Lagakos, William S.; Guan, Xudong; Ho, Shiu-Ying; Sawicki, Luciana Rodriguez; Corsico, Betina; Kodukula, Sarala; Murota, Kaeko; Stark, Ruth E.; Storch, Judith

    2013-01-01

    Liver fatty acid-binding protein (LFABP; FABP1) is expressed both in liver and intestinal mucosa. Mice null for LFABP were recently shown to have altered metabolism of not only fatty acids but also monoacylglycerol, the two major products of dietary triacylglycerol hydrolysis (Lagakos, W. S., Gajda, A. M., Agellon, L., Binas, B., Choi, V., Mandap, B., Russnak, T., Zhou, Y. X., and Storch, J. (2011) Am. J. Physiol. Gastrointest. Liver Physiol. 300, G803–G814). Nevertheless, the binding and transport of monoacylglycerol (MG) by LFABP are uncertain, with conflicting reports in the literature as to whether this single chain amphiphile is in fact bound by LFABP. In the present studies, gel filtration chromatography of liver cytosol from LFABP−/− mice shows the absence of the low molecular weight peak of radiolabeled monoolein present in the fractions that contain LFABP in cytosol from wild type mice, indicating that LFABP binds sn-2 MG in vivo. Furthermore, solution-state NMR spectroscopy demonstrates two molecules of sn-2 monoolein bound in the LFABP binding pocket in positions similar to those found for oleate binding. Equilibrium binding affinities are ∼2-fold lower for MG compared with fatty acid. Finally, kinetic studies examining the transfer of a fluorescent MG analog show that the rate of transfer of MG is 7-fold faster from LFABP to phospholipid membranes than from membranes to membranes and occurs by an aqueous diffusion mechanism. These results provide strong support for monoacylglycerol as a physiological ligand for LFABP and further suggest that LFABP functions in the efficient intracellular transport of MG. PMID:23658011

  15. Altered localisation of the copper efflux transporters ATP7A and ATP7B associated with cisplatin resistance in human ovarian carcinoma cells

    Copper homeostasis proteins ATP7A and ATP7B are assumed to be involved in the intracellular transport of cisplatin. The aim of the present study was to assess the relevance of sub cellular localisation of these transporters for acquired cisplatin resistance in vitro. For this purpose, localisation of ATP7A and ATP7B in A2780 human ovarian carcinoma cells and their cisplatin-resistant variant, A2780cis, was investigated. Sub cellular localisation of ATP7A and ATP7B in sensitive and resistant cells was investigated using confocal fluorescence microscopy after immunohistochemical staining. Co-localisation experiments with a cisplatin analogue modified with a carboxyfluorescein-diacetate residue were performed. Cytotoxicity of the fluorescent cisplatin analogue in A2780 and A2780cis cells was determined using an MTT-based assay. The significance of differences was analysed using Student's t test or Mann-Whitney test as appropriate, p values of < 0.05 were considered significant. In the sensitive cells, both transporters are mainly localised in the trans-Golgi network, whereas they are sequestrated in more peripherally located vesicles in the resistant cells. Altered localisation of ATP7A and ATP7B in A2780cis cells is likely to be a consequence of major abnormalities in intracellular protein trafficking related to a reduced lysosomal compartment in this cell line. Changes in sub cellular localisation of ATP7A and ATP7B may facilitate sequestration of cisplatin in the vesicular structures of A2780cis cells, which may prevent drug binding to genomic DNA and thereby contribute to cisplatin resistance. Our results indicate that alterations in sub cellular localisation of transport proteins may contribute to cisplatin resistance in vitro. Investigation of intracellular protein localisation in primary tumour cell cultures and tumour tissues may help to develop markers of clinically relevant cisplatin resistance. Detection of resistant tumours in patients may in turn

  16. Altered localisation of the copper efflux transporters ATP7A and ATP7B associated with cisplatin resistance in human ovarian carcinoma cells

    Reedijk Jan

    2008-06-01

    Full Text Available Abstract Background Copper homeostasis proteins ATP7A and ATP7B are assumed to be involved in the intracellular transport of cisplatin. The aim of the present study was to assess the relevance of sub cellular localisation of these transporters for acquired cisplatin resistance in vitro. For this purpose, localisation of ATP7A and ATP7B in A2780 human ovarian carcinoma cells and their cisplatin-resistant variant, A2780cis, was investigated. Methods Sub cellular localisation of ATP7A and ATP7B in sensitive and resistant cells was investigated using confocal fluorescence microscopy after immunohistochemical staining. Co-localisation experiments with a cisplatin analogue modified with a carboxyfluorescein-diacetate residue were performed. Cytotoxicity of the fluorescent cisplatin analogue in A2780 and A2780cis cells was determined using an MTT-based assay. The significance of differences was analysed using Student's t test or Mann-Whitney test as appropriate, p values of Results In the sensitive cells, both transporters are mainly localised in the trans-Golgi network, whereas they are sequestrated in more peripherally located vesicles in the resistant cells. Altered localisation of ATP7A and ATP7B in A2780cis cells is likely to be a consequence of major abnormalities in intracellular protein trafficking related to a reduced lysosomal compartment in this cell line. Changes in sub cellular localisation of ATP7A and ATP7B may facilitate sequestration of cisplatin in the vesicular structures of A2780cis cells, which may prevent drug binding to genomic DNA and thereby contribute to cisplatin resistance. Conclusion Our results indicate that alterations in sub cellular localisation of transport proteins may contribute to cisplatin resistance in vitro. Investigation of intracellular protein localisation in primary tumour cell cultures and tumour tissues may help to develop markers of clinically relevant cisplatin resistance. Detection of resistant tumours

  17. Synthetic in vitro circuits

    Hockenberry, Adam J.; Jewett, Michael C.

    2012-01-01

    Inspired by advances in the ability to construct programmable circuits in living organisms, in vitro circuits are emerging as a viable platform for designing, understanding, and exploiting dynamic biochemical circuitry. In vitro systems allow researchers to directly access and manipulate biomolecular parts without the unwieldy complexity and intertwined dependencies that often exist in vivo. Experimental and computational foundations in DNA, DNA/RNA, and DNA/RNA/protein based circuitry have g...

  18. Effect of simulated processing on the antioxidant capacity and in vitro protein digestion of fruit juice-milk beverage model systems.

    He, Zhiyong; Yuan, Bo; Zeng, Maomao; Tao, Guanjun; Chen, Jie

    2015-05-15

    The effects of simulated processing (pH adjustment and thermal treatment) on the antioxidant capacity and in vitro protein digestion of fruit juice-milk beverage (FJMB) models consisting of whey protein (WP), and chlorogenic acid (CHA) or catechin (CAT) were investigated. Results indicated that CAT was more susceptible to processing than CHA, and showed a significant (p 0.05) by pasteurization, whereas sterilization initially accelerated WP digestion but did not change its overall digestibility. PMID:25577106

  19. Effect of domestic processing on the cooking time, nutrients, antinutrients and in vitro protein digestibility of the African yambean (Sphenostylis stenocarpa).

    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 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. PMID:9139303

  20. In vitro mutation breeding for seed protein, alpha amylase activity and herbicide resistance in bulrush millet (Peenisetum nigritarum)

    In vitro mutation breeding in bulrush millet (Penissetum nigritarum) was investigated using chemical mutagenesis followed by selection. Mutations were induced by soaking dry viable seeds at room temperature in 8 mM ethylmethanesulphonate (EMS) for 15 hours or in 64 mM EMS for 3, 6, 9, 12, 15 and 24 hours. After treatment and rinsing in running water, the seeds were germinated in Petri dishes and later transplanted to loamy sand oil and grown to maturity in the greenhouse. Screening and analysis of 2500 M2 plants yielded a broad spectrum of mutations, including leaf variation and agronomically useful attributes such as improved and early germinability (+15% and +10 hours, respectively), and a higher seed protein content, alpha amylase activity and herbicide resistance. EMS did not reduce cell viability, but it produced a high frequency of mutations, accompanied by a relatively low frequency of chromosomal aberrations. The appropriate dosage and duration for mutation breeding was 8 mM for 15 hours or 64 mM for 3 hours. (author). 9 refs, 2 figs, 4 tabs

  1. Effects of protein source, vitamin E and phenazine ethosulfate on developmental competence and quality of porcine embryos cultured in vitro.

    Gajda, Barbara; Bryła, Magdalena; Smorag, Zdzisław

    2008-01-01

    Effects of fetal calf serum (FCS) or bovine serum albumin (BSA), with or without vitamin E (vit. E) or phenazine ethosulfate (PES) supplementation on developmental competence and quality of cultured porcine embryos were examined. The experiment was done on zygotes and 2-cell embryos obtained from superovulated gilts. Morphologically normal zygotes were cultured in vitro in NCSU-23 medium supplemented with: experiment 1-0.004 g/ml BSA, 10% FCS, protein-free (control); experiment 2-0 (control), 25, 50 or 100 microM vit. E; experiment 3-0 (control), 0.025, 0.05 or 0.075 microM PES. Embryo quality criteria were developmental competence (cleavage, morula and blastocyst rates), total cell number per blastocyst and degree of apoptosis as assessed by TUNEL staining. Presence of BSA in culture medium increased significantly morula and blastocysts production as compared to FCS (P DNA fragmented nucleus index as compared to the BSA (P < 0.05 and P < 0.01, respectively) and FCS (P < 0.5) group. Supplementation in culture medium of 100 microM vit. E increased blastocyst production as compared to control and 50 microM vit-E (P < 0.05). Both the number of cells per and percentage of TUNEL positive nuclei per blastocyst were slightly lower in PES treated than control groups. PMID:19055026

  2. Role and mechanism of uncoupling protein 2 on the fatty acid-induced dysfunction of pancreatic alpha cells in vitro

    SU Jie-ying; LI Hong-liang; YANG Wen-ying; XIAO Jian-zhong; DU Rui-qin; SHEN Xiao-xia; CAI Zhe; ZHANG Lan; SHU Jun

    2010-01-01

    Background Uncoupling protein (UCP) 2 is related to the dysfunction of beta cells induced by fatty acids. However,whether UCP2 has similar effects on alpha cell is still not clear. This study aimed to investigate the effects of UCP2 and its possible mechanisms in lipotoxicity-induced dysfunction of pancreatic alpha cells.Methods The alpha TC1-6 cells were used in this study to evaluate the effects of palmitate and/or UCP2 inhibit factors on the glucagon secretory function, glucagon content, the glucagon mRNA level and the nitrotyrosine level in the supernatant. Meantime, the expression levels of UCP2 and peroxisome proliferator-activated receptor-γ coactivator-1 alpha (PGC-1 alpha) were measured by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Furthermore, the possible relationship between UCP2 and insulin signal transduction pathway was analyzed.Results Palmitate stimulated alpha cell glucagon secretion and the expression of UCP2 and PGC-1 alpha, which could be partially decreased by the inhibition of UCP2. Palmitate increased nitrotyrosine level and suppressed insulin signal transduction pathway in alpha cells. Inhibition of UCP2 influenced the effects of free fatty acid on alpha cells and may relate to glucagon secretion.Conclusion UCP2 played an important role on alpha cell dysfunction induced by free fatty acid in vitro, which may be related to its effects on oxidative stress and insulin signal transduction pathway.

  3. Synthesis of nano-bioactive glass-ceramic powders and its in vitro bioactivity study in bovine serum albumin protein

    Nabian, Nima; Jahanshahi, Mohsen; Rabiee, Sayed Mahmood

    2011-07-01

    Bioactive glasses and ceramics have proved to be able to chemically bond to living bone due to the formation of an apatite-like layer on its surface. The aim of this work was preparation and characterization of bioactive glass-ceramic by sol-gel method. Nano-bioglass-ceramic material was crushed into powder and its bioactivity was examined in vitro with respect to the ability of hydroxyapatite layer to form on the surface as a result of contact with bovine serum albumin (BSA) protein. The obtained nano-bioactive glass-ceramic was analyzed before and after contact with BSA solution. This study used scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray powder diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) analysis to examine its morphology, crystallinity and composition. The TEM images showed that the NBG particles size were 10-40 nm. Bioactivity of nanopowder was confirmed by SEM and XRD due to the presence of a rich bone-like apatite layer. Therefore, this nano-BSA-bioglass-ceramic composite material is promising for medical applications such as bone substitutes and drug carriers.

  4. Topological and evolutional relationships between HCV core protein and hepatic lipid vesicles: Studies in vitro and in conditionally transgenic mice

    Ming-Ling Chang; Jeng-Chang Chen; Chau-Ting Yeh; I-Shyan Sheen; Dar-In Tai; Ming-Yu Chang; Cheng-Tang Chiu; Deng-Yn Lin; D Montgomery Bissell

    2007-01-01

    AIM: To investigate a specific association between hepatic steatosis and hepatitis C virus (HCV) core.METHODS: HeLa cells and primary mouse hepatocytes were transfected with HCV core plasmid, and conditional transgenics in which hepatic over-expression of HCV core is regulated by the tetracycline-off system, were developed. The expression of the HCV core was assessed over one to six months after withdrawal of doxycycline (dox) by immunohistochemistry (IHC)and Western blotting and by sequential liver biopsy.Hepatic steatosis was evaluated using oil red stain.8-hydroxydeoxyguanosine (8-OHdG) stains and caspase levels were conducted to clarify hepatic oxidative stress and apoptosis rate. Serum aminotransferase was checked.RESULTS: The transfected hepatocytes had globular cores under the lipid vesicles. In transgenic mice on control diet, core expression was robust, localized to the cytoplasmic vesicle membrane and strongly associated with microvesicular steatosis, which was gradually replaced by macrovesicular steatosis. However, both steatosis and core positive hepatocytes diminished with time. Increases in aminotransferase, caspase and 8-OHdG were associated with peak core expression.CONCLUSION: The core protein was readily detected and morphologically associated with steatosis in individual hepatocytes both in vitro and in vivo. In vivo,oxidative stress caused by the core potentially reduced the number of core positive hepatocytes and in parallel the level of steatosis. To our knowledge, this is the first animal model that directly shows topological relationship between HCV core and hepatic lipid vesicles.

  5. Phytic acid, in vitro protein digestibility, dietary fiber, and minerals of pulses as influenced by processing methods.

    Chitra, U; Singh, U; Rao, P V

    1996-06-01

    The objective of this project was to determine the effect of various types of processing on selected nutrition related parameters of commonly consumed Indian pulses and soybean. Germination reduced the phytic acid content of chickpea and pigeonpea seeds by over 60%, and that of mung bean, urd bean, and soybean by about 40%. Fermentation reduced phytic acid contents by 26-39% in all these legumes with the exception of pigeonpea in which it was reduced by more than 50%. Autoclaving and roasting were more effective in reducing phytic acid in chickpea and pigeonpea than in urd bean, mung bean, and soybean. Germination and fermentation greatly increased the in vitro protein digestibility (IVPD). IVPD was only slightly increased by roasting and autoclaving of all legumes. Germination and fermentation also remarkably decreased the total dietary fiber (TDF) in all legumes. Autoclaving and roasting resulted in slight increases in TDF values. All the processing treatments had little effect on calcium, magnesium and iron contents. PMID:8983057

  6. Effects of electron beam irradiation on chemical composition, antinutritional factors, ruminal degradation and in vitro protein digestibility of canola meal

    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.

  7. Effects of electron beam irradiation on chemical composition, antinutritional factors, ruminal degradation and in vitro protein digestibility of canola meal

    Taghinejad-Roudbaneh, M.; Ebrahimi, S. R.; Azizi, S.; Shawrang, P.

    2010-12-01

    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.

  8. Effects of different tannin-rich extracts and rapeseed tannin monomers on methane formation and microbial protein synthesis in vitro.

    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

  9. Studies on Single Cell Culture in vitro in Wheat--The variation of grain protein content and its fractions from regenerated plants

    1999-01-01

    On the basis of previous studies dealing with the variation of major agronomic and yield characteristics of regenerated plants derived from single cell culture in vitro of common wheat (Triticum aestivum L.Cultivar NE 7742), the grain protein content and its fractions from regenerated plants with stable agronomic characteristics were studied from 1992 to 1995. The results showed that the variation of grain protein content and its fractions in somaclones from single cell culture in vitro were very significant and the range was very wide (11.53~17.70%). Several types of variation were found in the studies, especially the type with higher protein content than that of cultivar NE 7742 (non-culture parent). Among them, -20.69% of lines the grain protein content was significantly higher than that of NE 7742 and combined with high yielding potential. The tendency of variation of the four protein fractions showed that the variation of albumin was not obvious and maintained the same level as NE7742, the content of gliadin increased in some somaclones and decreased in others. However, the percentages both globulin and glutenin tended to increase. The variation of total amount of structural protein and the ratio between globulin and glutenin tended to increase. The variation of total amount of structural protein and the ratio between globulin and albumm was mainly influenced by globulin under the condition of culture in vitro. The variation of total amount of storage protein and the ratio between gliadin and glutenin was mainly affected by glutenin. The results mentioned above demonstrated that the induction and screening of somaclonal variation could be an effective way in wheat improvement in combining high protein content with high yield.

  10. Influence of EMAP II, IFN-α2b and its medicinal preparations on the MGMT protein amount in human cells in vitro

    Lylo V. V.; Ruban T. P.; Macewicz L. L.; Kornelyuk A. I.; Chernykh S. I.; Lukash L. L.

    2014-01-01

    Aim. To study the effect of EMAP II, IFN-α2b and its medicinal preparations on the amount of O6-methylguanine-DNA methyltransferase (MGMT) protein in human cells in vitro. Methods. The human cells of 4BL and Hep-2 lines were treated with the purified recombinant proteins EMAP II, IFN-α2b and its commercial me dicinal preparations. Changes in the MGMT gene expression were studied at a protein level by Western blot analysis. Results. Treatment of Hep-2 and 4BL cells with EMAP II at the concentr...

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

    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 2...

  12. Degradation in vitro of [3H]lysine and [14C]leucine-labelled protein by proteases from developing normal and opaque-2 Zea mays endosperm

    During later stages of kernel development total lysine content per endosperm in normal and opaque-2 maize endosperms decreased in spite of net increase in total protein. The proteolytic enzymes increased during development until 25 d after pollination and later declined in both normal and opaque-2 endosperms. Release of lysine in vitro from [3H]lysine and [14C]leucine-labelled normal maize endosperm proteins by proteases from developing normal maize endosperms was higher than in opaque-2 endosperms towards later stages of maturity. Present study suggests that lysine-liberating proteases present in endosperms also contribute to decrease in lysine and consequently the quality of protein. (author)

  13. Adipose tissue-deprived stem cells acquire cementoblast features treated with dental follicle cell conditioned medium containing dentin non-collagenous proteins in vitro

    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.

  14. Adipose tissue-deprived stem cells acquire cementoblast features treated with dental follicle cell conditioned medium containing dentin non-collagenous proteins in vitro

    Highlights: → In this study we examine the effects of dental follicle cell conditioned medium (DFCCM) containing dentin non-collagenous proteins (dNCPs) on differentiation of ADSCs. → We examined that ADSCs treated with dNCPs/DFCCM underwent morphological changes and significantly lost their proliferative capacity. → dNCPs/DFCCM enhanced the mineralization behaviour and mineralization-related marker expression of ADSCs. → 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.

  15. Effects of vanillin, quillaja saponin, and essential oils on in vitro fermentation and protein-degrading microorganisms of the rumen.

    Patra, Amlan K; Yu, Zhongtang

    2014-01-01

    This study investigated the effects of vanillin on methanogenesis and rumen fermentation, and the responses of ruminal protein-degrading bacteria to vanillin (at concentrations of 0, 0.76 and 1.52 g/L), essential oils (clove oil, 1 g/L; origanum oil, 0.50 g/L, and peppermint oil, 1 g/L), and quillaja saponin (at concentration of 0 and 6 g/L) in vitro. Methane production, degradabilities of feed substrate, and ammonia concentration decreased linearly with increasing doses of vanillin. Concentration of total volatile fatty acids also decreased, whereas proportion of butyrate tended to increase linearly with increasing doses of vanillin. Protozoa population decreased, but abundances of Ruminococcus flavefaciens, Prevotella bryantii, Butyrivibrio fibrisolvens, Prevotella ruminicola, Clostridium aminophilum, and Ruminobacter amylophilus increased with increasing doses of vanillin. Origanum and clove oils resulted in lower ammonia concentrations compared to control and peppermint oil. All the tested essential oils decreased abundances of protozoa, Selenomonas ruminantium, R. amylophilus, P. ruminicola and P. bryantii, with the largest decrease resulted from origanum oil followed by clove oil and peppermint oil. The abundances of Megasphaera elsdenii, C. aminophilum, and Clostridium sticklandii were deceased by origanum oil while that of B. fibrisolvens was lowered by both origanum and clove oils. Saponin decreased ammonia concentration and protozoal population, but increased the abundances of S. ruminantium, R. amylophilus, P. ruminicola, and P. bryantii, though the magnitude was small (less than one log unit). The results suggest that reduction of ammonia production by vanillin and saponin may not be caused by direct inhibition of major known proteolytic bacteria, and essential oils can have different inhibitory effects on different proteolytic bacteria, resulting in varying reduction in ammonia production. PMID:23624710

  16. MicroRNA let-7b targets important cell cycle molecules in malignant melanoma cells and interferes with anchorage-independent growth

    Julia Schultz; Peter Lorenz; Gerd Gross; Saleh Ibrahim; Manfred Kunz

    2008-01-01

    A microRNA expression screen was performed analyzing 157 different microRNAs in laser-microdissected tissues from benign melanocytic nevi (n=10) and primary malignant melanomas (n=10),using quantitative real-time PCR.Differential expression was found for 72 microRNAs.Members of the let-7 family of microRNAs were significantly downregulated in primary melanomas as compared with benign nevi,suggestive for a possible role of these molecules as tumor suppressors in malignant melanoma.Interestingly,similar findings had been described for lung and colon cancer.Overexpression of let-7b in melanoma cells in vitro downregulated the expression of cyclins D1,D3,and A,and cyclin-dependent kinase (Cdk) 4,all of which had been described to play a role in melanoma development.The effect oflet-7b on protein expression was due to targeting of 3'-untranslated regions (3'UTRs) of individual mRNAs,as exemplified by reporter gene analyses for cyclin DI.In line with its downmodulating effects on cell cycle regulators,let-7b inhibited cell cycle progression and anchorage-independent growth of melanoma cells.Taken together,these findings not only point to new regulatory mechanisms of early melanoma development,but also may open avenues for future targeted therapies of this tumor.

  17. Transcription by Methanothermobacter thermautotrophicus RNA Polymerase In Vitro Releases Archaeal Transcription Factor B but Not TATA-Box Binding Protein from the Template DNA

    Xie, Yunwei; Reeve, John N.

    2004-01-01

    Transcription initiation in Archaea requires the assembly of a preinitiation complex containing the TATA- box binding protein (TBP), transcription factor B (TFB), and RNA polymerase (RNAP). The results reported establish the fate of Methanothermobacter thermautotrophicus TBP and TFB following transcription initiation by M. thermautotrophicus RNAP in vitro. TFB is released after initiation, during extension of the transcript from 4 to 24 nucleotides, but TBP remains bound to the template DNA. ...

  18. High-Mobility Group Chromatin Proteins 1 and 2 Functionally Interact with Steroid Hormone Receptors To Enhance Their DNA Binding In Vitro and Transcriptional Activity in Mammalian Cells

    Boonyaratanakornkit, Viroj; Melvin, Vida; Prendergast, Paul; Altmann, Magda; Ronfani, Lorenza; Marco E. Bianchi; Taraseviciene, Laima; Nordeen, Steven K.; Allegretto, Elizabeth A.; Edwards, Dean P.

    1998-01-01

    We previously reported that the chromatin high-mobility group protein 1 (HMG-1) enhances the sequence-specific DNA binding activity of progesterone receptor (PR) in vitro, thus providing the first evidence that HMG-1 may have a coregulatory role in steroid receptor-mediated gene transcription. Here we show that HMG-1 and the highly related HMG-2 stimulate DNA binding by other steroid receptors, including estrogen, androgen, and glucocorticoid receptors, but have no effect on DNA binding by se...

  19. Recombinant human interleukin-2 reverses in vitro-deficient cell-mediated immune responses to tuberculin purified protein derivative by lymphocytes of tuberculous patients.

    Shiratsuchi, H; Okuda, Y.; Tsuyuguchi, I

    1987-01-01

    In vitro lymphocyte proliferative response to purified protein derivative of tuberculin (PPD) was investigated in patients with tuberculosis. Peripheral blood lymphocytes (PBL) from patients with advanced, refractory tuberculosis showed a significantly depressed response compared with the response of PBL from patients with newly diagnosed tuberculosis (P less than 0.01). A further characterization of this low responsiveness to PPD revealed that PBL from these advanced tuberculous patients fai...

  20. In Vitro Resistance of Staphylococcus aureus to Thrombin-Induced Platelet Microbicidal Protein Is Associated with Alterations in Cytoplasmic Membrane Fluidity

    Bayer, Arnold S.; Prasad, Rajendra; Chandra, Jyotsna; Koul, Anjni; Smriti, M.; Varma, Archana; Skurray, Ronald A.; Firth, Neville; Brown, Melissa H.; Koo, Su-Pin; Yeaman, Michael R.

    2000-01-01

    Platelet microbicidal proteins (PMPs) are small, cationic peptides which possess potent microbicidal activities against common bloodstream pathogens, such as Staphylococcus aureus. We previously showed that S. aureus strains exhibiting resistance to thrombin-induced PMP (tPMP-1) in vitro have an enhanced capacity to cause human and experimental endocarditis (T. Wu, M. R. Yeaman, and A. S. Bayer, Antimicrob. Agents Chemother. 38:729–732, 1994; A. S. Bayer et al., Antimicrob. Agents Chemother. ...

  1. Influence of Processing on Dietary Fiber, Tannin and <i>in Vitro</i> Protein Digestibility of Pearl Millet

    Florence Suma Pushparaj; Asna Urooj

    2011-01-01

    From the nutritional point of view, data on dietary fiber content, tannin and in vitro protein digestibility of processed millet is of importance, because millets are never eaten raw. Effects of commonly used traditional methods on dietary fiber, tannin content and %IVPD of two locally available pearl millet varieties (Kalukombu and Maharashtra Rabi Bajra) were investigated. The millet was subjected to various processing methods like milling (whole flour, semi refined flour and bran rich frac...

  2. Drosophila Sgs3 TATA: effects of point mutations on expression in vivo and protein binding in vitro with staged nuclear extracts.

    Giangrande, A.; Mettling, C; Martin, M.; Ruiz, C.; Richards, G

    1989-01-01

    The Drosophila salivary gland secretion protein gene, Sgs3, has a consensus TATA sequence and gives rise to abundant stage and tissue-specific transcripts. Two TATA point mutations (TAAA and TAGA) reduce transcript levels approximately 50-fold when assayed in transgenic flies. This effect is reflected in vitro, in DNase I footprint and gel retardation assays where we observed TATA-probe-specific complexes that are not seen with TAAA, TAGA or non-specific probes. The binding patterns observed ...

  3. In vitro expression levels of cell-cycle checkpoint proteins are associated with cellular DNA repair capacity in peripheral blood lymphocytes: a multivariate analysis

    Fan, You-Hong; Hu, Zhibin; Li, Chunying; Wang, Li-E; Guo, Zhaozheng; Qiao, Yawei; Zhang, Li; Zhang, Wei; Mao, Li; Wei, Qingyi

    2007-01-01

    DNA repair should occur after cells sense DNA damage signals and undergo cell-cycle arrest to provide sufficient time for DNA repair, and suboptimal DNA repair capacity (DRC) in peripheral lymphocytes has been suggested as a cancer susceptibility marker. Numerous studies showed a functional link between DNA damage sensing, cell-cycle checkpoint and DNA repair. We hypothesized that in vitro cell-cycle checkpoint-related protein expression levels in stimulated lymphocytes predict DRC levels. To...

  4. Application of Escherichia coli phage K1E DNA-dependent RNA polymerase for in vitro RNA synthesis and in vivo protein production in Bacillus megaterium.

    Stammen, Simon; Schuller, Franziska; Dietrich, Sylvia; Gamer, Martin; Biedendieck, Rebekka; Jahn, Dieter

    2010-09-01

    Gene "7" of Escherichia coli phage K1E was proposed to encode a novel DNA-dependent RNA polymerase (RNAP). The corresponding protein was produced recombinantly, purified to apparent homogeneity via affinity chromatography, and successfully employed for in vitro RNA synthesis. Optimal assay conditions (pH 8, 37 degrees C, 10 mM magnesium chloride and 1.3 mM spermidine) were established. The corresponding promoter regions were identified on the phage genome and summarized in a sequence logo. Surprisingly, next to K1E promoters, the SP6 promoter was also recognized efficiently in vitro by K1E RNAP, while the T7 RNAP promoter was not recognized at all. Based on these results, a system for high-yield in vitro RNA synthesis using K1E RNAP was established. The template plasmid is a pUC18 derivative, which enables blue/white screening for positive cloning of the target DNA. Production of more than 5 microg of purified RNA per microgram plasmid DNA was achieved. Finally, in vivo protein production systems for Bacillus megaterium were established based on K1E and SP6 phage RNAP transcription. Up to 61.4 mg g (CDW) (-1) (K1E RNAP) of the reporter protein Gfp was produced in shaking flask cultures of B. megaterium. PMID:20596705

  5. BAY 61-3606, CDKi, and Sodium Butyrate Treatments Modulate p53 Protein Level and Its Site-Specific Phosphorylation in Human Vestibular Schwannomas In Vitro

    Rohan Mitra

    2014-01-01

    Full Text Available This study is done to evaluate the effect of spleen tyrosine kinase inhibitor (BAY 61-3606, cyclin-dependent kinase inhibitor (CDKi, and sodium butyrate (Na-Bu on the level and phosphorylation of p53 protein and its binding to murine double minute 2 (MDM2 homologue in human vestibular schwannomas (VS. Primary cultures of the tumor tissues were treated individually with optimum concentrations of these small molecules in vitro. The results indicate modulation of p53 protein status and its binding ability to MDM2 in treated samples as compared to the untreated control. The three individual treatments reduced the level of total p53 protein. These treatments also decreased Ser392 and Ser15 phosphorylated p53 in tumor samples of young patients and Ser315 phosphorylated p53 in old patients. Basal level of Thr55 phosphorylated p53 protein was present in all VS samples and it remained unchanged after treatments. The p53 protein from untreated VS samples showed reduced affinity to MDM2 binding in vitro and it increased significantly after treatments. The MDM2/p53 ratio increased approximately 3-fold in the treated VS tumor samples as compared to the control. The differential p53 protein phosphorylation status perhaps could play an important role in VS tumor cell death due to these treatments that we reported previously.

  6. LAS PROTEÍNAS β3 Y PDI AISLADAS DE PLAQUETAS HUMANAS SE UNEN CON EL ROTAVIRUS ECwt IN VITRO β3 and PDI proteins isolated from human platelets bind with ECwt rotavirus in vitro

    Diana Mayorga

    2010-01-01

    policlonal contra β3 y establecer que β3 y PDI se unen in vitro, luego de incubar las proteínas aisladas con el rotavirus ECwt, e in vivo, después de incubar el rotavirus con las vellosidades aisladas del intestino delgado de ratón lactante de la cepa ICR.Background. Commercial integrin β3 is currently not available and commercial PDI is too expensive, which is making access difficult to these proteins needed for conducting experiments aimed at the establishment of possible interactions between integrin β3 and PDI and wild type rotavirus strains. Objective. To explore a methodology allowing isolation of proteins β3 and PDI from human platelets to be used as antigens in the generation of rabbit polyclonal antibodies useful in the assessment of interactions between these proteins and rotavirus ECwt. Materials and methods. Proteins β3 and PDI from human platelet lysates were separated using preparative electrophoresis under reducing conditions and then eluted. Interactions of these proteins with rotavirus ECwt were analyzed using co-immunoprecipitation, Western blotting and capture ELISA. Results. Proteins from human platelet lysates were separated by preparative electrophoresis under reducing conditions. The identification of proteins β3 and PDI present in a gel slice was performed through their reaction with commercial antibodies in a Western blotting analysis. Protein purity was established after electroelution from a gel slice. Polyclonal antibodies against protein β3 were generated in rabbit. Incubation of eluted proteins β3 and PDI with rotavirus ECwt showed in co-immunoprecipitation and ELISA assays that these proteins bound virus in vitro. The same binding was showed to occur when rotavirus was incubated with isolated small intestinal villi from suckling mice. Conclusions. Relatively high amounts of proteins β3 and PDI were partially purified from human platelets by preparative electrophoresis. The isolation of these proteins allowed the generation of

  7. Transfer of Ho Endonuclease and Ufo1 to the Proteasome by the UbL-UbA Shuttle Protein, Ddi1, Analysed by Complex Formation In Vitro

    Voloshin, Olga; Bakhrat, Anya; Herrmann, Sharon; Raveh, Dina

    2012-01-01

    The F-box protein, Ufo1, recruits Ho endonuclease to the SCFUfo1 complex for ubiquitylation. Both ubiquitylated Ho and Ufo1 are transferred by the UbL-UbA protein, Ddi1, to the 19S Regulatory Particle (RP) of the proteasome for degradation. The Ddi1-UbL domain binds Rpn1 of the 19S RP, the Ddi1-UbA domain binds ubiquitin chains on the degradation substrate. Here we used complex reconstitution in vitro to identify stages in the transfer of Ho and Ufo1 from the SCFUfo1 complex to the proteasome...

  8. Transfer of Ho Endonuclease and Ufo1 to the Proteasome by the UbL-UbA Shuttle Protein, Ddi1, Analysed by Complex Formation In Vitro

    Olga Voloshin; Anya Bakhrat; Sharon Herrmann; Dina Raveh

    2012-01-01

    The F-box protein, Ufo1, recruits Ho endonuclease to the SCF(Ufo1) complex for ubiquitylation. Both ubiquitylated Ho and Ufo1 are transferred by the UbL-UbA protein, Ddi1, to the 19S Regulatory Particle (RP) of the proteasome for degradation. The Ddi1-UbL domain binds Rpn1 of the 19S RP, the Ddi1-UbA domain binds ubiquitin chains on the degradation substrate. Here we used complex reconstitution in vitro to identify stages in the transfer of Ho and Ufo1 from the SCF(Ufo1) complex to the protea...

  9. Phosphorylation of the regulatory beta-subunit of protein kinase CK2 by checkpoint kinase Chk1: identification of the in vitro CK2beta phosphorylation site

    Kristensen, Lars P; Larsen, Martin Røssel; Højrup, Peter; Issinger, Olaf-Georg; Guerra, Barbara

    The regulatory beta-subunit of protein kinase CK2 mediates the formation of the CK2 tetrameric form and it has functions independent of CK2 catalytic subunit through interaction with several intracellular proteins. Recently, we have shown that CK2beta associates with the human checkpoint kinase Chk......1. In this study, we show that Chk1 specifically phosphorylates in vitro the regulatory beta-subunit of CK2. Chymotryptic peptides and mutational analyses have revealed that CK2beta is phosphorylated at Thr213. Formation of a stable complex between CK2beta and Chk1 is not affected by the...

  10. Physiological and biochemical effects of morphactin IT 3233 on callus and tumour tissues of Nicotiana tabacum L. cultured in vitro. II. Protein synthesis and respiratory activity

    Z. Chirek

    2015-05-01

    Full Text Available It was found that the inhibition of callus tissue growth in Nicotiana tabacum L. cultured in vitro by the application of morphactin IT 3233 is associated with a rise of the protein level in spite of a 50 per cent depression of its synthesis. Respiratory activity of these tissues is also lowered. Tumour tissues, on the other hand, show only light changes in the parameters studied. It would seem that morphactin depresses a metabolic activity in the callus tissues, and probably causes deposition of a, large quantity of metabolically inactive proteins.

  11. Influence of Yeast Fermented Cassava Chip Protein (YEFECAP) and Roughage to Concentrate Ratio on Ruminal Fermentation and Microorganisms Using In vitro Gas Production Technique.

    Polyorach, S; Wanapat, M; Cherdthong, A

    2014-01-01

    The objective of this study was to determine the effects of protein sources and roughage (R) to concentrate (C) ratio on in vitro fermentation parameters using a gas production technique. The experimental design was a 2×5 factorial arrangement in a completely randomized design (CRD). Factor A was 2 levels of protein sources yeast fermented cassava chip protein (YEFECAP) and soybean meal (SBM) and factor B was 5 levels of roughage to concentrate (R:C) ratio at 80:20, 60:40, 40:60, 20:80, and 0:100, respectively. Rice straw was used as a roughage source. It was found that gas production from the insoluble fraction (b) of YEFECAP supplemented group was significantly higher (p0.05) on ether in vitro digestibility of dry matter (IVDMD) and organic (IVOMD) while R:C ratio affected the IVDMD and IVOMD (pzoospores, methanogens and protozoal population remained unchanged (p>0.05) as compared between the two sources of protein. Moreover, fungal zoospores and total bacteria population were significantly increased (p<0.01) while, F. succinogenes, R. flavefaciens, R. albus, methanogens and protozoal population were decreased (p<0.01) with decreasing R:C ratio. In conclusion, YEFECAP has a potential for use as a protein source for improving rumen fermentation efficiency in ruminants. PMID:25049924

  12. Replication-Competent Influenza A and B Viruses Expressing a Fluorescent Dynamic Timer Protein for In Vitro and In Vivo Studies.

    Michael Breen

    Full Text Available Influenza A and B viruses (IAV and IBV, respectively cause annual seasonal human respiratory disease epidemics. In addition, IAVs have been implicated in occasional pandemics with inordinate health and economic consequences. Studying influenza viruses in vitro or in vivo requires the use of laborious secondary methodologies to identify infected cells. To circumvent this requirement, replication-competent infectious influenza viruses expressing an easily traceable fluorescent reporter protein can be used. Timer is a fluorescent protein that undergoes a time-dependent color emission conversion from green to red. The rate of spectral change is independent of Timer protein concentration and can be used to chronologically measure the duration of its expression. Here, we describe the generation of replication-competent IAV and IBV where the viral non-structural protein 1 (NS1 was fused to the fluorescent dynamic Timer protein. Timer-expressing IAV and IBV displayed similar plaque phenotypes and growth kinetics to wild-type viruses in tissue culture. Within infected cells, Timer's spectral shift can be used to measure the rate and cell-to-cell spread of infection using fluorescent microscopy, plate readers, or flow cytometry. The progression of Timer-expressing IAV infection was also evaluated in a mouse model, demonstrating the feasibility to characterize IAV cell-to-cell infections in vivo. By providing the ability to chronologically track viral spread, Timer-expressing influenza viruses are an excellent option to evaluate the in vitro and in vivo dynamics of viral infection.

  13. Characterization of the invariable residue 51 mutations of human immunodeficiency virus type 1 capsid protein on in vitro CA assembly and infectivity

    Höglund Stefan

    2007-09-01

    Full Text Available Abstract Background The mature HIV-1 conical core formation proceeds through highly regulated protease cleavage of the Gag precursor, which ultimately leads to substantial rearrangements of the capsid (CAp24 molecule involving both inter- and intra-molecular contacts of the CAp24 molecules. In this aspect, Asp51 which is located in the N-terminal domain of HIV-1 CAp24 plays an important role by forming a salt-bridge with the free imino terminus Pro1 following proteolytic cleavage and liberation of the CAp24 protein from the Pr55Gag precursor. Thus, previous substitution mutation of Asp51 to alanine (D51A has shown to be lethal and that this invariable residue was found essential for tube formation in vitro, virus replication and virus capsid formation. Results We extended the above investigation by introducing three different D51 substitution mutations (D51N, D51E, and D51Q into both prokaryotic and eukaryotic expression systems and studied their effects on in vitro capsid assembly and virus infectivity. Two substitution mutations (D51E and D51N had no substantial effect on in vitro capsid assembly, yet they impaired viral infectivity and particle production. In contrast, the D51Q mutant was defective both for in vitro capsid assembly and for virus replication in cell culture. Conclusion These results show that substitutions of D51 with glutamate, glutamine, or asparagine, three amino acid residues that are structurally related to aspartate, could partially rescue both in vitro capsid assembly and intra-cellular CAp24 production but not replication of the virus in cultured cells.

  14. In Vitro Infectivity Assessment by Drug Susceptibility Comparison of Recombinant Leishmania major Expressing Enhanced Green Fluorescent Protein or EGFP-Luciferase Fused Genes with Wild-Type Parasite

    Sadeghi, Somayeh; Seyed, Negar; Etemadzadeh, Mohammad-Hossein; Abediankenari, Saeid; Rafati, Sima; Taheri, Tahereh

    2015-01-01

    Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitro infectivity rates with comparable parasite-induced levels of NO following interferon-γ/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency. PMID:26323836

  15. Influence of A7-B7 Disulfide Bond Deletion on the Refolding and Structure of Proinsulin%A7-B7二硫键缺失对胰岛素原重折叠及结构的影响

    刘颖; 唐建国

    2003-01-01

    为研究A7-B7二硫键在胰岛素原结构和折叠中的作用, 构建了A7-B7二硫键缺失的胰岛素原突变体, 研究了其与野生型胰岛素原在体外重折叠产率、自由巯基氧化速度、CD谱、受体及抗体结合活性, 以及对胰蛋白酶酶切敏感性的差别. 结果表明, A7-B7二硫键缺失可导致胰岛素原α-螺旋明显减少以及对胰蛋白酶的酶切敏感性显著增加, 其对胰岛素原结构的影响主要导致了受体结合活性的大幅度降低. 突变体在体外重折叠1 h后巯基氧化速率较野生型明显减慢, 但其最终折叠产率与野生型相当. 由此提出一个胰岛素原折叠的可能途径, 即A链链内二硫键最先形成, 然后是两对链间二硫键. 且A20-B19二硫键比A7-B7二硫键很可能先形成, 在折叠中更重要.%To probe the role of [A7-B7] disulfide bond in the structure and folding of proinsulin, [A7, B7Ser]-proinsulin was prepared. The differences in the in vitro refolding, oxidation of free thiol groups, circular dichroism (CD) spectra, antibody and receptor binding assays, and sensitivity to tryptic digestion between the mutant and the wild type proteins were studied. The deletion of [A7-B7] disulfide bond in proinsulin resulted in a significant decrease of α-helix content of the molecule and a great increase in sensitivity to tryptic digestion. The [A7-B7] disulfide bond deleted proinsulin showed a great loss of its receptor binding activity. The in vitro refolding study indicated that the rate of the oxidation of free thiol groups in the mutant was a little slower at the later stage as compared to the native molecule, but the deletion of [A7-B7] disulfide bond had little effect on the refolding yield. A possible proinsulin folding pathway way was proposed. During the folding, the intra-A chain disulfide bond forms first and very fast. The formation of the [A20-B19] disulfide bond is more crucial than [A7-B7] disulfide bond and forms very likely

  16. Effects of bovine serum proteins in culture medium on post-warming survival of bovine blastocysts developed in vitro.

    Ohboshi, S; Etoh, T; Sakamoto, K; Fujihara, N; Yoshida, T; Tomogane, H

    1997-04-15

    Experiments were conducted to investigate the factors affecting the survival of bovine blastocysts produced in vitro after cryopreservation by vitrification. Zygotes were obtained by in vitro maturation and fertilization of oocytes. Embryos used in this study were developed in vitro at Day 7 and 8 (Day 0 = insemination day) in modified synthetic oviduct fluid medium supplemented with calf serum or BSA. Embryos were cryopreserved in a two-step protocol consisting of exposure to 10% ethylene glycol for 5 min, followed by the original vitrification solution (designated as VS) consisting of 40% (v/v) ethylene glycol, 6% (w/v) polyethylene glycol and 0.5 M sucrose in phosphate-buffered saline for 1 min. After warming, embryos were cultured in modified TCM-199 for an in vitro survival assay. The highest survival rate was obtained from the warmed embryos developed at Day 7 in medium supplemented with BSA (82.6%), and there were significant differences between results with calf scrum and BSA treatment (42.4 and 70.7%, respectively; P < 0.01). However, there were no significant differences in the cell numbers of embryos among the treatments. These results suggest that the survival of embryos developed in medium with BSA is superior to that of embryos developed in medium containing calf serum, although the cell numbers of the embryos developed under both media were similar. PMID:16728072

  17. In vitro culture and structural differences in the major immunoreactive protein gp36 of geographically distant Ehrlichia canis isolates

    Zweygarth, E.; Cabezas Cruz, Alejandro; Josemans, A.I.; Oosthuizen, M.C.; Matjila, P.T.; Lis, K.; Broniszewska, M.; Schöl, H.; Ferrolho, J.; Grubhoffer, Libor; Passos, L.M.F.

    2014-01-01

    Roč. 5, č. 4 (2014), s. 423-431. ISSN 1877-959X Institutional support: RVO:60077344 Keywords : Ehrlichia canis * In vitro culture * IDE8 tick cells * DH82 * 16S rRNA * gp36 Subject RIV: FN - Epidemiology, Contagious Diseases ; Clinical Immunology Impact factor: 2.718, year: 2014

  18. Development and characterization of murine monoclonal antibody specific for the P1.4 PorA proteins from strain B:4:P1.(7b.4. of Neisseria meningitidis

    María Elena Pérez

    2011-08-01

    Full Text Available Neisseria meningitidis isolates are conventionally classified by serosubtyping that characterizes the reactivities of the PorA outer membrane protein variable-region epitopes with monoclonal antibodies. Porins are outer membrane proteins (OMPs of N. meningitidis serogroup B and have attracted study principally for two reasons: their use in the classification of meningococcal isolates into serotype and subtype and as potential components of vaccines against this important pathogen. New murine hybridomas, secreting specific monoclonal antibodies against PorA serotype P1.4 of N. meningitidis serogroup B, were generated using conventional hybridoma procedures. The monoclonal antibodies obtained were characterized by Western blot and whole cell ELISA, using reference strains from different N. meningitidis serotypes and subtypes. All monoclonal antibodies belong to isotype IgG1. Others hybridomas producing MAbs against PorB and FrpB were also obtained.

  19. Controlled spatial and conformational display of immobilised bone morphogenetic protein-2 and osteopontin signalling motifs regulates osteoblast adhesion and differentiation in vitro

    McCaskie Andrew W

    2010-05-01

    Full Text Available Abstract Background The interfacial molecular mechanisms that regulate mammalian cell growth and differentiation have important implications for biotechnology (production of cells and cell products and medicine (tissue engineering, prosthetic implants, cancer and developmental biology. We demonstrate here that engineered protein motifs can be robustly displayed to mammalian cells in vitro in a highly controlled manner using a soluble protein scaffold designed to self assemble on a gold surface. Results A protein was engineered to contain a C-terminal cysteine that would allow chemisorption to gold, followed by 12 amino acids that form a water soluble coil that could switch to a hydrophobic helix in the presence of alkane thiols. Bioactive motifs from either bone morphogenetic protein-2 or osteopontin were added to this scaffold protein and when assembled on a gold surface assessed for their ability to influence cell function. Data demonstrate that osteoblast adhesion and short-term responsiveness to bone morphogenetic protein-2 is dependent on the surface density of a cell adhesive motif derived from osteopontin. Furthermore an immobilised cell interaction motif from bone morphogenetic protein supported bone formation in vitro over 28 days (in the complete absence of other osteogenic supplements. In addition, two-dimensional patterning of this ligand using a soft lithography approach resulted in the spatial control of osteogenesis. Conclusion These data describe an approach that allows the influence of immobilised protein ligands on cell behaviour to be dissected at the molecular level. This approach presents a durable surface that allows both short (hours or days and long term (weeks effects on cell activity to be assessed. This widely applicable approach can provide mechanistic insight into the contribution of immobilised ligands in the control of cell activity.

  20. Magnolol Affects Cellular Proliferation, Polyamine Biosynthesis and Catabolism-Linked Protein Expression and Associated Cellular Signaling Pathways in Human Prostate Cancer Cells in vitro

    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.

  1. A comparative in vitro study of the digestibility of heat- and high pressure-induced gels prepared from industrial milk whey proteins

    He, Jin-Song; Mu, Tai-Hua; Wang, Juan

    2013-06-01

    We undertook this study to compare the digestibility of heat- and high pressure-induced gels produced from whey protein isolate (WPI). To simulate in vivo gastrointestinal digestion of WPI gels, a pepsin-trypsin digestion system was used. The in vitro protein digestibility of WPI gels induced by high pressure (400 MPa and 30 min; P-gel) and those induced by heat (80°C and 30 min; H-gel) was compared using a protein concentration of 0.14 g mL-1. The in vitro protein digestibility of P-gels was significantly greater than that of H-gels (p<0.05). The size-exclusion chromatography profiles of the hydrolysates showed that the P-gel generated more and smaller peptides than natural WPI and H-gels. Furthermore, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed some soluble disulfide-mediated aggregation in the P-gel, while there was more insoluble aggregation in the H-gel than the P-gel. The P-gel was more sensitive to proteinase than the H-gel, which was related to the content of S-S bonds, and this in turn could be attributed to the differences in the gelation mechanism between the H-gel and P-gel.

  2. The change in heat shock protein expression in avermectin induced neurotoxicity of the pigeon (Columba livia) both in vivo and in vitro.

    Li, Ming; Wang, Xian-Song; Xu, Feng-Ping; Liu, Shuang; Xu, Shi-Wen; Li, Shu

    2014-12-01

    The expression of heat shock proteins (Hsps) commonly increases to provide neuroprotection when brain tissues are under stress conditions. Residues of avermectins (AVMs) have neurotoxic effects on a number of non-target organisms. The aim of this study was to investigate the effects of AVM exposure on the expression levels of Hsp 60, Hsp 70 and Hsp 90 for pigeon (Columba livia) neurons both in vivo and in vitro. The results showed that in general, the mRNA and protein levels of Hsps were increased in treated groups relative to control groups after AVM exposure for 30d, 60d and 90d in the cerebrum, cerebellum and optic lobe in vivo. However, AVM exposure had no significant effects on the transcription expression of Hsps for 90d in the optic lobe and decreased the translation expression of Hsps significantly for 90d in the optic lobe. In vitro, the LC50 of avermectin for King pigeon neurons is between 15μgL(-1) and 20μgL(-1). Following AVM (2.5-20μgL(-1)) exposure, the mRNA expression of the 3 Hsps was up-regulated to different degrees. Compared with the control groups, a significant decrease, a remarkable increase and a non-significant change was found in the protein expression of Hsp 60, Hsp 70 and Hsp 90 separately following AVM (2.5-20μgL(-1)) exposure. Based on these results, we conclude that AVM exposure can induce a protective stress response in pigeons by means of promoting the mRNA and protein expression of Hsps under in vivo and in vitro conditions, thus easing the neurotoxic effects of AVM to some extent. PMID:25202854

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

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

    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 bro...

  4. Adrenomedullin promotes differentiation of oligodendrocyte precursor cells into myelin-basic-protein expressing oligodendrocytes under pathological conditions in vitro

    Takakuni Maki; Yoko Takahashi; Nobukazu Miyamoto; Liang, Anna C.; Masafumi Ihara; Eng H Lo; Ken Arai

    2015-01-01

    Oligodendrocytes, which are the main cell type in cerebral white matter, are generated from their precursor cells (oligodendrocyte precursor cells: OPCs). However, the differentiation from OPCs to oligodendrocytes is disturbed under stressed conditions. Therefore, drugs that can improve oligodendrocyte regeneration may be effective for white matter-related diseases. Here we show that a vasoactive peptide adrenomedullin (AM) promotes the in vitro differentiation of OPCs under pathological cond...

  5. Potensi Protein Reseptor Fertilisasi Zona Pelusida Kambing Sebagai Kandidat Imunokontrasepsi dengan Fertilisasi in vitro pada Sapi (POTENTIAL OF FERTILIZATION RECEPTOR PROTEIN GOAT ZONA PELLUCIDA AS CANDIDATE OF IMMUNOCONTRACEPTION BY USING IN VITRO FER

    Sri Mulyati; Imam Mustofa; Laba Mahaputra

    2013-01-01

    Studies on the role of zona pellucida glycoprotein-3 (ZP3) in immunocontraception have been conductedin many species including goats (gZP3). Our previous study indicated that gZP3 effective in preventingpregnancy in mice. The aim of this study was to prove the evidence of cross reaction in gZP3 to preventfertilization in cow in vitro. Antibody of gZP3 was produced in mice. Immunized mice serum was analyzedusing ELISA technique and dot blot method. Sperm from frozen semen were processed then i...

  6. Specific in vitro cleavage of Mason-Pfizer monkey virus capsid protein: evidence for a potential role of retroviral protease in early stages of infection

    Processing of Gag polyproteins by viral protease (PR) leads to reorganization of immature retroviral particles and formation of a ribonucleoprotein core. In some retroviruses, such as HIV and RSV, cleavage of a spacer peptide separating capsid and nucleocapsid proteins is essential for the core formation. We show here that no similar spacer peptide is present in the capsid-nucleocapsid (CA-NC) region of Mason-Pfizer monkey virus (M-PMV) and that the CA protein is cleaved in vitro by the PR within the major homology region (MHR) and the NC protein in several sites at the N-terminus. The CA cleavage product was also identified shortly after penetration of M-PMV into COS cells, suggesting that the protease-catalyzed cleavage is involved in core disintegration

  7. Optimization and characterization of an in vitro bovine mammary cell culture system to study regulation of milk protein synthesis and mammary differentiation

    A long term bovine mammary cell culture system that maintains normal mammary cell function was established and optimized to study milk protein synthesis and secretion and mammary differentiation. This culture system used bovine mammary acini isolated from developing or lactating mammary gland by enzymatic dissociation, and cryopreserved until thawed and plated for growth in vitro for these studies. Cells in M199 with lactogenic hormones ± fetal calf serum (FCS) were cultured on plastic, 100ul and 500ul type I collagen, and Matrigel, or embedded within type I collagen. Cell morphology, cell number, and total TCA-precipitable 35S-labelled proteins were monitored. Milk protein (αs,1-casein, lactoferrin (LF), α-lactalbumin, and β-lactoglobulin) secretion and intracellular levels were determined by an ELISA assay

  8. Expression of INHβA and INHβB proteins in porcine oocytes cultured in vitro is dependent on the follicle size

    Kempisty, Bartosz; Piotrowska, Hanna; Rybska, Marta; Woźna, Magdalena; Antosik, Paweł; Bukowska, Dorota; Zawierucha, Piotr; CIESIÓŁKA, Sylwia; Jaśkowski, Jędrzej M.; Nowicki, Michał; Brüssow, Klaus-Peter; Zabel, Maciej

    2013-01-01

    Summary The current study aimed to investigate differential expression of inhibin βA (INHβA) and inhibin βB (INHβB) in porcine oocytes before or after in vitro maturation (IVM) isolated from follicles of various sizes. Porcine oocytes isolated from large, medium and small follicles (40 from each) were used to study the INHβA and INHβB protein expression pattern using western blot analysis before or after 44 h of oocyte IVM. An increased expression of INHβA was found in oocytes collected from ...

  9. Application of an in vitro-amplification assay as a novel pre-screening test for compounds inhibiting the aggregation of prion protein scrapie

    Matthias Schmitz; Maria Cramm; Franc Llorens; Niccolò Candelise; Dominik Müller-Cramm; Daniela Varges; Schulz-Schaeffer, Walter J.; Saima Zafar; Inga Zerr

    2016-01-01

    In vitro amplification assays, such as real-time quaking-induced conversion (RT-QuIC) are used to detect aggregation activity of misfolded prion protein (PrP) in brain, cerebrospinal fluid (CSF) and urine samples from patients with a prion disease. We believe that the method also has a much broader application spectrum. In the present study, we applied RT-QuIC as a pre-screening test for substances that potentially inhibit the aggregation process of the cellular PrP (PrPC) to proteinase (PK)-...

  10. Heterologous Expression of MeLEA3: A 10 kDa Late Embryogenesis Abundant Protein of Cassava, Confers Tolerance to Abiotic Stress in Escherichia coli with Recombinant Protein Showing In Vitro Chaperone Activity.

    Barros, Nicolle L F; da Silva, Diehgo T; Marques, Deyvid N; de Brito, Fabiano M; dos Reis, Savio P; de Souza, Claudia R B

    2015-01-01

    Late embryogenesis abundant (LEA) proteins are small molecular weight proteins involved in acquisition of tolerance to drought, salinity, high temperature, cold, and freezing stress in many plants. Previous studies revealed a cDNA sequence coding for a 10 kDa atypical LEA protein, named MeLEA3, predicted to be located into mitochondria with potential role in salt stress response of cassava (Manihot esculenta Crantz). Here we aimed to produce the recombinant MeLEA3 protein by heterologous expression in Escherichia coli and evaluate the tolerance of bacteria expressing this protein under abiotic stress. Our result revealed that the recombinant MeLEA3 protein conferred a protective function against heat and salt stress in bacterial cells. Also, the recombinant MeLEA3 protein showed in vitro chaperone activity by protection of NdeI restriction enzyme activity under heat stress. PMID:25990084

  11. Quercetin Enhances the Antitumor Activity of Trichostatin A through Upregulation of p53 Protein Expression In Vitro and In Vivo

    Chan, Shu-Ting; Yang, Nae-Cherng; Huang, Chin-Shiu; Liao, Jiunn-Wang; Yeh, Shu-Lan

    2013-01-01

    This study investigated the effects of quercetin on the anti-tumor effect of trichostatin A (TSA), a novel anticancer drug, in vitro and in vivo and the possible mechanisms of these effects in human lung cancer cells. We first showed that quercetin (5 µM) significantly increased the growth arrest and apoptosis in A549 cells (expressing wild-type p53) induced by 25 ng/mL of (82.5 nM) TSA at 48 h by about 25% and 101%, respectively. However, such enhancing effects of quercetin (5 µM) were not s...

  12. Mass spectrometry data for in vitro protein profiles in early and late stages of Douglas-fir xylogenesis.

    Dziedzic, Jowita A; McDonald, Armando G

    2016-06-01

    A Douglas-fir tissue culture system was developed [1] that could be induced to differentiate into tracheary elements (fibers) making it possible to monitor xylogenesis in vitro by a proteomics approach. Two proteomes, one from an early and one from a late stage of fiber differentiation process were analyzed and compared. Obtained mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [2] with the dataset identifiers PXD001484 and DOI:10.6019/ PXD001484 [3]. PMID:27408915

  13. Cytosolic Proteins From Tobacco Pollen Tubes That Crosslink Microtubules and Actin Filaments In Vitro Are Metabolic Enzymes

    Romagnoli, Silvia; Faleri, Claudia; Bini, Luca; Baskin, Tobias I.; Cresti, Mauro

    2010-01-01

    In plant cells, many processes require cooperative action of both microtubules and actin filaments, but proteins mediating interactions between these cytoskeletal members are mostly undiscovered. Here, we attempt to identify such proteins by affinity purification. Cytosol from Nicotiana tabacum (tob

  14. Functional exchangeability of the nuclear localization signal (NLS of capsid protein between PCV1 and PCV2 in vitro: Implications for the role of NLS in viral replication

    He Yongqiang

    2011-07-01

    Full Text Available Abstract Background Porcine circovirus type 2 (PCV2 is believed to be the primary causative agent of postweaning multisystemic wasting syndrome (PMWS. It is supposed that capsid protein of PCV may contribute to replication control via interaction between Cap and Rep in the nucleoplasm. In this study, we described the construction and in vitro characterization of NLS-exchanged PCV DNA clones based on a PMWS-associated PCV2b isolate from China to determine the role of ORF2 NLS in PCV replication. Results The PCV1, PCV2, PCV2-NLS1 and PCV1-NLS2 DNA clone were generated by ligating a copy of respective genome in tandem with a partial duplication. The PCV2-NLS1 and PCV1-NLS2 DNA clone contained a chimeric genome in which the ORF2 NLS was exchanged. The four DNA clones were all confirmed to be infectious in vitro when transfected into PK-15 cells, as PCV capsid protein were expressed in approximately 10-20% of the transfected cells. The in vitro growth characteristics of the DNA clones were then determined and compared. All the recovered progeny viruses gave rise to increasing infectious titers during passages and were genetically stable by genomic sequencing. The chimeric PCV1-NLS2 and PCV2-NLS1 viruses had the final titers of about 104.2 and 103.8 TCID50/ml, which were significantly lower than that of PCV1 and PCV2 (105.6 and 105.0 TCID50/ml, respectively. When the ORF2 NLS exchanged, the mutant PCV2 (PCV2-NLS1 still replicated less efficiently and showed lower infectious titer than did PCV1 mutant (PCV1-NLS2, which was consistent with the distinction between wild type PCV1 and PCV2. Conclusions Recovery of the chimeiric PCV1-NLS2 and PCV2-NLS1 progeny viruses indicate that the nuclear localization signal sequence of capsid protein are functionally exchangeable between PCV1 and PCV2 with respect to the role of nuclear importing and propagation. The findings also reveal that ORF2 NLS play an accessory role in the replication of PCV. However, we found

  15. Isolation of an acidic protein from cholesterol gallstones, which inhibits the precipitation of calcium carbonate in vitro.

    Shimizu, S.; Sabsay, B; Veis, A.; Ostrow, J D; Rege, R V; Dawes, L G

    1989-01-01

    In seeking to identify nucleating/antinucleating proteins involved in the pathogenesis of cholesterol gallstones, a major acidic protein was isolated from each of 13 samples of cholesterol gallstones. After the stones were extracted with methyl t-butyl ether to remove cholesterol, and methanol to remove bile salts and other lipids, they were demineralized with EDTA. The extracts were desalted with Sephadex-G25, and the proteins separated by PAGE. A protein was isolated, of molecular weight be...

  16. Comparative Features of Copper ATPases ATP7A and ATP7B Heterologously Expressed in COS-1 Cells

    Liu, Yueyong; Pilankatta, Rajendra; Hatori, Yuta; Lewis, David; Inesi, Giuseppe

    2010-01-01

    ATP7A and ATP7B are P-type ATPases required for copper homeostasis and involved in the etiology of Menkes and Wilson diseases. We used heterologous expression of ATP7A or ATP7B in COS-1 cells infected with adenovirus vectors to characterize differential features pertinent to each protein expressed in the same mammalian cell type, rather than to extrinsic factors related to different cells sustaining expression. Electrophoretic analysis of the expressed protein, before and after purification, ...

  17. The Level of DING Proteins Is Increased in HIV-Infected Patients: In Vitro and In Vivo Studies

    Djeghader, Ahmed; Aragonès, Gerard; Darbinian, Nune; Elias, Mikael; Gonzalez, Daniel; García-Heredia, Anabel; Beltrán-Debón, Raúl; Kaminski, Rafal; Gotthard, Guillaume; Hiblot, Julien; Rull, Anna; Rohr, Olivier; Schwartz, Christian; Alonso-Villaverde, Carlos; Joven, Jorge; Camps, Jordi; Chabriere, Eric

    2012-01-01

    DING proteins constitute an interesting family, owing to their intriguing and important activities. However, after a decade of research, little is known about these proteins. In humans, at least five different DING proteins have been identified, which were implicated in important biological processes and diseases, including HIV. Indeed, recent data from different research groups have highlighted the anti-HIV activity of some DING representatives. These proteins share the ability to inhibit the transcriptional step of HIV-1, a key step of the viral cycle that is not yet targeted by the current therapies. Since such proteins have been isolated from humans, we undertook a comprehensive study that focuses on the relationship between these proteins and HIV-infection in an infectious context. Hence, we developed a home-made ELISA for the quantification of the concentration of DING proteins in human serum. Using this method, we were able to determine the concentration of DING proteins in healthy and HIV-infected patients. Interestingly, we observed a significant increase of the concentration of DING proteins in non treated and treated HIV-infected patients compared to controls. In addition, cell cultures infected with HIV also show an increased expression of DING proteins, ruling out the possible role of antiretroviral treatment in the increase of the expression of DING proteins. In conclusion, results from this study show that the organism reacts to HIV-infection by an overexpression of DING proteins. PMID:22427948

  18. The Identification of Three Sizes of Core Proteins during the Establishment of Persistent Hepatitis C Virus Infection in vitro

    Qingjiao Liao; Jiansheng Tian; Yang Wu; Xulin Chen

    2013-01-01

    Similar to Hepatitis C virus (HCV) infection in humans,HCVcc infection can also result in persistent and chronic infection.The core protein is a variable protein and exists in several sizes.Some sizes of core proteins have been reported to be related to chronic HCV infection.To study the possible role of the core protein in persistent HCV infection,a persistent HCVcc infection was established,and the expression of the core protein was analysed over the course of the infection.The results show that there are three sizes of core proteins (p24,p21 and p19) expressed during the establishment of persistent HCVcc infection.Of these,the p21 core protein is the mature form of the HCV core protein.The p24 core protein is the phosphorylated form of p21.The p19 core protein appears to be a functional by-product generated during the course of infection.These three core proteins are all localized in the cytoplasm and can be encapsidated into the HCV virion.The appearance of the p19 and p24 core proteins might be related to acute HCVcc infection and chronic infection respectively and may play an important role in the pathology of a HCV infection.

  19. Lack of detectable allergenicity in genetically modified maize containing "Cry" proteins as compared to native maize based on in silico & in vitro analysis.

    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.

  20. In vitro solubility of meat and bone meal protein with different pepsin concentrations Solubilidade in vitro da proteína de farinhas de carne e ossos com diferentes concentraçoes de pepsina

    Cláudio Bellaver

    2000-06-01

    Full Text Available In vitro protein digestibility of protein sources has been correlated with in vivo digestibility values. However, factors like protein origin, enzyme used and its concentration, pH and processing have been related with the significance of the correlation between the estimates. To address only the enzyme concentration factor, this paper had the objective of testing pepsin at 0.2, 0.02, 0.002 and 0.0002% using the standard AOAC (1995 procedure. Two meat and bone meals (MBM with low and high crude protein (CP content were used to determine the coefficient of solubility of CP in pepsin and HCl (CSCPPEPH. Centrifugation was used to establish the nitrogen (N in the soluble phase, instead of filtration and analysis of N in the residue. The variance analysis and a non-linear asymptotic model were adjusted. The CSCPPEPH under different pepsin concentrations for the two MBM showed higher solubility discrimination with low pepsin concentration. The level of 0.0002% pepsin is better to predict the CP soluble in MBM. This finding implies the assumption that 0.2% pepsin found in the AOAC is not correct for the purpose of determining the range of solubility in high and low CP content in MBM.A digestibilidade in vitro da proteína de fontes protéicas tem sido correlacionada com a digestibilidade in vivo. Entretanto, fatores como a origem protéica, enzima usada e sua concentração, pH e processamento têm sido relacionados com a significância da correlação entre as estimativas. Para atuar somente no fator da concentração enzimática, este trabalho teve por objetivo testar a pepsina nas concentrações de 0,2, 0,02, 0,002 e 0,0002%, utilizando o procedimento padrão do AOAC (1995. Duas farinhas de carne e ossos com baixo ou alto teor de proteína (PB foram usadas para determinar o coeficiente de solubilidade da PB em pepsina e HCl (CSCPPEPH. Centrifugação foi usada para obter o nitrogênio (N na fase solúvel, em vez da filtração e análise do N no

  1. Cell-penetrating DNA-binding protein as a safe and efficient naked DNA delivery carrier in vitro and in vivo

    Kim, Eun-Sung; Yang, Seung-Woo [Department of Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Hong, Dong-Ki; Kim, Woo-Taek [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Ho-Guen [Department of Pathology, Yonsei Medical School, Seoul 120-752 (Korea, Republic of); Lee, Sang-Kyou, E-mail: sjrlee@yonsei.ac.kr [Department of Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2010-01-29

    Non-viral gene delivery is a safe and suitable alternative to viral vector-mediated delivery to overcome the immunogenicity and tumorigenesis associated with viral vectors. Using the novel, human-origin Hph-1 protein transduction domain that can facilitate the transduction of protein into cells, we developed a new strategy to deliver naked DNA in vitro and in vivo. The new DNA delivery system contains Hph-1-GAL4 DNA-binding domain (DBD) fusion protein and enhanced green fluorescent protein (EGFP) reporter plasmid that includes the five repeats of GAL4 upstream activating sequence (UAS). Hph-1-GAL4-DBD protein formed complex with plasmid DNA through the specific interaction between GAL4-DBD and UAS, and delivered into the cells via the Hph-1-PTD. The pEGFP DNA was successfully delivered by the Hph-1-GAL4 system, and the EGFP was effectively expressed in mammalian cells such as HeLa and Jurkat, as well as in Bright Yellow-2 (BY-2) plant cells. When 10 {mu}g of pEGFP DNA was intranasally administered to mice using Hph-1-GAL4 protein, a high level of EGFP expression was detected throughout the lung tissue for 7 days. These results suggest that an Hph-1-PTD-mediated DNA delivery strategy may be an useful non-viral DNA delivery system for gene therapy and DNA vaccines.

  2. IN VITRO CO-STIMULATORY ACTIVITY OF HUMAN B7.2(IgV+C)PROTEIN PRODUCED BY ENGINEERED BACTERIA

    2001-01-01

    Objective To express human B7.2 extracellular domain with prokaryote expression system and to evaluate its biological activity in vitro. Methods PCR was used to amplify the extracellular region of human B7. 2which contained both the IgV and IgC domains. The recombinant PGEX-4T-3/hB7. 2 (IgV+C) was obtained by cloning the PCR product into a prokaryote expression plasmid PGEX-4T-3 and was transformed into the host strain of DH5-α. The fusion protein consisted of GST and hB7.2(IgV+C) was identified by SDS-PAGE and Western blotting.T cell activation was observed by exposing purified T lymphocytes to the fusion protein and [3H]-TdR incorporation with the presence of the first signal imitated by anti-CD3 antibody. Results The fusion protein GST-hB7.2 (IgV+C) was produced and detected in inclusive body form from engineered bacterial cells. With the first signal existed,T lymphocytes proliferated when it was co-stimulated by the fusion protein. Conclusion These results indicated that the functional human B7.2(IgV+C) fusion protein can be produced in bacterial cells and the fusion protein displays the co-stimulatory activity in T lymphocytes activation.

  3. Immobilized Cytochrome P450 2C9 (CYP2C9): Applications for Metabolite Generation, Monitoring Protein-Protein Interactions, and Improving In-vivo Predictions Using Enhanced In-vitro Models

    Wollenberg, Lance A.

    Cytochrome P450 (P450) enzymes are a family of oxoferroreductase enzymes containing a heme moiety and are well known to be involved in the metabolism of a wide variety of endogenous and xenobiotic materials. It is estimated that roughly 75% of all pharmaceutical compounds are metabolized by these enzymes. Traditional reconstituted in-vitro incubation studies using recombinant P450 enzymes are often used to predict in-vivo kinetic parameters of a drug early in development. However, in many cases, these reconstituted incubations are prone to aggregation which has been shown to affect the catalytic activity of an enzyme. Moreover, the presence of other isoforms of P450 enzymes present in a metabolic incubation, as is the case with microsomal systems, may affect the catalytic activity of an enzyme through isoform-specific protein-protein interactions. Both of these effects may result in inaccurate prediction of in-vivo drug metabolism using in-vitro experiments. Here we described the development of immobilized P450 constructs designed to elucidate the effects of aggregation and protein-protein interactions between P450 isoforms on catalytic activities. The long term objective of this project is to develop a system to control the oligomeric state of Cytochrome P450 enzymes to accurately elucidate discrepancies between in vitro reconstituted systems and actual in vivo drug metabolism for the precise prediction of metabolic activity. This approach will serve as a system to better draw correlations between in-vivo and in-vitro drug metabolism data. The central hypothesis is that Cytochrome P450 enzymes catalytic activity can be altered by protein-protein interactions occurring between Cytochrome P450 enzymes involved in drug metabolism, and is dependent on varying states of protein aggregation. This dissertation explains the details of the construction and characterization of a nanostructure device designed to control the state of aggregation of a P450 enzyme. Moreover

  4. Evaluation of the antioxidant capacity of a milk protein matrix in vitro and in vivo in women aged 50-70 years.

    Power-Grant, Orla; McCormack, William G; Ramia De Cap, Maximiliano; Amigo-Benavent, Miryam; Fitzgerald, Richard J; Jakeman, Phil

    2016-01-01

    Bovine milk proteins have emerged as a novel, dairy-based source of dietary antioxidants and a component of a nutritional strategy to maintain muscle mass during ageing. The aim of this study was to characterise the in vitro antioxidant capacity (AOC) of a milk-based protein matrix (MPM) before and after simulated gastrointestinal digestion (SGID) and determine whether plasma AOC was similarly modified in vivo following acute ingestion of the MPM in healthy 50-70 years old women. To achieve this, the AOC of the MPM was measured by the oxygen radical absorbance capacity (ORAC) assay prior to and following SGID. In parallel, plasma obtained from women prior to and for 3 h following ingestion of the MPM was analysed ex vivo for change in AOC to evaluate the translation in vivo. SGID of the MPM increased AOC by ∼ 35% (27,365 ± 2152 versus 42,592 ± 2299 μmol TE/100 g dw; p AOC by ∼ 23% (10,952 ± 751 to 13,519 ± 800 μmol TE/L; p AOC of an MPM subjected to simulated digest in vitro and the change in plasma AOC following ingestion of the MPM sampled ex vivo from healthy elderly women. PMID:26960816

  5. Effect of 5-azacytidine on the Protein Expression of Porcine Bone Marrow Mesenchymal Stem Cells in vitro

    Neng-Sheng Ye; Rong-Li Zhang; Yan-Feng Zhao; Xue Feng; Yi-Ming Wang; Guo-An Luo

    2006-01-01

    Bone marrow-derived mesenchymal stem cells (MSCs) are pluripotent stem cells that show a vital potential in the clinical application for cell transplantation. In the present paper, proteomic techniques were used to approach the protein profiles associated with porcine bone marrow MSCs and investigate the regulation of MSC proteins on the effect of 5-azacytidine (5-aza). Over 1,700 protein species were separated from MSCs according to gel analysis. Compared with the expression profiling of control MSCs, there were 11 protein spots up-regulated and 26 downregulated in the protein pattern of 5-aza-treated cells. A total of 21 proteins were successfully identified by MALDI-TOF-MS analysis, among which some interesting proteins, such as alpha B-crystallin, annexin A2, and stathmin 1, had been reported to involve in cell proliferation and differentiation through different signaling pathways. Our data should be useful for the future study of MSC differentiation and apoptosis.

  6. Antioxidant activity of Cod (Gadus morhua) protein hydrolysates: in vitro assays and evaluation in 5% fish oil-in-water emulsion.

    Farvin, K H Sabeena; Lystbæk Andersen, Lisa; Hauch Nielsen, Henrik; Jacobsen, Charlotte; Jakobsen, Greta; Johansson, Inez; Jessen, Flemming

    2014-04-15

    Cod protein hydrolysates were fractionated according to the molecular mass into three fractions of >5 kDa, 3-5 kDa and hydrolysates and the fractions were investigated, both in in vitro assays (DPPH radical scavenging activity, reducing power, Fe²⁺ chelating activity and inhibition of lipid oxidation in liposome model system), and in 5% fish oil-in-water emulsions. The hydrolysate, were able to protect fish oil against oxidation in an iron induced oxidation system. However, none of the peptide fractions were effective in preventing tocopherol loss and showed no tocopherol sparing property. Volatile oxidation products showed an interaction between the aldehydes and the protein/peptides added in the emulsions, and this needs further investigation. PMID:24295714

  7. EFFECTS OF SYSTEMIC FLUCONAZOLE THERAPY ON IN VITRO ADHESION OF CANDIDA ALBICANS TO BUCCAL EPITHELIAL CELLS AND CHANGES OF THE CELL SURFACE PROTEINS OF THE EPITHELIAL CELLS

    吴绍熙; 郭宁如; 侯幼红

    1996-01-01

    This paper presented the effects of systemic fluconazole therapy via intravenous (IV) and oral (PO) administrations on the adhesion of Candida albicans (C. albicans) to the huccal epithelial ceils (BEC) from five treated patients with three candidosis, one mucornlycosis and one sporotrichosis and at the same time,an analysis of the cell surface proteins involving candidal adherent receptor in the BEC of the patients in the course of 7 days were exposed to 3H-leucine radiolabaled C. atbicans for in vitro eandidal adherent assay,and the BEC from first intake day and the last intake day of the patients were extracted by dithiothreitol(DTT)-iodoacetamide treatment for SDS-PAGE. These results indicate that the systemic iluconazole therapy resuks in the inhibitory effect of candldal adhesion to BEC of treated patients to prevent them from oral candidosis for a prolonged time, which is based on the absent surface protein (35KDa) of the BEC.

  8. Keratin 8 phosphorylation in vitro by cAMP-dependent protein kinase occurs within the amino- and carboxyl-terminal end domains.

    Ando, S; Tokui, T; Yano, T; Inagaki, M

    1996-04-01

    We reported earlier that phosphorylation in vitro of keratin filaments reconstituted from rat type I keratin 18 and type II keratin 8 by cAPM-dependent protein kinase induces disassembly of the keratin filament structure. Keratin 8 rather than keratin 18 was the major target of the kinase. We have now identified the sites on rat keratin 8 for cAMP-dependent protein kinase. Sequential analysis of the purified phosphoropeptides, together with the known primary sequence, revealed that four major sites, Ser-12, Ser-23, Ser-36, and Ser-50, and three minor sites, Ser-8, Ser-33, Ser-42, are located in the amino-terminal head domain, while three minor sites, Ser-416, Ser-423 and Ser-425 locate in the carboxyl-terminal tail domain. PMID:8660345

  9. Double-stranded RNA-dependent protein kinase is required for bone calcification in MC3T3-E1 cells in vitro.

    Yoshida, Kaya; Okamura, Hirohiko; Amorim, Bruna Rabelo; Ozaki, Akiko; Tanaka, Hiroaki; Morimoto, Hiroyuki; Haneji, Tatsuji

    2005-11-15

    In this study, we demonstrated that double-stranded RNA-dependent protein kinase (PKR) is required for the calcification of osteoblasts via the signal transducers and activators of transcription 1alpha (STAT1alpha) signaling in vitro. A dominant-negative mutant PKR cDNA, in which the amino acid lysine at 296 was replaced with arginine and which does not have catalytic activity, was transfected into mouse osteoblastic MC3T3-E1 cells; thereby, we established cells that stably expressed the PKR mutant gene (PKR-K/R). Phosphorylation of PKR was not stimulated by polyinosic-polycytidylic acid in the mutant cells. The PKR-K/R mutant cells exhibited up-regulated cell growth and had low alkaline phosphatase (ALP) activity. The PKR-K/R mutant cells were not able to form bone nodules in vitro. In the PKR-K/R mutant cells, runt-related gene 2 (Runx2)-mediated transcription decreased compared with the levels in the control cells. The expression of STAT1alpha protein increased and the protein was translocated to the nucleus in the PKR-K/R mutant cells. When the expression of STAT1alpha protein in PKR mutant cells was suppressed using RNAi, the activity of Runx2-mediated transcription recovered to the control level. Our results indicate that PKR is a stimulator of Runx2 transcription and is a negative modulator of STAT1alpha expression. Our findings also suggest that PKR plays important roles in the differentiation and calcification of osteoblasts by modulating STAT1alpha and/or Runx2 expression. PMID:16216244

  10. Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study.

    Stanczyk, Paulina J; Lai, F Anthony; Zissimopoulos, Spyros

    2016-01-01

    Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca(2+) release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by β-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins. PMID:27500320

  11. Influence of EMAP II, IFN-α2b and its medicinal preparations on the MGMT protein amount in human cells in vitro

    Lylo V. V.

    2014-11-01

    Full Text Available Aim. To study the effect of EMAP II, IFN-α2b and its medicinal preparations on the amount of O6-methylguanine-DNA methyltransferase (MGMT protein in human cells in vitro. Methods. The human cells of 4BL and Hep-2 lines were treated with the purified recombinant proteins EMAP II, IFN-α2b and its commercial me dicinal preparations. Changes in the MGMT gene expression were studied at a protein level by Western blot analysis. Results. Treatment of Hep-2 and 4BL cells with EMAP II at the concentrations of 0.02 mg/ml and 2 mg/ml respectively led to induction of the MGMT gene expression. EMAP II at the concentrations of 0.2–20 g/ml caused decrease of the MGMT protein amount in Hep-2 cells. The regulating activity of EMAP II was also observed for MARP (anti-Methyltransferase Antibody Recognizable Protein. IFN-α2b and Laferon-PharmBiotek with the activity of 200 and 2000 IU/ml were shown to cause an increase of the MGMT protein amount in Hep-2 cells. Conclusions. The purified recombinant proteins EMAP II and IFN-α2b which are substrates for the medicinal preparations influenced on the amount of MGMT protein in the human cell cultures in a concentration-dependent manner. At the same time the effect of medicinal preparations differs from that of the purified protein IFN-α2b. Possibly it depends on the presence of stabilizing components in their compositions.

  12. In vitro assembly properties of mutant and chimeric intermediate filament proteins: insight into the function of sequences in the rod and end domains of IF

    The factors and mechanisms regulating assembly of intermediate filament (IF) proteins to produce filaments with their characteristic 10 nm diameter are not fully understood. All IF proteins contain a central rod domain flanked by variable head and tail domains. To elucidate the role that different domains of IF proteins play in filament assembly, we used negative staining and electron microscopy (EM) to study the in vitro assembly properties of purified bacterially expressed IF proteins, in which specific domains of the proteins were either mutated or swapped between a cytoplasmic (mouse neurofilament-light (NF-L) subunit) and nuclear intermediate filament protein (human lamin A). Our results indicate that filament formation is profoundly influenced by the composition of the assembly buffer. Wild type (wt) mouse NF-L formed 10 nm filaments in assembly buffer containing 175 mM NaCl, whereas a mutant deleted of 18 NH2-terminal amino acids failed to assemble under similar conditions. Instead, the mutant assembled efficiently in buffers containing CaCl2 ≥ 6 mM forming filaments that were 10 times longer than those formed by wt NF-L, although their diameter was significantly smaller (6-7 nm). These results suggest that the 18 NH2-terminal sequence of NF-L might serve two functions, to inhibit filament elongation and to promote lateral association of NF-L subunits. We also demonstrate that lengthening of the NF-L rod domain, by inserting a 42 aa sequence unique to nuclear IF proteins, does not compromise filament assembly in any noticeable way. Our results suggests that the known inability of nuclear lamin proteins to assemble into 10 nm filaments in vitro cannot derive solely from their longer rod domain. Finally, we demonstrate that the head domain of lamin A can substitute for that of NF-L in filament assembly, whereas substitution of both the head and tail domains of lamins for those of NF-L compromises assembly. Therefore, the effect of lamin A 'tail' domain alone

  13. Purification and in vitro activities of the native nitrogen fixation control proteins NifA and NifL.

    Austin, S; Buck, M; Cannon, W; Eydmann, T.; Dixon, R

    1994-01-01

    The prokaryotic enhancer-binding protein NifA stimulates transcription at a distance by binding to sequences upstream of nitrogen fixation (nif) promoters and catalyzing the formation of open promoter complexes by RNA polymerase containing the alternative sigma factor, sigma 54. The activity of NifA in vivo is modulated by the negative regulatory protein NifL in response to environmental oxygen and fixed nitrogen. To date, a detailed biochemical analysis of these proteins from the model diazo...

  14. Effect of pH 5 enzyme from liver on the protein synthesis by mammary gland subcellular fractions in vitro

    The effect of pH 5 enzyme fraction of liver on the protein synthesizing activity of the subcellular fractions of the mammary gland has been investigated. Results indicate that (1) lactating liver pH 5 enzyme stimulates protein synthesis which is enhanced by the addition of ATP-generating system and (2) the enzyme fractions from the non-lactating liver inhibits the protein synthesis by mammary fractions, but in some cases like mitochondrial and supernatant fractions of mammary it elevates the synthesis when supplemented with ATP-generating system. Chlorella protein hydrolysate-14C was used as a tracer and rabits were used as experimental animals. (M.G.B.)

  15. Role of Lactobacillus reuteri cell and mucus-binding protein A (CmbA) in adhesion to intestinal epithelial cells and mucus in vitro.

    Jensen, Hanne; Roos, Stefan; Jonsson, Hans; Rud, Ida; Grimmer, Stine; van Pijkeren, Jan-Peter; Britton, Robert A; Axelsson, Lars

    2014-04-01

    Lactobacillus reuteri, a symbiotic inhabitant of the gastrointestinal tract in humans and animals, is marketed as a probiotic. The ability to adhere to intestinal epithelial cells and mucus is an interesting property with regard to probiotic features such as colonization of the gastrointestinal tract and interaction with the host. Here, we present a study performed to elucidate the role of sortase (SrtA), four putative sortase-dependent proteins (SDPs), and one C-terminal membrane-anchored cell surface protein of Lactobacillus reuteri ATCC PTA 6475 in adhesion to Caco-2 cells and mucus in vitro. This included mutagenesis of the genes encoding these proteins and complementation of mutants. A null mutation in hmpref0536_10255 encoding srtA resulted in significantly reduced adhesion to Caco-2 cells and mucus, indicating involvement of SDPs in adhesion. Evaluation of the bacterial adhesion revealed that of the five putative surface protein mutants tested, only a null mutation in the hmpref0536_10633 gene, encoding a putative SDP with an LPxTG motif, resulted in a significant loss of adhesion to both Caco-2 cells and mucus. Complementation with the functional gene on a plasmid restored adhesion to Caco-2 cells. However, complete restoration of adhesion to mucus was not achieved. Overexpression of hmpref0536_10633 in strain ATCC PTA 6475 resulted in an increased adhesion to Caco-2 cells and mucus compared with the WT strain. We conclude from these results that, among the putative surface proteins tested, the protein encoded by hmpref0536_10633 plays a critical role in binding of Lactobacillus reuteri ATCC PTA 6475 to Caco-2 cells and mucus. Based on this, we propose that this LPxTG motif containing protein should be referred to as cell and mucus binding protein A (CmbA). PMID:24473252

  16. Changes in nucleic acid and protein levels during in vitro germ¬nation and elongation of Lilium longifforum cv. "Ace" polleni

    William V. Dashek

    2014-02-01

    Full Text Available While changes in nucleic acid and protein levels during germination and subsequent tube elongation have been determined for a number of pollens, they have not been extensively examined for in vitro grown Lilium longiflorum, cv. `Ace' pollen. Nucleic acids and proteins were extracted with cold trichloroacetic acrid (TCA, cold-hot TCA or cold TCA and potassium hydroxide-perchloric acid (KOH-HClO4. Following extraction, RNA, DNA and total protein were assayed colorimetrically with orcinol, diphenylamine and Folin-Phenol reagents, respectively. Extraction of 500 x g supernatants with KOH-HClO4, yielded less RNA than either of the TCA-extraction procedures which gave similar nucleic acids and protein recoveries. Whereas total protein levels decreased initially and then increased during 36 h, RNA and DNA levels rose throughout the time-course. Precipitation and quaritiation of nucleic acids and protein from homogenized and soaicated 500 x g pellets resulted in time-dependent alterations in levels of macromolecules which differed from those for 500 x g supernatants. Whereas DNA and RNA levels increased and then decreased over 36 h, total protein levels remained constant for 12 h and then declined during the : next 24 h. Addition of the data obtained for 500 x g supernatants to those for 500 x g pellets revealed that total protein levels increased 2.4 times for the first 12 h and thereafter remained constant, that RNA levels increased 9.8 times for the first 12 h and then levelled off and that the DNA content rose more than 5 times over 36 h.

  17. Antioxidant Activity of Fish Protein Hydrolysates in in vitro Assays and in Oil-in-Water Emulsions

    Farvin, Sabeena; Andersen, Lisa Lystbæk; Jacobsen, Charlotte;

    The aim of this study was to screen different protein hydrolysates with respect to their antioxidative properties in order to select the most promising extracts for further evaluation in oil-in-water emulsions. Three fractions of protein hydrolysates (Crude, >5kDa and 5kDa, 3-5kDa and...

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

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

    2016-01-01

    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,

  19. Incorporation of albumin fusion proteins into fibrin clots in vitro and in vivo: comparison of different fusion motifs recognized by factor XIIIa

    Sheffield William P

    2011-12-01

    Full Text Available Abstract Background The transglutaminase activated factor XIII (FXIIIa acts to strengthen pathological fibrin clots and to slow their dissolution, in part by crosslinking active α2-antiplasmin (α2AP to fibrin. We previously reported that a yeast-derived recombinant fusion protein comprising α2AP residues 13-42 linked to human serum albumin (HSA weakened in vitro clots but failed to become specifically incorporated into in vivo clots. In this study, our aims were to improve both the stability and clot localization of the HSA fusion protein by replacing α2AP residues 13-42 with shorter sequences recognized more effectively by FXIIIa. Results Expression plasmids were prepared encoding recombinant HSA with the following N-terminal 23 residue extensions: H6NQEQVSPLTLLAG4Y (designated XL1; H6DQMMLPWAVTLG4Y (XL2; H6WQHKIDLPYNGAG4Y (XL3; and their 17 residue non-His-tagged equivalents (XL4, XL5, and XL6. The HSA moiety of XL4- to XL6-HSA proteins was C-terminally His-tagged. All chimerae were efficiently secreted from transformed Pichia pastoris yeast except XL3-HSA, and following nickel chelate affinity purification were found to be intact by amino acid sequencing, as was an N-terminally His-tagged version of α2AP(13-42-HSA. Of the proteins tested, XL5-HSA was cross-linked to biotin pentylamine (BPA most rapidly by FXIIIa, and was the most effective competitor of α2AP crosslinking not only to BPA but also to plasma fibrin clots. In the mouse ferric chloride vena cava thrombosis model, radiolabeled XL5-HSA was retained in the clot to a greater extent than recombinant HSA. In the rabbit jugular vein stasis thrombosis model, XL5-HSA was also retained in the clot, in a urea-insensitive manner indicative of crosslinking to fibrin, to a greater extent than recombinant HSA. Conclusions Fusion protein XL5-HSA (DQMMLPWAVTLG4Y-HSAH6 was found to be more active as a substrate for FXIIIa-mediated transamidation than seven other candidate fusion proteins in

  20. In vitro and in vivo evaluation of bone formation using solid freeform fabrication-based bone morphogenic protein-2 releasing PCL/PLGA scaffolds

    The aim of this study was to develop novel polycaprolactone/poly(lactic-co-glycolic acid) (PCL/PLGA) scaffolds with a heparin–dopamine (Hep–DOPA) conjugate for controlled release of bone morphogenic protein-2 (BMP-2) to enhance osteoblast activity in vitro and also bone formation in vivo. PCL/PLGA scaffolds were prepared by a solid freeform fabrication method. The PCL/PLGA scaffolds were functionalized with Hep–DOPA and then BMP-2 was sequentially coated onto the Hep–DOPA/PCL/PLGA scaffolds. The characterization and surface elemental composition of all scaffolds were evaluated by scanning electron microscope and x-ray photoelectron spectroscopy. The osteoblast activities on all scaffolds were assessed by cell proliferation, alkaline phosphatase (ALP) activity and calcium deposition in vitro. To demonstrate bone formation in vivo, plain radiograph, micro-computed tomography (micro-CT) evaluation and histological studies were performed after the implantation of all scaffolds on a rat femur defect. Hep–DOPA/PCL/PLGA had more controlled release of BMP-2, which was quantified by enzyme-linked immunosorbent assay, compared with Hep/PCL/PLGA. The in vitro results showed that osteoblast-like cells (MG-63 cells) grown on BMP-2/Hep–DOPA/PCL/PLGA had significantly enhanced ALP activity and calcium deposition compared with those on BMP-2/Hep/PCL/PLGA and PCL/PLGA. In addition, the plain radiograph, micro-CT evaluation and histological studies demonstrated that the implanted BMP-2/Hep–DOPA/PCL/PLGA on rat femur had more bone formation than BMP-2/Hep/PCL/PLGA and PCL/PLGA in vivo. (paper)

  1. In vitro binding of anthrax protective antigen on bacteriophage T4 capsid surface through Hoc-capsid interactions: A strategy for efficient display of large full-length proteins

    An in vitro binding system is described to display large full-length proteins on bacteriophage T4 capsid surface at high density. The phage T4 icosahedral capsid features 155 copies of a nonessential highly antigenic outer capsid protein, Hoc, at the center of each major capsid protein hexon. Gene fusions were engineered to express the 83-kDa protective antigen (PA) from Bacillus anthracis fused to the N-terminus of Hoc and the 130-kDa PA-Hoc protein was expressed in Escherichia coli and purified. The purified PA-Hoc was assembled in vitro on hoc - phage particles. Binding was specific, stable, and of high affinity. This defined in vitro system allowed manipulation of the copy number of displayed PA and imposed no significant limitation on the size of the displayed antigen. In contrast to in vivo display systems, the in vitro approach allows all the capsid binding sites to be occupied by the 130-kDa PA-Hoc fusion protein. The PA-T4 particles were immunogenic in mice in the absence of an adjuvant, eliciting strong PA-specific antibodies and anthrax lethal toxin neutralizing antibodies. The in vitro display on phage T4 offers a novel platform for potential construction of customized vaccines against anthrax and other infectious diseases

  2. A novel killer protein from Pichia kluyveri isolated from an Algerian soil: purification and characterization of its in vitro activity against food and beverage spoilage yeasts.

    Labbani, Fatima-Zohra Kenza; Turchetti, Benedetta; Bennamoun, Leila; Dakhmouche, Scheherazad; Roberti, Rita; Corazzi, Lanfranco; Meraihi, Zahia; Buzzini, Pietro

    2015-04-01

    A novel killer protein (Pkkp) secreted by a Pichia kluyveri strain isolated from an Algerian soil was active against food and beverage spoilage yeasts of the genera Dekkera, Kluyveromyces, Pichia, Saccharomyces, Torulaspora, Wickerhamomyces and Zygosaccharomyces. After purification by gel filtration chromatography Pkkp revealed an apparent molecular mass of 54 kDa with SDS-PAGE. Minimum inhibitory concentrations (MICs) of purified Pkkp exhibited a high in vitro activity against Dekkera bruxellensis (MICs from 64,000- to 256,000-fold lower than that exhibited by potassium metabisulphite) and Saccharomyces cerevisiae (MICs from 32,000- to 64,000- fold lower than potassium sorbate). No in vitro synergistic interactions (calculated by FIC index - Σ FIC) were observed when Pkkp was used in combination with potassium metabisulphite, potassium sorbate, or ethanol. Pkkp exhibited a dose-response effect against D. bruxellensis and S. cerevisiae in a low-alcoholic drink and fruit juice, respectively. The results of the present study suggest that Pkkp could be proposed as a novel food-grade compound useful for the control of food and beverage spoilage yeasts. PMID:25618417

  3. In vitro Stability of Heat Shock Protein 27 in Serum and Plasma Under Different Pre-analytical Conditions: Implications for Large-Scale Clinical Studies.

    Zimmermann, Matthias; Traxler, Denise; Simader, Elisabeth; Bekos, Christine; Dieplinger, Benjamin; Lainscak, Mitja; Ankersmit, Hendrik Jan; Mueller, Thomas

    2016-07-01

    The effects of storage temperatures, repeated freeze-thaw cycles, or delays in separating plasma or serum from blood samples are largely unknown for heat shock protein 27 (HSP27). We evaluated (1) the imprecision of the HSP27 assay used in this study; (2) the in vitro stability of HSP27 in blood samples stored at 4°C for up to 6 hr with immediate and delayed serum/plasma separation from cells; and (3) the in vitro stability of HSP27 in blood samples stored at -80°C after repeated freeze-thaw cycles. The ELISA to detect HSP27 in this study showed a within-run CV of total CV of <15%. After 4-6 hr of storage at 4°C, HSP27 concentrations remained stable when using serum tubes irrespective of sample handling, but HSP27 concentrations decreased by 25-45% when using EDTA plasma tubes. Compared with baseline HSP27, one freeze-thaw cycle had no effect on serum concentrations. However, plasma concentrations increased by 3.1-fold after one freeze-thaw cycle and by 7.3-fold after five freeze-thaw cycles. In conclusion, serum is an appropriate biological sample type for use in epidemiological and large-scale clinical studies. PMID:27139608

  4. In vitro neutrophil migration requires protein kinase c-delta (δ-PKC) mediated MARCKS (Myristoylated Alanine Rich C-Kinase Substrate) phosphorylation

    Sung, Eui Jae; Adler, Kenneth B.; Jones, Samuel L.

    2015-01-01

    Dysregulated release of neutrophil reactive oxygen species and proteolytic enzymes contributes to both acute and chronic inflammatory diseases. Therefore, molecular regulators of these processes are potential targets for new anti-inflammatory therapies. We have shown previously that MARCKS (Myristoylated Alanine Rich C-Kinase Substrate), a well-known PKC substrate protein, is a key regulator of neutrophil functions. In the current study we investigate the role of PKC-mediated MARCKS phosphorylation in neutrophil migration and adhesion in vitro. We report that treatment of human neutrophils with the δ-PKC inhibitor rottlerin significantly attenuates fMLF induced MARCKS phosphorylation (IC50 = 5.709 μM), adhesion (IC50 = 8.4 uM) and migration (IC50 = 6.7 uM); while α-, β- and ζ-PKC inhibitors had no significant effect. We conclude that δ-PKC mediated MARCKS phosphorylation is essential for human neutrophil migration and adhesion in vitro. These results implicate δ-PKC mediated MARCKS phosphorylation as a key step in the inflammatory response of neutrophils. PMID:25515270

  5. The movement protein of barley yellow dwarf virus-GAV self-interacts and forms homodimers in vitro and in vivo.

    Xia, Zongliang; Cao, Rufei; Sun, Kaile; Zhang, Hua

    2012-07-01

    The 17-kDa movement protein (MP) of the GAV strain of barley yellow dwarf virus (BYDV-GAV) can bind the viral RNA and target to the nucleus. However, much less is known about the active form of the MP in planta. In this study, the ability of the MP to self-interact was analyzed by yeast two-hybrid assay and bimolecular fluorescence complementation. The BYDV-GAV MP has a strong potential to self-interact in vitro and in vivo, and self-interaction was mediated by the N-terminal domain spanning the second α-helix (residues 17-39). Chemical cross-linking and heterologous MP expression from a pea early browning virus (PEBV) vector further showed that MP self-interacts to form homodimers in vitro and in planta. Interestingly, the N-terminal domain necessary for MP self-interaction has previously been identified as important for nuclear targeting. Based on these findings, a functional link between MP self-interaction and nuclear targeting is discussed. PMID:22437255

  6. Expression of 17beta- and 3beta-hydroxysteroid dehydrogenases and steroidogenic acute regulatory protein in non-luteinizing bovine granulosa cells in vitro.

    Sahmi, M; Nicola, E S; Silva, J M; Price, C A

    2004-08-31

    Granulosa cells of small follicles differentiate in vitro in serum-free medium, resulting in increased estradiol secretion and abundance of mRNA encoding cytochrome P450aromatase (P450arom). We tested the hypothesis that differentiation in vitro also involves increased expression of 3beta- and 17beta-hydroxysteroid dehydrogenases (HSD) in the absence of steroidogenic acute regulatory protein (StAR) expression, as has been observed in vivo. Granulosa cells from small (basal layer of the membrana granulosa) did not affect steroidogenesis. We conclude that under the present cell culture system granulosa cells do not luteinize, and show expression of key steroidogenic enzymes in patterns similar to those occurring in differentiating follicles in vivo. Further, the data suggest that 17beta-HSD may be as important as P450arom in regulating estradiol secretion, and that 3beta-HSD is more important than P450scc as a regulator of progesterone secretion in non-luteinizing granulosa cells. PMID:15279910

  7. Soluble proteins delivered to dendritic cells via pH-sensitive liposomes induce primary cytotoxic T lymphocyte responses in vitro

    1992-01-01

    Effective immunity to many infectious agents, particularly viruses, requires a CD8+ cytotoxic T lymphocyte (CTL) response. Understanding how to achieve CTL induction with soluble proteins is important for vaccine development since such antigens are usually not processed appropriately to induce CTL. In the present report, we have demonstrated that a potent primary CTL response against a soluble protein can be achieved by delivering antigen in pH-sensitive liposomes to dendritic cells (DC) eith...

  8. Allergen extracts and recombinant proteins: comparison of efficiency of in vitro allergy diagnostics using multiplex assay on a biological microchip

    Smoldovskaya, Olga; Feyzkhanova, Guzel; Arefieva, Alla; Voloshin, Sergei; Ivashkina, Olga; Reznikov, Yuriy; Rubina, Alla

    2016-01-01

    Background Immunological test systems for diagnostics of type I hypersensitivity involve the following types of antigens: whole allergen extracts, individual highly purified proteins and their recombinant analogues. The goal of this study was to compare the results obtained with whole allergen extracts (birch pollen, cat dander, and timothy grass pollen) and their respective recombinant proteins in biochip-based immunoassay. Methods Multiplex fluorescent immunoassay of 139 patients’ blood ser...

  9. Standard in vitro assays for protein-nucleic acid interactions--gel shift assays for RNA and DNA binding.

    Mitchell, Sarah F; Lorsch, Jon R

    2014-01-01

    The characterization of protein-nucleic acid interactions is necessary for the study of a wide variety of biological processes. One straightforward and widely used approach to this problem is the electrophoretic mobility shift assay (EMSA), in which the binding of a nucleic acid to one or more proteins changes its mobility through a nondenaturing gel matrix. Usually, the mobility of the nucleic acid is reduced, but examples of increased mobility do exist. This type of assay can be used to investigate the affinity of the interaction between the protein and nucleic acid, the specificity of the interaction, the minimal binding site, and the kinetics of the interaction. One particular advantage of EMSA is the ability to analyze multiple proteins, or protein complexes, binding to nucleic acids. This assay is relatively quick and easy and utilizes equipment available in most laboratories; however, there are many variables that can only be determined empirically; therefore, optimization is necessary and can be highly dependent upon the system. The protocol described here is for the poly(A)-binding protein (PABP) binding to an unstructured RNA probe of 43 bases. While this may be a useful protocol for some additional assays, it is recommended that both reaction conditions and gel running conditions be tailored to the individual interaction to be probed. PMID:24674072

  10. Size control of in vitro synthesized magnetite crystals by the MamC protein of Magnetococcus marinus strain MC-1.

    Valverde-Tercedor, C; Montalbán-López, M; Perez-Gonzalez, T; Sanchez-Quesada, M S; Prozorov, T; Pineda-Molina, E; Fernandez-Vivas, M A; Rodriguez-Navarro, A B; Trubitsyn, D; Bazylinski, Dennis A; Jimenez-Lopez, C

    2015-06-01

    Magnetotactic bacteria are a diverse group of prokaryotes that share the unique ability of biomineralizing magnetosomes, which are intracellular, membrane-bounded crystals of either magnetite (Fe3O4) or greigite (Fe3S4). Magnetosome biomineralization is mediated by a number of specific proteins, many of which are localized in the magnetosome membrane, and thus is under strict genetic control. Several studies have partially elucidated the effects of a number of these magnetosome-associated proteins in the control of the size of magnetosome magnetite crystals. However, the effect of MamC, one of the most abundant proteins in the magnetosome membrane, remains unclear. In this present study, magnetite nanoparticles were synthesized inorganically in free-drift experiments at 25 °C in the presence of different concentrations of the iron-binding recombinant proteins MamC and MamCnts (MamC without its first transmembrane segment) from the marine, magnetotactic bacterium Magnetococcus marinus strain MC-1 and three commercial proteins [α-lactalbumin (α-Lac), myoglobin (Myo), and lysozyme (Lyz)]. While no effect was observed on the size of magnetite crystals formed in the presence of the commercial proteins, biomimetic synthesis in the presence of MamC and MamCnts at concentrations of 10-60 μg/mL resulted in the production of larger and more well-developed magnetite crystals (~30-40 nm) compared to those of the control (~20-30 nm; magnetite crystals grown protein-free). Our results demonstrate that MamC plays an important role in the control of the size of magnetite crystals and could be utilized in biomimetic synthesis of magnetite nanocrystals. PMID:25874532

  11. Conditioned medium from irradiated bovine pulmonary artery endothelial cells stimulates increased protein synthesis by irradiated bovine lung fibroblasts in vitro

    Pulmonary fibrosis, a potentially fatal consequence of radiation exposure, occurs by unknown mechanisms. The hypothesis that endothelial cells, injured by radiation, could alter the biochemical function of lung fibroblasts, was tested by exposing cultures of bovine pulmonary artery endothelial cells to 0 or 5 Gy radiation and then incubating them in fresh medium for 48 h. This endothelial cell conditioned medium (ECCM) was then applied to irradiated or nonirradiated cultures of bovine lung fibroblasts. Forty-eight hours later the fibroblasts were analyzed for their ability to synthesize DNA and protein. The ECCM from injured cells stimulated fibroblast protein synthesis twofold to threefold in irradiated fibroblasts without increasing DNA synthesis. It also stimulated a significant but less marked increase in protein synthesis in nonirradiated fibroblasts. Two-dimensional gel electrophoresis revealed this increased synthesis to be expressed in less than 10% of the 1100 separable fibroblast proteins. This study shows that endothelial cells injured by radiation produce factors that stimulate injured fibroblasts to markedly increase their synthesis of certain intracellular proteins, while not stimulating fibroblast replication

  12. Alpha-phellandrene-induced DNA damage and affect DNA repair protein expression in WEHI-3 murine leukemia cells in vitro.

    Lin, Jen-Jyh; Wu, Chih-Chung; Hsu, Shu-Chun; Weng, Shu-Wen; Ma, Yi-Shih; Huang, Yi-Ping; Lin, Jaung-Geng; Chung, Jing-Gung

    2015-11-01

    Although there are few reports regarding α-phellandrene (α-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α-PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α-PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α-PA on total cell viability and the results indicated that α-PA induced cell death. Comet assay and 4,6-diamidino-2-phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α-PA induced DNA damage and condensation in a concentration-dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α-PA induced DNA damage in WEHI-3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α-PA increased p-p53, p-H2A.X, 14-3-3-σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA-PK, and BRCA-1. PMID:24861204

  13. Analysis of the effects of blue light on morphofunctional status of in vitro cultured blastocysts from mice carrying gene of enhanced green fluorescent protein (EGFP).

    Sakharova, N Yu; Mezhevikina, L M; Smirnov, A A; Vikhlyantseva, E F

    2014-05-01

    We studied the effect of blue light (440-490 nm) on the development of late blastocysts of mice carrying the gene of enhanced green fluorescent protein (EGFP). Exposure to blue light for 20 min reduced adhesive properties of blastocysts and their capacity to form primary colonies consisting of the cells of inner cell mass, trophoblast, and extraembryonic endoderm. The negative effects of blue light manifested in morphological changes in the primary colonies and impairment of differentiation and migration of cells of the trophoblast and extraembryonic endoderm. The problems of cell-cell interaction and inductive influences of the inner cell mass on other cell subpopulations are discussed. EGFP blastocysts were proposed as the model for evaluation of the mechanisms underlying the effects of blue light as the major negative factor of visible light used in in vitro experiments on mammalian embryos. PMID:24913583

  14. The impact of heating and soaking on the in vitro enzymatic hydrolysis of protein varies in different species of tropical legumes.

    Torres, Julieta; Rutherfurd, Shane M; Muñoz, Luz S; Peters, Michael; Montoya, Carlos A

    2016-03-01

    The effects of different thermal (raw, autoclaving or boiling for 5 and 20min) and soaking (with or without) treatments on the degree of hydrolysis (DH) of protein were investigated for selected legumes (Canavalia brasiliensis; Lablab purpureus; pink, red and white colour hulls Vigna unguiculata). Each legume preparation underwent in vitro simulated gastrointestinal tract digestion comprising either pepsin (120min) or pepsin/pancreatin (120/240min) digestion. The DH was determined based on the amount of free amino groups released. Autoclaving for 5min increased the pepsin/pancreatin DH for all the unsoaked and soaked legumes (+20% to 46% units) except Canavalia, while boiling for 5min only increased DH for two soaked legumes (+12% to 28% units). Extending boiling from 5 to 20min increased the DH for three soaked legumes (+5% to 29% units). In conclusion, autoclaving, in general, extensively increased the sequential pepsin/pancreatin DH, while boiling only increased it for selected legumes. PMID:26471569

  15. In Vitro Proliferation and Anti-Apoptosis of the Papain-Generated Casein and Soy Protein Hydrolysates towards Osteoblastic Cells (hFOB1.19

    Xiao-Wen Pan

    2015-06-01

    Full Text Available Casein and soy protein were digested by papain to three degrees of hydrolysis (DH 7.3%–13.3%, to obtain respective six casein and soy protein hydrolysates, aiming to clarify their in vitro proliferation and anti-apoptosis towards a human osteoblastic cell line (hFOB1.19 cells. Six casein and soy protein hydrolysates at five levels (0.01–0.2 mg/mL mostly showed proliferation as positive 17β-estradiol did, because they conferred the osteoblasts with cell viability of 100%–114% and 104%–123%, respectively. The hydrolysates of higher DH values had stronger proliferation. Casein and soy protein hydrolysates of the highest DH values altered cell cycle progression, and enhanced cell proportion of S-phase from 50.5% to 56.5% and 60.5%. The two also antagonized etoposide- and NaF-induced osteoblast apoptosis. In apoptotic prevention, apoptotic cells were decreased from 31.6% to 22.6% and 15.6% (etoposide treatment, or from 19.5% to 17.7% and 12.4% (NaF treatment, respectively. In apoptotic reversal, soy protein hydrolysate decreased apoptotic cells from 13.3% to 11.7% (etoposide treatment, or from 14.5% to 11.0% (NaF treatment, but casein hydrolysate showed no reversal effect. It is concluded that the hydrolysates of two kinds had estradiol-like action on the osteoblasts, and soy protein hydrolysates had stronger proliferation and anti-apoptosis on the osteoblasts than casein hydrolysates.

  16. Short communication. Effects of adding different protein and carbohydrates sources on chemical composition and in vitro gas production of corn stover silage

    L. A. Mejía-Uribe

    2013-05-01

    Full Text Available The use of protein-rich by-products based in swine manure (SM, poultry waste (PW or chemicals compounds as urea (U, as well as energy products like molasses (M and bakery by-product (BB, is a viable method to produce good quality silage. In addition, the use of a bacterial additive can improve the fermentation characteristics of silage. The objective of this study was to determine chemical composition, in vitro gas production (GP and dry matter disappearance (DMd, using different sources of protein and energy in silage. The silages were made using SM, PW or U as protein sources and M or BB as energy source, with corn stover and with or without a bacterial additive. The organic matter (OM content was higher (p < 0.001 in silages with UBB, UM and SMBB compared with the rest of the treatments; meanwhile crude protein content was higher (p < 0.001 in silages with U. The addition of a bacterial additive increased (p < 0.05 OM content and decreased (p < 0.05 fiber content. Total GP was higher (p < 0.05 in silages containing BB, but DMd was higher (p < 0.05 in silages with U and SMBB. The inclusion of a bacterial additive decreased (p < 0.05 GP and DMd. The use of alternative sources of protein such as poultry and swine manure or urea, and of by-products of sugar industry and bakery is an alternative for silages based on corn stover. The results show that when properly formulated, the silages can provide more than 16% of crude protein and have DMd values above 60%.

  17. Conserved Cysteine Residue in the DNA-Binding Domain of the Bovine Papillomavirus Type 1 E2 Protein Confers Redox Regulation of the DNA- Binding Activity in Vitro

    McBride, Alison A.; Klausner, Richard D.; Howley, Peter M.

    1992-08-01

    The bovine papillomavirus type 1 E2 open reading frame encodes three proteins involved in viral DNA replication and transcriptional regulation. These polypeptides share a carboxyl-terminal domain with a specific DNA-binding activity; through this domain the E2 polypeptides form dimers. In this study, we demonstrate the inhibition of E2 DNA binding in vitro by reagents that oxidize or otherwise chemically modify the free sulfydryl groups of reactive cysteine residues. However, these reagents had no effect on DNA-binding activity when the E2 polypeptide was first bound to DNA, suggesting that the free sulfydryl group(s) may be protected by DNA binding. Sensitivity to sulfydryl modification was mapped to a cysteine residue at position 340 in the E2 DNA-binding domain, an amino acid that is highly conserved among the E2 proteins of different papillomaviruses. Replacement of this residue with other amino acids abrogated the sensitivity to oxidation-reduction changes but did not affect the DNA-binding property of the E2 protein. These results suggest that papillomavirus DNA replication and transcriptional regulation could be modulated through the E2 proteins by changes in the intracellular redox environment. Furthermore, a motif consisting of a reactive cysteine residue carboxyl-terminal to a lysine residue in a basic region of the DNA-binding domain is a feature common to a number of transcriptional regulatory proteins that, like E2, are subject to redox regulation. Thus, posttranslational regulation of the activity of these proteins by the intracellular redox environment may be a general phenomenon.

  18. Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro

    Lio, Yi-Ching; Mazin, Alexander V.; Kowalczykowski, Stephen C.; Chen, David J.

    2003-01-01

    The human Rad51 protein is essential for DNA repair by homologous recombination. In addition to Rad51 protein, five paralogs have been identified: Rad51B/Rad51L1, Rad51C/Rad51L2, Rad51D/Rad51L3, XRCC2, and XRCC3. To further characterize a subset of these proteins, recombinant Rad51, Rad51B-(His)(6), and Rad51C proteins were individually expressed employing the baculovirus system, and each was purified from Sf9 insect cells. Evidence from nickel-nitrilotriacetic acid pull-down experiments demonstrates a highly stable Rad51B.Rad51C heterodimer, which interacts weakly with Rad51. Rad51B and Rad51C proteins were found to bind single- and double-stranded DNA and to preferentially bind 3'-end-tailed double-stranded DNA. The ability to bind DNA was elevated with mixed Rad51 and Rad51C, as well as with mixed Rad51B and Rad51C, compared with that of the individual protein. In addition, both Rad51B and Rad51C exhibit DNA-stimulated ATPase activity. Rad51C displays an ATP-independent apparent DNA strand exchange activity, whereas Rad51B shows no such activity; this apparent strand exchange ability results actually from a duplex DNA destabilization capability of Rad51C. By analogy to the yeast Rad55 and Rad57, our results suggest that Rad51B and Rad51C function through interactions with the human Rad51 recombinase and play a crucial role in the homologous recombinational repair pathway.

  19. Carbonyl reductase inactivation may contribute to mouse lung tumor promotion by electrophilic metabolites of butylated hydroxytoluene: protein alkylation in vivo and in vitro.

    Shearn, Colin T; Fritz, Kristofer S; Meier, Brent W; Kirichenko, Oleg V; Thompson, John A

    2008-08-01

    Promotion of lung tumors in mice by the food additive butylated hydroxytoluene (BHT) is mediated by electrophilic metabolites produced in the target organ. Identifying the proteins alkylated by these quinone methides (QMs) is a necessary step in understanding the underlying mechanisms. Covalent adducts of the antioxidant enzymes peroxiredoxin 6 and Cu,Zn superoxide dismutase were detected previously in lung cytosols from BALB/c mice injected with BHT, and complimentary in vitro studies demonstrated that QM alkylation causes inactivation and enhances oxidative stress. In the present work, adducts of another protective enzyme, carbonyl reductase (CBR), were detected by Western blotting and mass spectrometry in mitochondria from lungs of mice one day after a single injection of BHT and throughout a 28-day period of weekly injections required to achieve tumor promotion. BHT treatment was accompanied by the accumulation of protein carbonyls in lung cytosol from sustained oxidative stress. Studies in vitro demonstrated that CBR activity in lung homogenates was susceptible to concentration- and time-dependent inhibition by QMs. Recombinant CBR underwent irreversible inhibition during QM exposure, and mass spectrometry was utilized to identify alkylation sites at Cys 51, Lys 17, Lys 189, Lys 201, His 28, and His 204. Except for Lys 17, all of these adducts were eliminated as a cause of enzyme inhibition either by chemical modification (cysteine) or site-directed mutagenesis (lysines and histidines). The data demonstrated that Lys 17 is the critical alkylation target, consistent with the role of this basic residue in NADPH binding. These data support the possibility that CBR inhibition occurs in BHT-treated mice, thereby compromising one pathway for inactivating lipid peroxidation products, particularly 4-oxo-2-nonenal. These data, in concert with previous evidence for the inactivation of antioxidant enzymes, provide a molecular basis to explain lung inflammation leading to

  20. Quercetin enhances the antitumor activity of trichostatin A through upregulation of p53 protein expression in vitro and in vivo.

    Shu-Ting Chan

    Full Text Available This study investigated the effects of quercetin on the anti-tumor effect of trichostatin A (TSA, a novel anticancer drug, in vitro and in vivo and the possible mechanisms of these effects in human lung cancer cells. We first showed that quercetin (5 µM significantly increased the growth arrest and apoptosis in A549 cells (expressing wild-type p53 induced by 25 ng/mL of (82.5 nM TSA at 48 h by about 25% and 101%, respectively. However, such enhancing effects of quercetin (5 µM were not significant in TSA-exposed H1299 cells (a p53 null mutant or were much lower than in A549 cells. In addition, quercetin significantly increased TSA-induced p53 expression in A549 cells. Transfection of p53 siRNA into A549 cells significantly but not completely diminished the enhancing effects of quercetin on TSA-induced apoptosis. Furthermore, we demonstrated that quercetin enhanced TSA-induced apoptosis through the mitochondrial pathway. Transfection of p53 siRNA abolished such enhancing effects of quercetin. However, quercetin increased the acetylation of histones H3 and H4 induced by TSA in A549 cells, even with p53 siRNA transfection as well as in H1299 cells. In a xenograft mouse model of lung cancer, quercetin enhanced the antitumor effect of TSA. Tumors from mice treated with TSA in combination with quercetin had higher p53 and apoptosis levels than did those from control and TSA-treated mice. These data indicate that regulation of the expression of p53 by quercetin plays an important role in enhancing TSA-induced apoptosis in A549 cells. However, p53-independent mechanisms may also contribute to the enhancing effect of quercetin.

  1. Quercetin enhances the antitumor activity of trichostatin A through upregulation of p53 protein expression in vitro and in vivo.

    Chan, Shu-Ting; Yang, Nae-Cherng; Huang, Chin-Shiu; Liao, Jiunn-Wang; Yeh, Shu-Lan

    2013-01-01

    This study investigated the effects of quercetin on the anti-tumor effect of trichostatin A (TSA), a novel anticancer drug, in vitro and in vivo and the possible mechanisms of these effects in human lung cancer cells. We first showed that quercetin (5 µM) significantly increased the growth arrest and apoptosis in A549 cells (expressing wild-type p53) induced by 25 ng/mL of (82.5 nM) TSA at 48 h by about 25% and 101%, respectively. However, such enhancing effects of quercetin (5 µM) were not significant in TSA-exposed H1299 cells (a p53 null mutant) or were much lower than in A549 cells. In addition, quercetin significantly increased TSA-induced p53 expression in A549 cells. Transfection of p53 siRNA into A549 cells significantly but not completely diminished the enhancing effects of quercetin on TSA-induced apoptosis. Furthermore, we demonstrated that quercetin enhanced TSA-induced apoptosis through the mitochondrial pathway. Transfection of p53 siRNA abolished such enhancing effects of quercetin. However, quercetin increased the acetylation of histones H3 and H4 induced by TSA in A549 cells, even with p53 siRNA transfection as well as in H1299 cells. In a xenograft mouse model of lung cancer, quercetin enhanced the antitumor effect of TSA. Tumors from mice treated with TSA in combination with quercetin had higher p53 and apoptosis levels than did those from control and TSA-treated mice. These data indicate that regulation of the expression of p53 by quercetin plays an important role in enhancing TSA-induced apoptosis in A549 cells. However, p53-independent mechanisms may also contribute to the enhancing effect of quercetin. PMID:23342112

  2. The effect of interleukin-13 (IL-13 and interferon-γ (IFN-γ on expression of surfactant proteins in adult human alveolar type II cells in vitro

    Mason Robert J

    2010-11-01

    Full Text Available Abstract Background Surfactant proteins are produced predominantly by alveolar type II (ATII cells, and the expression of these proteins can be altered by cytokines and growth factors. Th1/Th2 cytokine imbalance is suggested to be important in the pathogenesis of several adult lung diseases. Recently, we developed a culture system for maintaining differentiated adult human ATII cells. Therefore, we sought to determine the effects of IL-13 and IFN-γ on the expression of surfactant proteins in adult human ATII cells in vitro. Additional studies were done with rat ATII cells. Methods Adult human ATII cells were isolated from deidentified organ donors whose lungs were not suitable for transplantation and donated for medical research. The cells were cultured on a mixture of Matrigel and rat-tail collagen for 8 d with differentiation factors and human recombinant IL-13 or IFN-γ. Results IL-13 reduced the mRNA and protein levels of surfactant protein (SP-C, whereas IFN-γ increased the mRNA level of SP-C and proSP-C protein but not mature SP-C. Neither cytokine changed the mRNA level of SP-B but IFN-γ slightly decreased mature SP-B. IFN-γ reduced the level of the active form of cathepsin H. IL-13 also reduced the mRNA and protein levels of SP-D, whereas IFN-γ increased both mRNA and protein levels of SP-D. IL-13 did not alter SP-A, but IFN-γ slightly increased the mRNA levels of SP-A. Conclusions We demonstrated that IL-13 and IFN-γ altered the expression of surfactant proteins in human adult ATII cells in vitro. IL-13 decreased SP-C and SP-D in human ATII cells, whereas IFN-γ had the opposite effect. The protein levels of mature SP-B were decreased by IFN-γ treatment, likely due to the reduction in active form cathpesin H. Similarly, the active form of cathepsin H was relatively insufficient to fully process proSP-C as IFN-γ increased the mRNA levels for SP-C and proSP-C protein, but there was no increase in mature SP-C. These observations

  3. Let-7b promotes alpaca hair growth via transcriptional repression of TGFβR I.

    Yan, Shen; Yu, Zhang; Ning, Liu; Hai-Dong, Wang; Jian-Shan, Xie; Shu-Yuan, Gao; Jia-Qi, Cheng; Xiu-Ju, Yu; Ting, Wang; Chang-Sheng, Dong; Xiao-Yan, He

    2016-02-10

    The young male alpaca ear and the back skins were used to investigate the effect of transforming growth factor receptor-β I (TGFβR I) on alpaca hair follicles and hair growth. The expression level and location of TGFβR I in alpaca ear and dorsal skin were detected through real-time quantitative PCR (RT-PCR) and paraffin section immunohistochemical technique (ICC-P). The results shown TGFβR I was lower expression in back skin compared to ear skin and the mean density of the positive reaction in ear skin was significantly higher than back skin. The targeted relationship with let-7b was detected using the dual-luciferase reporter vector of TGFβR I, which showed a significant target relationship between let-7b and TGFβR I. After transfection with let-7b eukaryotic expression vector, the relative mRNA expression of TGFβR I in alpaca skin fibroblasts did not differ, while the relative protein level was significantly decreased. In summary, a higher TGFβR I expression level in the ear skin suggests that TGFβR I may inhibit coat hair elongation. Further studies showed TGFβR I protein was downregulated by let-7b through transcriptional repression. PMID:26611528

  4. Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer

    Chen, Yi; Pirisi, Lucia [Department of Pathology, Microbiology and Immunology, School of Medicine, University of South Carolina, Columbia, SC (United States); Creek, Kim E., E-mail: creekk@sccp.sc.edu [Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC (United States)

    2013-09-15

    We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cell proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer. - Highlights: • Ski oncoprotein levels increase during progression of HPV16-transformed cells. • Ski and phospho-Ski protein levels are cell cycle dependent. • Ski knock-down in HPV16-transformed keratinocytes inhibited cell proliferation. • Cervical cancer samples overexpress Ski.

  5. Challenge with innate and protein antigens induces CCR7 expression by microglia in vitro and in vivo

    Dijkstra, I. M.; de Haas, A. H.; Brouwer, N.; Boddeke, H. W. G. M.; Biber, K.

    2006-01-01

    Since activated microglia are able to phagocytose damaged cells and subsequently express major histocompatibility complex class II (MHC-II) and co-stimulatory proteins, they are considered to function as antigen presenting cells (APCs) in the central nervous system. The maturation and migratory pote

  6. Distinct in vitro interaction pattern of dopamine receptor subtypes with adaptor proteins involved in post-endocytotic receptor targeting

    Heydorn, Arne; Søndergaard, Birgitte P; Hadrup, Niels;

    2004-01-01

    The mechanisms underlying targeted sorting of endocytosed receptors for recycling to the plasma membrane or degradation in lysosomes are poorly understood. In this report, the C-terminal tails of the five dopamine receptors (D1-D5) were expressed as glutathione S-transferase (GST) fusion proteins...

  7. Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer

    We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cell proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer. - Highlights: • Ski oncoprotein levels increase during progression of HPV16-transformed cells. • Ski and phospho-Ski protein levels are cell cycle dependent. • Ski knock-down in HPV16-transformed keratinocytes inhibited cell proliferation. • Cervical cancer samples overexpress Ski

  8. In silico and in vitro studies of cytotoxic activity of different peptides derived from vesicular stomatitis virus G protein

    Fereshte Ghandehari

    2015-01-01

    Conclusion: The results confirmed that P26 and P7 peptides might induce membrane damage and initiate apoptosis. The present study suggested that P26 and P7 peptides could be appropriate candidates for further studies as cytotoxic agents and modifications in the residue at positions 70-280 might potentially produce a more efficient VSVG protein in gene therapy.

  9. Size control of in vitro synthesized magnetite crystals by the MamC protein of Magnetococcus marinus strain MC-1

    Valverde-Tercedor, C; Montalbán-López, M; Perez-Gonzalez, T; Sanchez-Quesada, M S; Prozorov, T; Pineda-Molina, E; Fernandez-Vivas, M A; Rodriguez-Navarro, A B; Trubitsyn, D; Bazylinski, Dennis A; Jimenez-Lopez, C

    2015-01-01

    Magnetotactic bacteria are a diverse group of prokaryotes that share the unique ability of biomineralizing magnetosomes, which are intracellular, membrane-bounded crystals of either magnetite (Fe3O4) or greigite (Fe3S4). Magnetosome biomineralization is mediated by a number of specific proteins, man

  10. Calcium dependent formation of tubular assemblies by recombinant S-layer proteins in vivo and in vitro

    Korkmaz, Nuriye; Ostermann, Kai; Roedel, Gerhard, E-mail: nuriye_korkmaz@yahoo.com, E-mail: kai.ostermann@tu-dresden.de, E-mail: gerhard.roedel@tu-dresden.de [Institut fuer Genetik, Technische Universitaet Dresden, Zellescher Weg 20b, 01217 Dresden (Germany)

    2011-03-04

    Surface layer proteins have the appealing property to self-assemble in nanosized arrays in solution and on solid substrates. In this work, we characterize the formation of assembly structures of the recombinant surface layer protein SbsC of Geobacillus stearothermophilus ATTC 12980, which was tagged with enhanced green fluorescent protein and expressed in the yeast Saccharomyces cerevisiae. The tubular structures formed by the protein in vivo are retained upon bursting the cells by osmotic shock; however, their average length is decreased. During dialysis, monomers obtained by treatment with chaotropic chemicals recrystallize again to form tube-like structures. This process is strictly dependent on calcium (Ca{sup 2+}) ions, with an optimal concentration of 10 mM. Further increase of the Ca{sup 2+} concentration results in multiple non-productive nucleation points. We further show that the lengths of the S-layer assemblies increase with time and can be controlled by pH. After 48 h, the average length at pH 9.0 is 4.13 {mu}m compared to 2.69 {mu}m at pH 5.5. Successful chemical deposition of platinum indicates the potential of recrystallized mSbsC-eGFP structures for nanobiotechnological applications.

  11. Characterization of the in vitro binding and inhibition kinetics of primary amine oxidase/vascular adhesion protein-1 by glucosamine.

    Olivieri, Aldo

    2012-04-01

    Primary-amine oxidase (PrAO) catalyzes the oxidative deamination of endogenous and exogenous primary amines and also functions, in some tissues, as an inflammation-inducible endothelial factor, known as vascular adhesion protein-1. VAP-1 mediates the slow rolling and adhesion of lymphocytes to endothelial cells in a number of inflammatory conditions, including inflammation of the synovium.

  12. In silico and in vitro analyses of the angiotensin-I converting enzyme inhibitory activity of hydrolysates generated from crude barley (Hordeum vulgare) protein concentrates.

    Gangopadhyay, Nirupama; Wynne, Kieran; O'Connor, Paula; Gallagher, Eimear; Brunton, Nigel P; Rai, Dilip K; Hayes, Maria

    2016-07-15

    Angiotensin-I-converting enzyme (ACE-I) plays a key role in control of hypertension, and type-2 diabetes mellitus, which frequently co-exist. Our current work utilised in silico methodologies and peptide databases as tools for predicting release of ACE-I inhibitory peptides from barley proteins. Papain was the enzyme of choice, based on in silico analysis, for experimental hydrolysis of barley protein concentrate, which was performed at the enzyme's optimum conditions (60 °C, pH 6.0) for 24 h. The generated hydrolysate was subjected to molecular weight cut-off (MWCO) filtration, following which the non-ultrafiltered hydrolysate (NUFH), and the generated 3 kDa and 10 kDa MWCO filtrates were assessed for their in vitro ACE-I inhibitory activities. The 3 kDa filtrate (1 mg/ml), that demonstrated highest ACE-I inhibitory activity of 70.37%, was characterised in terms of its peptidic composition using mass spectrometry and 1882 peptides derived from 61 barley proteins were identified, amongst which 15 peptides were selected for chemical synthesis based on their predicted ACE-I inhibitory properties. Of the synthesized peptides, FQLPKF and GFPTLKIF were most potent, demonstrating ACE-I IC50 values of 28.2 μM and 41.2 μM respectively. PMID:26948626

  13. Developmental and hyperthermia-induced expression of the heat shock proteins HSP60 and HSP70 in tissues of the housefly Musca domestica: an in vitro study

    Sunita Sharma

    2007-01-01

    Full Text Available The expression pattern of two major chaperones, the heat shock proteins (HSPs HSP60 and HSP70 was studied in vitro in tissues of the housefly Musca domestica during larval and adult stages of development to identify their immunological relatives and understand their functional significance in normal cellular activities and during thermal stress. Fluorographs of labeled polypeptides and western blots demonstrated that both HSPs are expressed constitutively and heat-induced in all the larval and adult cell types examined. The pattern of whole tissue immunocytochemical staining using anti-HSP60 and anti-HSP70 antibodies corresponded well with the observations from western blots or fluorographs. In developing oocytes, both constitutive and heat inducible expression of HSP60 were regulated in an oocyte stage-specific manner. In unstressed ovaries the expression of these proteins was less pronounced in early stage oocytes (1st - 8th than at later stages (9th and onward. The heat shock, however, induced both HSP70 and HSP60 to a significantly high level in early stage oocytes (1st-8th as compared to their respective controls. Our findings indicate the involvement of the HSP60 and HSP70 proteins in the development, growth and differentiation of both somatic and germ line tissues. Furthermore, the enhanced co-expression of HSP70 and HSP60 upon heat shock in various larval and adult cell types suggests the possible role of HSP60 in thermoprotection.

  14. Polydatin up-regulates clara cell secretory protein to suppress phospholipase A2 of lung induced by LPS in vivo and in vitro

    Jie Chen

    2011-07-01

    Full Text Available Abstract Background Lung injury induced by lipopolysaccharide (LPS remains one of the leading causes of morbidity and mortality in children. The damage to membrane phospholipids leads to the collapse of the bronchial alveolar epithelial barrier during acute lung injury (ALI/acute respiratory distress syndrome (ARDS. Phospholipase A2 (PLA2, a key enzyme in the hydrolysis of membrane phospholipids, plays an important traumatic role in pulmonary inflammation, and Clara cell secretory protein (CCSP is an endogenous inhibitor of PLA2. Our previous study showed that polydatin (PD, a monocrystalline extracted from a traditional Chinese medicinal herb (Polygonum cuspidatum Sieb, et Zucc, reduced PLA2 activity and sPLA2-IIA mRNA expression and mitigated LPS-induced lung injury. However, the potential mechanism for these effects has not been well defined. We have continued to investigate the effect of PD on LPS-induced expression of CCSP mRNA and protein in vivo and in vitro. Results Our results suggested that the CCSP mRNA level was consistent with its protein expression. CCSP expression was decreased in lung after LPS challenge. In contrast, PD markedly increased CCSP expression in a concentration-dependent manner. In particular, CCSP expression in PD-pretreated rat lung was higher than in rats receiving only PD treatment. Conclusion These results indicated that up-regulation of CCSP expression causing inhibition of PLA2 activation may be one of the crucial protective mechanisms of PD in LPS-induced lung injury.

  15. Both short intense and prolonged moderate in vitro stimulation reduce the mRNA expression of calcium-regulatory proteins in rat skeletal muscle

    Mänttäri, Satu; Ørtenblad, Niels; Madsen, Klavs;

    2013-01-01

    ) muscles at 4 h of recovery following in vitro stimulations (either short intensive (SHO) 60 Hz, 5 min, or prolonged moderate (PRO) 20 Hz, 40 min). Stimulation induced acute regulation of the mRNA level of Ca(2+)-regulating proteins in a manner that does not follow typical fibre-type-specific transitions....... In general, stimulation decreased mRNA content of all proteins studied. Most prominent down-regulation was observed for Cacna1 (26 and 32 % after SHO and PRO, respectively, in SOL; 19 % after SHO in EDL). SERCA1, SERCA2, CASQ1, CASQ2, and RyR1 mRNA content also decreased significantly in both muscles...... relative to resting control. Of notice is that hexokinase II mRNA content was increased in EDL and unchanged in SOL underlining the specificity of the down-regulation of mRNA of Ca(2+) regulatory proteins. The results demonstrate contraction-induced down-regulation of mRNAs for the main components of Ca(2...

  16. Effect of ATP and 2-oxoglutarate on the in vitro interaction between the NifA GAF domain and the GlnB protein of Azospirillum brasilense

    Sotomaior, P.; Araújo, L.M.; Nishikawa, C.Y.; Huergo, L.F.; Monteiro, R.A.; Pedrosa, F.O.; Chubatsu, L.S.; Souza, E.M. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil)

    2012-09-21

    Azospirillum brasilense is a diazotroph that associates with important agricultural crops and thus has potential to be a nitrogen biofertilizer. The A. brasilense transcription regulator NifA, which seems to be constitutively expressed, activates the transcription of nitrogen fixation genes. It has been suggested that the nitrogen status-signaling protein GlnB regulates NifA activity by direct interaction with the NifA N-terminal GAF domain, preventing the inhibitory effect of this domain under conditions of nitrogen fixation. In the present study, we show that an N-terminal truncated form of NifA no longer required GlnB for activity and lost regulation by ammonium. On the other hand, in trans co-expression of the N-terminal GAF domain inhibited the N-truncated protein in response to fixed nitrogen levels. We also used pull-down assays to show in vitro interaction between the purified N-terminal GAF domain of NifA and the GlnB protein. The results showed that A. brasilense GlnB interacts directly with the NifA N-terminal domain and this interaction is dependent on the presence of ATP and 2-oxoglutarate.

  17. Effect of ATP and 2-oxoglutarate on the in vitro interaction between the NifA GAF domain and the GlnB protein of Azospirillum brasilense

    Azospirillum brasilense is a diazotroph that associates with important agricultural crops and thus has potential to be a nitrogen biofertilizer. The A. brasilense transcription regulator NifA, which seems to be constitutively expressed, activates the transcription of nitrogen fixation genes. It has been suggested that the nitrogen status-signaling protein GlnB regulates NifA activity by direct interaction with the NifA N-terminal GAF domain, preventing the inhibitory effect of this domain under conditions of nitrogen fixation. In the present study, we show that an N-terminal truncated form of NifA no longer required GlnB for activity and lost regulation by ammonium. On the other hand, in trans co-expression of the N-terminal GAF domain inhibited the N-truncated protein in response to fixed nitrogen levels. We also used pull-down assays to show in vitro interaction between the purified N-terminal GAF domain of NifA and the GlnB protein. The results showed that A. brasilense GlnB interacts directly with the NifA N-terminal domain and this interaction is dependent on the presence of ATP and 2-oxoglutarate

  18. Nuclear medicine in vitro

    The subject is discussed under the following main headings: crystal scintillation counting; liquid scintillation counting; activation analysis; the in vitro nuclear medicine laboratory; blood volume in clinical practice B12 and folate deficiency; radionuclide studies associated with abnormalities of iron; basic principles of competitive radioassay; plasma cortisol; radioimmunoassays for T3 and T4; radioimmunoassay of estrogens; determination of androgens in biological fluids; radioimmunoassay of digitalis glycosides; growth hormone; thyrotropin; gonadotropins; radioimmunoassay of gastrin; glucagon; radioisotopic measurements of insulin; radioimmunoassay of the calcium-regulating hormones; the renin-angiotensin system and aldosterone; tumor antigens; fat absorption; protein-losing enteropathy; Australia antigen; bacteriologic cultures and sensitivities; and future pathways

  19. Let-7b-mediated suppression of basigin expression and metastasis in mouse melanoma cells

    Fu, Tzu-Yen [Department of Animal Science, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Chang, Chia-Che [Institute of Biomedical Sciences, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Graduate Institute of Basic Medical Science, China Medical University, 91 Hsueh Shih Road, Taichung 40402, Taiwan (China); Lin, Chun-Ting [Department of Animal Science, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Lai, Cong-Hao [Institute of Biomedical Sciences, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Department of Life Sciences, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Peng, Shao-Yu; Ko, Yi-Ju [Department of Animal Science, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Tang, Pin-Chi, E-mail: pctang@dragon.nchu.edu.tw [Department of Animal Science, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China)

    2011-02-15

    Basigin (Bsg), also called extracellular matrix metalloproteinase inducer (EMMPRIN), is highly expressed on the surface of tumor cells and stimulates adjacent fibroblasts or tumor cells to produce matrix metalloproteinases (mmps). It has been shown that Bsg plays an important role in growth, development, cell differentiation, and tumor progression. MicroRNAs (miRNAs) are a class of short endogenous non-protein coding RNAs of 20-25 nucleotides (nt) that function as post-transcriptional regulators of gene expression by base-pairing to their target mRNAs and thereby mediate cleavage of target mRNAs or translational repression. In this study, let-7b, one of the let-7 family members, was investigated for its effect on the growth and invasiveness of the mouse melanoma cell line B16-F10. We have shown that let-7b can suppress the expression of Bsg in B16-F10 cells and also provided evidence that this suppression could result in the indirect suppression of mmp-9. The ability of B16-F10 cells transfected with let-7b to invade or migrate was significantly reduced. In addition, let-7b transfected B16-F10 cells displayed an inhibition of both cellular proliferation and colony formation. Furthermore, it was shown that the overexpression of let-7b in B16-F10 cells could reduce lung metastasis. Taken together, the present study identifies let-7b as a tumor suppressor that represses cancer cell proliferation and migration as well as tumor metastasis in mouse melanoma cells.

  20. Expression of L protein of vesicular stomatitis virus Indiana serotype from recombinant baculovirus in insect cells: requirement of a host factor(s) for its biological activity in vitro.

    Mathur, M.; Das, T.; Banerjee, A K

    1996-01-01

    The 241-kDa large (L) protein of vesicular stomatitis virus (VSV) Indiana serotype, a multifunctional catalytic subunit of the viral RNA polymerase, has been expressed in Spodoptera frugiperda cells infected with recombinant baculovirus BacPAK6-L containing the L gene under the control of a polyhedrin promoter. The recombinant L protein was biologically active and supported viral mRNA synthesis in vitro. When the expressed L protein was purified by phosphocellulose column chromatography, it e...

  1. pH and the effect of fluoride and zinc on protein and collagen biosynthesis in rabbit dental pulp in vitro

    The effect of pH in the range 6.6-8.2 on the incorporation of 14C-proline into the rabbit dental pulp incubated in vitro has been studied. The amount of label in the cold TCA-soluble pool, total protein, and hydroxyproline (i.e. collagen) of the pulp tissue increased linearly with pH in all three fractions. A similar increase was found in the amount of labeled total protein and collagen recovered from the incubation medium. The results indicated that the uptake of 14C-proline into the TCA-soluble pool was the pH-sensitive step, whereas the incorporation into protein, formation of hydroxyproline, and the release of labeled macromolecules into the medium were not affected to any measurable degree by the ambient pH. In this system, zinc (60 μM) had less effect on the incorporation of 14C-proline into the different fractions of the pulp tissue at pH 6.6 as compared with pH 7.4, whereas with fluoride (1.3 mM) an increased inhibition of the uptake of 14C-proline into the TCA-soluble pool and the incorporation into total protein was found upon lowering the pH to 6.8. The inhibitory effect of zinc and fluoride on the release of labeled total protein and collagen into the incubation medium was not affected when the pH was lowered. (author)

  2. Dose-dependent Inhibition of Gynecophoral Canal Protein Gene Expression in Vitro in the Schistosome (Schistosomajaponicum) by RNA Interference

    Guo-Feng CHENG; Jiao-Jiao LIN; Yi SHI; You-Xin JIN; Zhi-Qiang FU; Ya-Mei JIN; Yuan-Cong ZHOU; You-Min CAI

    2005-01-01

    The gynecophoral canal protein gene SjGCP of Schistosoma japonicum that is necessary for the pairing between the male and female worms is specifically expressed in the adult male worm. This protein is widely distributed in the adult female worm after pairing. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence were employed to analyze the relationship between the RNAi effect and dsRNA dosage in the parasites. The results revealed that the inhibition of SjGCP expression by siRNA is dose-dependent. RT-PCR analysis showed that the SjGCP transcript level was reduced by 75%when 100 nM dsRNA was applied.

  3. Fibrin glue is a candidate scaffold for long-term therapeutic protein expression in spontaneously differentiated adipocytes in vitro

    Adipose tissue is expected to provide a source of cells for protein replacement therapies via auto-transplantation. However, the conditioning of the environment surrounding the transplanted adipocytes for their long-term survival and protein secretion properties has not been established. We have recently developed a preparation procedure for preadipocytes, ceiling culture-derived proliferative adipocytes (ccdPAs), as a therapeutic gene vehicle suitable for stable gene product secretion. We herein report the results of our evaluation of using fibrin glue as a scaffold for the transplanted ccdPAs for the expression of a transduced gene in a three-dimensional culture system. The ccdPAs secreted the functional protein translated from an exogenously transduced gene, as well as physiological adipocyte proteins, and the long viability of ccdPAs (up to 84 days) was dependent on the fibrinogen concentrations. The ccdPAs spontaneously accumulated lipid droplets, and their expression levels of the transduced exogenous gene with its product were maintained for at least 56 days. The fibrinogen concentration modified the adipogenic differentiation of ccdPAs and their exogenous gene expression levels, and the levels of exogenously transduced gene expression at the different fibrinogen concentrations were dependent on the extent of adipogenic differentiation in the gel. These results indicate that fibrin glue helps to maintain the high adipogenic potential of cultured adipocytes after passaging in a 3D culture system, and suggests that once they are successfully implanted at the transplantation site, the cells exhibit increased expression of the transduced gene with adipogenic differentiation.

  4. Synthesis and in vitro antioxidant functions of protein hydrolysate from backbones of Rastrelliger kanagurta by proteolytic enzymes

    Sheriff, Sheik Abdulazeez; Sundaram, Balasubramanian; Ramamoorthy, Baranitharan; Ponnusamy, Ponmurugan

    2013-01-01

    Every year, a huge quantity of fishery wastes and by-products are generated by fish processing industries. These wastes are either underutilized to produce low market value products or dumped leading to environmental issues. Complete utilization of fishery wastes for recovering value added products would be beneficial to the society and individual. The fish protein hydrolysates and derived peptides of fishery resources are widely used as nutritional supplements, functional ingredients, and fl...

  5. The bone morphogenetic protein receptor-1A pathway is required for lactogenic differentiation of mammary epithelial cells in vitro

    Perotti, C.; Karayazi, Ö.; Moffat, S.; Shemanko, C. S.

    2012-01-01

    Bone morphogenetic proteins (BMPs) have been implicated in the control of proliferation, tissue formation, and differentiation. BMPs regulate the biology of stem and progenitor cells and can promote cellular differentiation, depending on the cell type and context. Although the BMP pathway is known to be involved in early embryonic development of the mammary gland via mesenchymal cells, its role in later epithelial cellular differentiation has not been examined. The majority of the mammary gla...

  6. Protein Kinase CK2 Expression Predicts Relapse Survival in ERα Dependent Breast Cancer, and Modulates ERα Expression in Vitro

    Marlon D. Williams

    2015-12-01

    Full Text Available The heterotetrameric protein kinase CK2 has been associated with oncogenic transformation, and our previous studies have shown that it may affect estrogenic signaling. Here, we investigate the role of the protein kinase CK2 in regulating ERα (estrogen receptor α signaling in breast cancer. We determined the correlation of CK2α expression with relapse free breast cancer patient survival utilizing Kaplan Meier Plotter (kmplot.com/analysis/ to mine breast cancer microarrays repositories. Patients were stratified according to ERα status, histological grade, and hormonal therapy. Luciferase reporter assays and flow cytometry were implemented to determine the impact of CK2 inhibition on ERE-mediated gene expression and expression of ERα protein. CK2α expression is associated with shorter relapse free survival among ERα (+ patients with grade 1 or 2 tumors, as well as among those patients receiving hormonal therapy. Biochemical inhibition of CK2 activity results in increased ER-transactivation as well as increased expression among ERα (+ and ERα (− breast cancer cell lines. These findings suggest that CK2 may contribute to estrogen-independent cell proliferation and breast tumor progression, and may potentially serve as a biomarker and pharmacological target in breast cancer.

  7. Human bone morphogenetic protein-2 gene transfer induces human mesenchymal stem cell proliferation and differentiation in vitro

    李军; 范清宇; 钱济先; 马保安; 周勇; 张明华

    2004-01-01

    Objective: To identify eukaryotic expression vector of human bone morphogenetic protein 2 pcDNA3/BMP2, verify its expression in transfected human mesenchymal stem cells (hMSCs) and the effect on hMSCs differentiation.Methods: The BMP2 gene was cloned into a eukaryotic expression vector pcDNA3. Transfected the recombinant into hMSCs by liposome. Immunnohistochemistry and in situ hybridization methods were used to identify the expression of BMP2 mRNA and protein; ALP and Von Kossa stains were performed to identify the BMP2 gene differentiated effect on the hMSCs. Results: The pcDNA3/BMP2 fragments were as large as theory. BMP2 mRNA and protein were expressed and synthesized both in 48 h and 4 weeks after transfection, the ALP and Ca deposit exhibition, which marked the osteogenic lineage of hMSCs,were enhanced and sped. Conclusion: Transfection of pcDNA3/BMP2 is able to provide transient and persistent expression in hMSCs, and promote the MSCs differentiation to osteogenic lineage.

  8. In vitro antioxidant activity of pine kernel protein hydrolysate%松仁蛋白酶解液体外抗氧化活性分析

    林秀芳; 吴晓红; 赵梓辰; 徐冰心; 高小棠; 刘敏

    2014-01-01

    采用两种胃肠消化酶(胃蛋白酶、胰蛋白酶)对松仁蛋白进行酶解实验,研究其胃蛋白酶酶解液、胰蛋白酶酶解液和胃蛋白酶-胰蛋白酶分步酶解酶解液的体外抗氧化活性。结果表明:胃蛋白酶酶解30 min的酶解液具有最强体外抗氧化活性,·OH清除率为67.43%,O2-·清除率为55.66%;胰蛋白酶酶解60 min的酶解液具有最强清除·OH能力,·OH清除率为68.79%;胰蛋白酶酶解120 min的酶解液具有最强清除O2-·能力,O2-·清除率为39.20%;胃蛋白酶-胰蛋白酶分步酶解松仁蛋白酶解液在酶解240 min 时,具有最强的体化抗氧化活性,此时· OH 清除率为79.11%,O2-·清除率为68.99%。%Enzymatic hydrolysis experiment of pine kernel protein was conducted by two kinds of gastroin-testinal digestive enzymes ( pepsin and trypsin ) , and in vitro antioxidant activities of enzymatic hydrolysates of pine kernel protein obtained by hydrolysis of pepsin, trypsin and pepsin -trypsin were studied. The results showed that after enzymatic hydrolysis for 30 min,the pepsin enzymatic hydrolysate of pine kernel protein had the strongest in vitro antioxidant activity,and the scavenging rates on ·OH radical and O2-·radical were 67. 43% and 55. 66% respectively;the trypsin enzymatic hydrolysate of pine kernel protein had the strongest scavenging capacity on ·OH radical after enzymatic hydrolysis for 60 min,and the scavenging rate on ·OH radical was 68. 79%,while it had the strongest scavenging ca-pacity on O2-· radical after enzymatic hydrolysis for 120 min,and the scavenging rate on O2-· radical was 39 . 20%;pepsin-trypsin step-by-step enzymatic hydrolysate of pine kernel protein had the stron-gest in vitro antioxidant activity after enzymatic hydrolysis for 240 min,and the scavenging rates on·OH radical and O2-·radical were 79. 11% and 68. 99% respectively.

  9. Discovering and validating unknown phospho-sites from p38 and HuR protein kinases in vitro by Phosphoproteomic and Bioinformatic tools

    López Elena

    2011-07-01

    Full Text Available Abstract Background The mitogen activated protein kinase (MAPK pathways are known to be deregulated in many human malignancies. Phosphopeptide identification of protein-kinases and site determination are major challenges in biomedical mass spectrometry (MS. P38 and HuR protein kinases have been reported extensively in the general principles of signalling pathways modulated by phosphorylation, mainly by molecular biology and western blotting techniques. Thus, although it has been demonstrated they are phosphorylated in different stress/stimuli conditions, the phosphopeptides and specific amino acids in which the phosphate groups are located in those protein kinases have not been shown completely. Methods We have combined different resins: (a IMAC (Immobilized Metal Affinity Capture, (b TiO2 (Titanium dioxide and (c SIMAC (Sequential Elution from IMAC to isolate phosphopeptides from p38 and HuR protein kinases in vitro. Different phosphopeptide MS strategies were carried out by the LTQ ion Trap mass spectrometer (Thermo: (a Multistage activation (MSA and (b Neutral loss MS3 (DDNLMS3. In addition, Molecular Dynamics (MD bioinformatic simulation has been applied in order to simulate, over a period of time, the effects of the presence of the extra phosphate group (and the associated negative charge in the overall structure and behaviour of the protein HuR. This study is supported by the Declaration of Helsinki and subsequent ethical guidelines. Results The combination of these techniques allowed for: (1 The identification of 6 unknown phosphopeptides of these protein kinases. (2 Amino acid site assignments of the phosphate groups from each identified phosphopeptide, including manual validation by inspection of all the spectra. (3 The analyses of the phosphopeptides discovered were carried out in four triplicate experiments to avoid false positives getting high reproducibility in all the isolated phosphopeptides recovered from both protein kinases. (4

  10. Screening Glycolipids Against Proteins in Vitro Using Picodiscs and Catch-and-Release Electrospray Ionization-Mass Spectrometry.

    Li, Jun; Fan, Xuxin; Kitova, Elena N; Zou, Chunxia; Cairo, Christopher W; Eugenio, Luiz; Ng, Kenneth K S; Xiong, Zi Jian; Privé, Gilbert G; Klassen, John S

    2016-05-01

    This work describes the application of the catch-and-release electrospray ionization-mass spectrometry (CaR-ESI-MS) assay, implemented using picodiscs (complexes comprised of saposin A and lipids, PDs), to screen mixtures of glycolipids (GLs) against water-soluble proteins to detect specific interactions. To demonstrate the reliability of the method, seven gangliosides (GM1, GM2, GM3, GD1a, GD1b, GD2, and GT1b) were incorporated, either individually or as a mixture, into PDs and screened against two lectins: the B subunit homopentamer of cholera toxin (CTB5) and a subfragment of toxin A from Clostridium difficile (TcdA-A2). The CaR-ESI-MS results revealed that CTB5 binds to six of the gangliosides (GM1, GM2, GM3, GD1a, GD1b, and GT1b), while TcdA-A2 binds to five of them (GM1, GM2, GM3, GD1a, and GT1b). These findings are consistent with the measured binding specificities of these proteins for ganglioside oligosaccharides. Screening mixtures of lipids extracted from porcine brain and a human epithelial cell line against CTB5 revealed binding to multiple GM1 isoforms as well as to fucosyl-GM1, which is a known ligand. Finally, a comparison of the present results with data obtained with the CaR-ESI-MS assay implemented using nanodiscs (NDs) revealed that the PDs exhibited similar or superior performance to NDs for protein-GL binding measurements. PMID:27049760

  11. Effects of anticonvulsant drugs on the synthesis of DNA and protein by human bone marrow cells in vitro

    Suspensions of human bone marrow cells were incubated with various concentrations of phenobarbitone or phenytoin sodium for 2 h, and the effects of this incubation on the subsequent incorporation of 3H-thymidine and 3H-leucine into DNA and protein, respectively, were studied. Both drugs caused a depression of 3H-thymidine incorporation and this phenomenon was not prevented by the addition of 100 μg of pteroylglutamic acid, folinic acid or 5-methyltetrahydrofolate per ml of marrow culture. The lowest concentration of drug which caused a statistically significant depression of 3H-thymidine incorporation was 200μg per ml for phenobarbitone and 50 μg per ml for phenytoin sodium. Both phenobarbitone and phenytoin sodium also caused an increase in the incorporation of 3H-leucine at concentrations of 50 and 20 μg per ml., respectively, suggesting the possibility that a stimulation of protein synthesis within erythropoietic cells may play an important role in the development of anticonvulsant-induced macrocytosis. (authod)

  12. Antioxidant and Chelating Activity of Nontoxic Jatropha curcas L. Protein Hydrolysates Produced by In Vitro Digestion Using Pepsin and Pancreatin

    Santiago Gallegos Tintoré

    2015-01-01

    Full Text Available The antioxidant and metal chelating activities in J. curcas protein hydrolysates have been determined. The hydrolysates were produced by treatment of a nontoxic genotype with the digestive enzymes pepsin and pancreatin and then were characterized by fast protein liquid chromatography and reverse phase chromatography. Peptidic fractions with higher radical scavenging activity were analysed by matrix-assisted laser desorption/ionization mass spectrometry. The antioxidant activity was determined by measuring inhibition of the oxidative degradation of β-carotene and by measuring the reactive oxygen species (ROS in Caco-2 cell cultures. Cu2+ and Fe2+ chelating activities were also determined. The hydrolysates inhibited the degradation of β-carotene and the formation of ROS in Caco-2 cells. The lower molecular weight peptidic fractions from FPLC had stronger antioxidant activity in cell cultures compared with the hydrolysates, which correlated with a higher content in antioxidant and chelating amino acids. These fractions were characterized by a large presence of peptides with different molecular masses. The hydrolysates exhibited both Cu2+ and Fe2+ chelating activity. It was concluded that J. curcas is a good source of antioxidant and metal chelating peptides, which may have a positive impact on the economic value of this crop, as a potential source of food functional components.

  13. Antiviral activity of recombinant porcine surfactant protein A against porcine reproductive and respiratory syndrome virus in vitro.

    Li, Lan; Zheng, Qisheng; Zhang, Yuanpeng; Li, Pengcheng; Fu, Yanfeng; Hou, Jibo; Xiao, Xilong

    2016-07-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) has caused significant economic losses in the swine industry worldwide. However, there is not an ideal vaccine to provide complete protection against PRRSV. Thus, the need for new antiviral strategies to control PRRSV still remains. Surfactant protein A (SP-A) belongs to the family of C-type lectins, which can exert antiviral activities. In this present study, we assessed the antiviral properties of recombinant porcine SP-A (RpSP-A) on PRRSV infection in Marc 145 cells and revealed its antiviral mechanism using a plaque assay, real-time qPCR, western blotting analysis and an attachment and penetration assay. Our results showed that RpSP-A could inhibit the infectivity of PRRSV in Marc 145 cells and could reduce the total RNA and protein level. The attachment assay indicated that RpSP-A in the presence of Ca(2+) could largely inhibit Marc 145 cell attachment; however, in the penetration assay, it was relatively inactive. Furthermore, our study suggested that virus progeny released from infected Marc145 cells were blocked by RpSP-A from infecting other cells. We conclude that RpSP-A has antiviral activity against PRRSV, most probably by blocking viral attachment and the cell-to-cell transmission pathway, and therefore, RpSP-A holds promise as a novel antiviral agent against PRRSV. PMID:27101074

  14. Retinoic acid differentially affects in vitro proliferation, differentiation and mineralization of two fish bone-derived cell lines: different gene expression of nuclear receptors and ECM proteins.

    Fernández, Ignacio; Tiago, Daniel M; Laizé, Vincent; Leonor Cancela, M; Gisbert, Enric

    2014-03-01

    Retinoic acid (RA), the main active metabolite of vitamin A, regulates vertebrate morphogenesis through signaling pathways not yet fully understood. Such process involves the specific activation of retinoic acid and retinoid X receptors (RARs and RXRs), which are nuclear receptors of the steroid/thyroid hormone receptor superfamily. Teleost fish are suitable models to study vertebrate development, such as skeletogenesis. Cell systems capable of in vitro mineralization have been developed for several fish species and may provide new insights into the specific cellular and molecular events related to vitamin A activity in bone, complementary to in vivo studies. This work aims at investigating the in vitro effects of RA (0.5 and 12.5 μM) on proliferation, differentiation and extracellular matrix (ECM) mineralization of two gilthead seabream bone-derived cell lines (VSa13 and VSa16), and at identifying molecular targets of its action through gene expression analysis. RA induced phenotypic changes and cellular proliferation was inhibited in both cell lines in a cell type-dependent manner (36-59% in VSa13 and 17-46% in VSa16 cells). While RA stimulated mineral deposition in VSa13 cell cultures (50-62% stimulation), it inhibited the mineralization of extracellular matrix in VSa16 cells (11-57% inhibition). Expression of hormone receptor genes (rars and rxrs), and extracellular matrix-related genes such as matrix and bone Gla proteins (mgp and bglap), osteopontin (spp1) and type I collagen (col1a1) were differentially regulated upon exposure to RA in proliferating, differentiating and mineralizing cultures of VSa13 and VSa16 cells. Altogether, our results show: (i) RA affects proliferative and mineralogenic activities in two fish skeletal cell types and (ii) that during phenotype transitions, specific RA nuclear receptors and bone-related genes are differentially expressed in a cell type-dependent manner. PMID:24291400

  15. Dynamic interfacial properties of human tear-lipid films and their interactions with model-tear proteins in vitro.

    Svitova, Tatyana F; Lin, Meng C

    2016-07-01

    This review summarizes the current state of knowledge regarding interfacial properties of very complex biological colloids, specifically, human meibum and tear lipids, and their interactions with proteins similar to the proteins found in aqueous part of human tears. Tear lipids spread as thin films over the surface of tear-film aqueous and play crucial roles in tear-film stability and overall ocular-surface health. The vast majority of papers published to date report interfacial properties of meibum-lipid monolayers spread on various aqueous sub-phases, often containing model proteins, in Langmuir trough. However, it is well established that natural human ocular tear lipids exist as multilayered films with a thickness between 30 and 100nm, that is very much disparate from 1 to 2nm thick meibum monolayers. We employed sessile-bubble tensiometry to study the dynamic interfacial and rheological properties of reconstituted multilayered human tear-lipid films. Small amounts (0.5-1μg) of human tear lipids were deposited on an air-bubble surface to produce tear-lipid films in thickness range 30-100nm corresponding to ocular lipid films. Thus, we were able to overcome major Langmuir-trough method limitations because ocular tear lipids can be safely harvested only in minute, sub-milligram quantities, insufficient for Langmuir through studies. Sessile-bubble method is demonstrated to be a versatile tool for assessing conventional synthetic surfactants adsorption/desorption dynamics at an air-aqueous solution interface. (Svitova T., Weatherbee M., Radke C.J. Dynamics of surfactant sorption at the air/water interface: continuous-flow tensiometry. J. Colloid Interf. Sci. 2003;261:1170-179). The augmented flow-sessile-bubble setup, with step-strain relaxation module for dynamic interfacial rheological properties and high-precision syringe pump to generate larger and slow interfacial area expansions-contractions, was developed and employed in our studies. We established that

  16. Mycoplasma hyopneumoniae-derived lipid-associated membrane proteins induce inflammation and apoptosis in porcine peripheral blood mononuclear cells in vitro.

    Bai, Fangfang; Ni, Bo; Liu, Maojun; Feng, Zhixin; Xiong, Qiyan; Shao, Guoqing

    2015-01-30

    Mycoplasma hyopneumoniae is the causative agent of swine enzootic pneumonia (EP), a disease that causes considerable economic losss in swine industry. Lipid-associated membrane proteins (LAMPs) of mycoplasma play important roles in causing mycoplasma diseases. The present study explores the pathogenic mechanisms of M. hyopneumoniae LAMPs by elucidating their role in modulating the inflammation, apoptosis, and relevant signaling pathways of peripheral blood mononuclear cells (PBMCs) of pig. LAMP treatment inhibited the growth of PBMCs. Up-regulation of cytokines, such as IL-6 and IL-1β, as well as increased production of nitric oxide (NO) and superoxide anion were all detected in the supernatant of LAMPs-treated PBMCs. Furthermore, flow cytometric analysis using dual staining with annexin-V-FITC and propidium iodide (PI) showed that LAMPs of M. hyopneumoniae induced a time-dependent apoptosis in lymphocyts and monocytes from PBMCs, which was blocked by NOS inhibitor or antioxidant. In addition, LAMPs induced the phosphorylation of p38, the ratio of pro-apoptotic Bax protein to anti-apoptotic Bcl-2, activation of caspase-3 and caspase-8, and poly ADP-ribose polymerase (PARP) cleavage in PBMCs. These findings demonstrated that M. hyopneumoniae LAMPs induced the production of proinflammatory cytokines, NO and reactive oxygen species (ROS), and apoptosis of PBMCs in vitro through p38 MAPK and Bax/Bcl-2 signaling pathways, as well as caspase activation. PMID:25481242

  17. Microtubule-associated protein 1B (MAP1B)-deficient neurons show structural presynaptic deficiencies in vitro and altered presynaptic physiology.

    Bodaleo, Felipe J; Montenegro-Venegas, Carolina; Henríquez, Daniel R; Court, Felipe A; Gonzalez-Billault, Christian

    2016-01-01

    Microtubule-associated protein 1B (MAP1B) is expressed predominantly during the early stages of development of the nervous system, where it regulates processes such as axonal guidance and elongation. Nevertheless, MAP1B expression in the brain persists in adult stages, where it participates in the regulation of the structure and physiology of dendritic spines in glutamatergic synapses. Moreover, MAP1B expression is also found in presynaptic synaptosomal preparations. In this work, we describe a presynaptic phenotype in mature neurons derived from MAP1B knockout (MAP1B KO) mice. Mature neurons express MAP1B, and its deficiency does not alter the expression levels of a subgroup of other synaptic proteins. MAP1B KO neurons display a decrease in the density of presynaptic and postsynaptic terminals, which involves a reduction in the density of synaptic contacts, and an increased proportion of orphan presynaptic terminals. Accordingly, MAP1B KO neurons present altered synaptic vesicle fusion events, as shown by FM4-64 release assay, and a decrease in the density of both synaptic vesicles and dense core vesicles at presynaptic terminals. Finally, an increased proportion of excitatory immature symmetrical synaptic contacts in MAP1B KO neurons was detected. Altogether these results suggest a novel role for MAP1B in presynaptic structure and physiology regulation in vitro. PMID:27425640

  18. In vivo and in vitro protein-DNA interactions at the distal oestrogen response element of the chicken vitellogenin gene: evidence for the same protein binding to this sequence in hen and rooster liver.

    McEwan, I J; Saluz, H P; Jost, J P

    1991-03-01

    The major egg white protein, vitellogenin, is synthesized in a tissue specific and oestradiol dependent manner in the liver of egg-laying hens. In this paper, we describe a detailed study of the protein-DNA interactions at the distal oestrogen response element (ERED) located 600 bp upstream of the start of transcription. In vivo footprinting of hepatocytes from adult hens and roosters with 0.5-0.0005% dimethylsulphate (DMS) revealed, at critical concentrations of DMS, protection of distinct guanosine residues within the ERED and adjacent downstream sequence in both cases. From this, it was concluded that there were proteins present in both tissues binding to this region in vivo. In vitro studies using missing base contact probing and proteolytic clipping band shift assays with hen and rooster liver nuclear extracts identified the ERE binding protein to be the same or very closely related in both tissues. Furthermore, the protein from rooster nuclear extracts bound to the ERE sequence even when the DNA was methylated at CpG dinucleotides, u.v. cross-linking experiments performed with bromodeoxyuridine substituted ERE, revealed that a nuclear protein with Mr of about 75,000-80,000 bound specifically to this sequence. These studies demonstrate that apart from the oestrogen receptor, at least one other protein can interact specifically with the chicken vitellogenin ERE, independently of hormonal expression of the gene. PMID:2009219

  19. Hybrids of the bHLH and bZIP protein motifs display different DNA-binding activities in vivo vs. in vitro.

    Hiu-Kwan Chow

    Full Text Available Minimalist hybrids comprising the DNA-binding domain of bHLH/PAS (basic-helix-loop-helix/Per-Arnt-Sim protein Arnt fused to the leucine zipper (LZ dimerization domain from bZIP (basic region-leucine zipper protein C/EBP were designed to bind the E-box DNA site, CACGTG, targeted by bHLHZ (basic-helix-loop-helix-zipper proteins Myc and Max, as well as the Arnt homodimer. The bHLHZ-like structure of ArntbHLH-C/EBP comprises the Arnt bHLH domain fused to the C/EBP LZ: i.e. swap of the 330 aa PAS domain for the 29 aa LZ. In the yeast one-hybrid assay (Y1H, transcriptional activation from the E-box was strong by ArntbHLH-C/EBP, and undetectable for the truncated ArntbHLH (PAS removed, as detected via readout from the HIS3 and lacZ reporters. In contrast, fluorescence anisotropy titrations showed affinities for the E-box with ArntbHLH-C/EBP and ArntbHLH comparable to other transcription factors (K(d 148.9 nM and 40.2 nM, respectively, but only under select conditions that maintained folded protein. Although in vivo yeast results and in vitro spectroscopic studies for ArntbHLH-C/EBP targeting the E-box correlate well, the same does not hold for ArntbHLH. As circular dichroism confirms that ArntbHLH-C/EBP is a much more strongly alpha-helical structure than ArntbHLH, we conclude that the nonfunctional ArntbHLH in the Y1H must be due to misfolding, leading to the false negative that this protein is incapable of targeting the E-box. Many experiments, including protein design and selections from large libraries, depend on protein domains remaining well-behaved in the nonnative experimental environment, especially small motifs like the bHLH (60-70 aa. Interestingly, a short helical LZ can serve as a folding- and/or solubility-enhancing tag, an important device given the focus of current research on exploration of vast networks of biomolecular interactions.

  20. Anticariogenic Activity and Haemolytic Study of Some Medicinal Plants Leaf Protein Extract against Six Oral pathogens in In vitro condition

    Hiral Soni

    2014-09-01

    Full Text Available Leaf extract of 12 medicinal plants were obtained by using 10mM sodium phosphate buffer and studied for their antibacterial activity against 6 cariogenic pathogens. Among them, the extract from Streblus asper showed the most intensive activity. Antibacterial activity of Streblus asper was investigated by the minimum inhibitory concentration (MIC test and the viable cell count method. MIC of Streblus asper against Candida albicans causing dental caries was determined to be 2.5 μg/ml. The kinetic study showed that Streblus asper completely inactivated C. albicans at the concentration 50μg/ml in 15hours.Hemolytic study of the Streblus asper plant leaf protein gives HC50 value at a very high range than that of the MIC values against C. albicans of the organisms and thus flourish its application as successful pharmaceutical drug in practice.

  1. In vitro effects of the extracellular protein of Renibacterium salmoninarum on phagocyte function in brook trout (Salvelinus fontinalis).

    Densmore, C L; Smith, S A; Holladay, S D

    1998-04-30

    Renibacterium salmoninarum is a facultative intracellular pathogen often found in host phagocytes where it appears to successfully avoid the host fish's immunological defenses. The objective of this investigation was to determine if soluble extracellular protein (ECP) produced by R. salmoninarum may contribute to the immunomodulation in bacterial kidney disease (BKD) via inhibition of phagocyte respiratory burst and/or phagocytosis mechanisms. Splenic cells from adult brook trout (Salvelinus fontinalis) were incubated with two different concentrations of ECP (0.1 mg/ml and 1.0 mg/ml) and viable R. salmoninarum. Splenic cell cultures were evaluated for respiratory burst activity via flow cytometry with the dichlorofluorescin diacetate (DCF-DA) assay and for phagocytosis via light microscopic assessment of microsphere engulfment. Respiratory burst activity was significantly inhibited in all treatment groups as compared to untreated fish, while no differences were noted in phagocytic activity. PMID:9646439

  2. Synthesis, characterization and target protein binding of drug-conjugated quantum dots in vitro and in living cells

    Choi, Youngseon; Kim, Minjung; Cho, Yoojin; Yun, Eunsuk; Song, Rita

    2013-02-01

    Elucidation of unknown target proteins of a drug is of great importance in understanding cell biology and drug discovery. There have been extensive studies to discover and identify target proteins in the cell. Visualization of targets using drug-conjugated probes has been an important approach to gathering mechanistic information of drug action at the cellular level. As quantum dot (QD) nanocrystals have attracted much attention as a fluorescent probe in the bioimaging area, we prepared drug-conjugated QD to explore the potential of target discovery. As a model drug, we selected a well-known anticancer drug, methotrexate (MTX), which has been known to target dihydrofolate reductase (DHFR) with high affinity binding (Kd = 0.54 nM). MTX molecules were covalently attached to amino-PEG-polymer-coated QDs. Specific interactions of MTX-conjugated QDs with DHFR were identified using agarose gel electrophoresis and fluorescence microscopy. Cellular uptake of the MTX-conjugated QDs in living CHO cells was investigated with regard to their localization and distribution pattern. MTX-QD was found to be internalized into the cells via caveolae-medicated endocytosis without significant sequestration in endosomes. A colocalization experiment of the MTX-QD conjugate with antiDHFR-TAT-QD also confirmed that MTX-QD binds to the target DHFR. This study showed the potential of the drug-QD conjugate to identify or visualize drug-target interactions in the cell, which is currently of great importance in the area of drug discovery and chemical biology.

  3. Synthesis, characterization and target protein binding of drug-conjugated quantum dots in vitro and in living cells

    Elucidation of unknown target proteins of a drug is of great importance in understanding cell biology and drug discovery. There have been extensive studies to discover and identify target proteins in the cell. Visualization of targets using drug-conjugated probes has been an important approach to gathering mechanistic information of drug action at the cellular level. As quantum dot (QD) nanocrystals have attracted much attention as a fluorescent probe in the bioimaging area, we prepared drug-conjugated QD to explore the potential of target discovery. As a model drug, we selected a well-known anticancer drug, methotrexate (MTX), which has been known to target dihydrofolate reductase (DHFR) with high affinity binding (Kd = 0.54 nM). MTX molecules were covalently attached to amino-PEG-polymer-coated QDs. Specific interactions of MTX-conjugated QDs with DHFR were identified using agarose gel electrophoresis and fluorescence microscopy. Cellular uptake of the MTX-conjugated QDs in living CHO cells was investigated with regard to their localization and distribution pattern. MTX–QD was found to be internalized into the cells via caveolae-medicated endocytosis without significant sequestration in endosomes. A colocalization experiment of the MTX–QD conjugate with antiDHFR-TAT-QD also confirmed that MTX–QD binds to the target DHFR. This study showed the potential of the drug-QD conjugate to identify or visualize drug–target interactions in the cell, which is currently of great importance in the area of drug discovery and chemical biology. (paper)

  4. Inhibition of protein kinase CK2 by condensed polyphenolic derivatives. An in vitro and in vivo study.

    Meggio, Flavio; Pagano, Mario A; Moro, Stefano; Zagotto, Giuseppe; Ruzzene, Maria; Sarno, Stefania; Cozza, Giorgio; Bain, Jenny; Elliott, Matthew; Deana, Arianna Donella; Brunati, Anna Maria; Pinna, Lorenzo A

    2004-10-12

    ATP site-directed inhibitors that can target individual kinases are powerful tools for use in signal transduction research, all the more so in the case of a pleiotropic, constitutively active protein kinase such as CK2, which is not turned on in response to specific stimuli. By screening a library of more than 200 derivatives of natural polyphenolic compounds, we have identified 16 molecules which inhibit CK2 with IC(50) values of protein kinases tested. Treatment of Jurkat cells with these compounds promotes inhibition of endogenous CK2 and induction of apoptosis. A correlation is observed between their efficacy as CK2 inhibitors (as judged from IC(50) values) and their capacity to induce cell death (DC(50) values). Mutations of the unique CK2alpha residues Val66 and/or Ile174 to alanine have a detrimental effect on inhibition by these compounds with 16-67-fold increases in IC(50) values. The combined usage of these reagents can be exploited to gain information about cellular functions mediated by CK2. PMID:15461466

  5. Effects of bone morphogenic protein 4 (BMP4) and its inhibitor, Noggin, on in vitro maturation and culture of bovine preimplantation embryos

    Fernandez-Martin Rafael; Pereira Michele M; Camargo Luiz SA; La Rosa Isabel; Paz Dante A; Salamone Daniel F

    2011-01-01

    Abstract Background BMP4 is a member of the transforming growth factor beta (TGFbeta) superfamily and Noggin is a potent BMP inhibitor that exerts its function by binding to BMPs preventing interactions with its receptors. The aim of this work was to investigate the role of BMP4 and Noggin, on oocytes in vitro maturation (m experiments) and embryos in vitro development (c experiments) of bovine. Methods For m experiments, COCs were collected from slaughterhouse ovaries and in vitro matured in...

  6. Recombinant protein of heptad-repeat HR212, a stable fusion inhibitor with potent anti-HIV action in vitro

    HR212, a recombinant protein expressed in Escherichia coli, has been previously reported to inhibit HIV-1 membrane fusion at low nanomolar level. Here we report that HR212 is effective in blocking laboratory strain HIV-1IIIB entry and replication with EC50 values of 3.92 ± 0.62 and 6.59 ± 1.74 nM, respectively, and inhibiting infection by clinic isolate HIV-1KM018 with EC50 values of 44.44 ± 10.20 nM, as well as suppressing HIV-1-induced cytopathic effect with an EC50 value of 3.04 ± 1.20 nM. It also inhibited HIV-2ROD and HIV-2CBL-20 entry and replication in the μM range. Notably, HR212 was highly effective against T20-resistant strains with EC50 values ranging from 5.09 to 7.75 nM. Unlike T20, HR212 showed stability sufficient to inhibit syncytia formation in a time-of-addition assay, and was insensitive to proteinase K digestion. These results suggest that HR212 has great potential to be further developed as novel HIV-1 fusion inhibitor for treatment of HIV/AIDS patients, particularly for those infected by T20-resistant variants

  7. A protein extract and a cysteine protease inhibitor enriched fraction from Jatropha curcas seed cake have in vitro anti-Toxoplasma gondii activity.

    Soares, A M S; Carvalho, L P; Melo, E J T; Costa, H P S; Vasconcelos, I M; Oliveira, J T A

    2015-06-01

    Toxoplasma gondii is a parasite of great medical and veterinary importance that has worldwide distribution and causes toxoplasmosis. There are few treatments available for toxoplasmosis and the search for plant extracts and compounds with anti-Toxoplasma activity is of utmost importance for the discovery of new active drugs. The objective of this study was to investigate the action of a protein extract and a protease inhibitor enriched fraction from J. curcas seed cake on developing tachyzoites of T. gondii-infected Vero cells. The protein extract (JcCE) was obtained after solubilization of the J. curcas seed cake with 100 mM sodium borate buffer, pH 10, centrifugation and dialysis of the resulting supernatant with the extracting buffer. JcCE was used for the in vitro assays of anti-Toxoplasma activity at 0.01, 0.1, 0.5, 1.5, 3.0 and 5.0 mg/ml concentration for 24 h. The results showed that JcCE reduced the percentage of infection and the number of intracellular parasites, but had no effect on the morphology of Vero cells up to 3.0 mg/mL. The cysteine protease inhibitor enriched fraction, which was obtained after chromatography of JcCE on Sephadex G-75 and presented a unique protein band following SDS-PAGE, reduced both the number of T. gondii infected cells and intracellular parasites. These results suggest that both JcCE and the cysteine protease inhibitor enriched fraction interfere with the intracellular growth of T. gondii. PMID:25816973

  8. Slowed transcription and rapid messenger RNA turnover contribute to a decline in synthesis of ovine trophoblast protein-1 during in vitro culture

    Ovine trophoblast protein-1 (oTP-1) is produced in massive amounts by conceptuses during the Day-12-20-period of early pregnancy. The rate of production of oTP-1 declines during culture of conceptuses, however, suggesting a role for critical intrauterine factors for continued production. The present study was conducted to define the mechanism responsible for the in vitro decline in oTP-1 synthesis. Over 24 h culture, synthesis of oTP-1 was initially rapid but then declined such that there was little increase in either the protein or its antiviral activity after 12 h. By contrast, the release of total protein into the medium continued at an approximately linear rate for the entire 24-h period of culture. By using in situ hybridization to tissue sections and dot-blot analysis of tissue extracts with labeled cDNA probes, it was shown that the quantity of oTP-1 mRNA fell 3- to 5-fold during culture, whereas the amount of actin mRNA remained relatively constant. To determine whether this selective fall in levels of oTP-1 mRNA resulted from decreased transcription rate and/or high turnover rate of existing mRNA, conceptuses were either provided continuously with [3H]uridine for 24 h or labeled for 9 h and then exposed to medium enriched in unlabeled uridine and cytidine for a further 15 h. Specific incorporation of 3H into oTP-1 mRNA was assessed by hybridization to excess oTP-1 cDNA immobilized on nitrocellulose membranes. In the continuous labeling study, 3H in total RNA increased at an approximately linear rate for at least 18 h, whereas the content of 3H in oTP-1 mRNA peaked at 9 h and declined about 10-fold by 24 h

  9. Effects of Momordica charantia on osmotic fragility and label red blood cells and plasmatic protein with 99m-Tc in vitro

    Magnata, Simey S.L.P. [Pernambuco Univ., Recife, PE (Brazil). Dept. de Energia Nuclear]. E-mail: sfmagnata@terra.com.br; Correia, Marilia B.L.; Brandao, Jose Odinilson C.; Souza, Grace M.L.; Catanho, Maria Teresa J.A. [Pernambuco Univ., Recife, PE (Brazil). Dept. de Biofisica e Radiobiologia; Terra, Daniele A.; Amorim, Lucia F. [Rio Grande do Norte Univ., Natal, RN (Brazil). Dept. de Fisiologia

    2005-07-01

    The use of natural products in the treatment physiopathology awaken the interest in the inquiry of the action mechanisms. The Momordica charantia, Melao de Sao Caetano, is used in the Caribbean and Orient for the diseases as stomatitis, cancer and diabetes. This work aims to verify the effect of the Momordica charantia's aqueous extract leaves on osmotic fragility and on labeling red blood cells (RBC) and plasmatic proteins with {sup 99m}Tc in vitro. To evaluate the osmotic fragility, samples of heparinized blood (500 mL) was incubed for 1 hour with brut extract (500 mL) in different concentrations (0; 10; 50 and 100% v/v); after centrifugation, the RCB were submitted the incubation (1 hour) with a gradient of NaCl (0;0,1;0,25;0,4;0,7 and 0.9%), the OD of supernatant was determined. With regards to label red blood cells and plasmatic proteins with {sup 99m}Tc in vitro was carried out by incubating of anticoagulant whole blood (500 mL) for 1 hour with brut extract (500 mL) in different concentrations (0; 10; 50 and 100% v/v). A stannous chloride solution of 1,2 {mu}g/mL was added the incubation for 60 minutes. After this the {sup 99m}Tc (3,7 MBq) was added and the incubation was continued for another 10 minutes. Those were centrifuged, precipitated with trichloroacetic acid 5% and mensured in a counter. The results shows that with regard to osmotic fragility, only the extract in the concentration of 100% provoked hemolysis. The Momordica charantia's extract is an agent who modify the fixation of {sup 99m}Tc in red blood cells. The results show with regard to osmotic fragility, only the extract in the quantity 100% provoked hemolysis. It is concluded that the Momordica charantia's extract is an agent who unchains the cellular fragility and {sup 99m}Tc fixation, showing a reduction effect. (author)

  10. Effects of Momordica charantia on osmotic fragility and label red blood cells and plasmatic protein with 99m-Tc in vitro

    The use of natural products in the treatment physiopathology awaken the interest in the inquiry of the action mechanisms. The Momordica charantia, Melao de Sao Caetano, is used in the Caribbean and Orient for the diseases as stomatitis, cancer and diabetes. This work aims to verify the effect of the Momordica charantia's aqueous extract leaves on osmotic fragility and on labeling red blood cells (RBC) and plasmatic proteins with 99mTc in vitro. To evaluate the osmotic fragility, samples of heparinized blood (500 mL) was incubed for 1 hour with brut extract (500 mL) in different concentrations (0; 10; 50 and 100% v/v); after centrifugation, the RCB were submitted the incubation (1 hour) with a gradient of NaCl (0;0,1;0,25;0,4;0,7 and 0.9%), the OD of supernatant was determined. With regards to label red blood cells and plasmatic proteins with 99mTc in vitro was carried out by incubating of anticoagulant whole blood (500 mL) for 1 hour with brut extract (500 mL) in different concentrations (0; 10; 50 and 100% v/v). A stannous chloride solution of 1,2 μg/mL was added the incubation for 60 minutes. After this the 99mTc (3,7 MBq) was added and the incubation was continued for another 10 minutes. Those were centrifuged, precipitated with trichloroacetic acid 5% and mensured in a counter. The results shows that with regard to osmotic fragility, only the extract in the concentration of 100% provoked hemolysis. The Momordica charantia's extract is an agent who modify the fixation of 99mTc in red blood cells. The results show with regard to osmotic fragility, only the extract in the quantity 100% provoked hemolysis. It is concluded that the Momordica charantia's extract is an agent who unchains the cellular fragility and 99mTc fixation, showing a reduction effect. (author)

  11. Visuospatial properties of caudal area 7b in Macaca fascicularis

    Hui-Hui JIANG; Ying-Zhou HU; Jian-Hong WANG; Yuan-Ye MA; Xin-Tian HU

    2013-01-01

    To proceed from sensation to movement,integration and transformation of information from different senses and reference frames are required.Several brain areas are involved in this transformation process,but previous neuroanatomical and neurophysiological studies have implicated the caudal area 7b as one particular component of this transformation system.In this study,we present the first quantitative report on the spatial coding properties of caudal area 7b.The results showed that neurons in this area had intermediate component characteristics in the transformation system; the area contained bimodal neurons,and neurons in this area encode spatial information using a hybrid reference frame.These results provide evidence that caudal area 7b may belong to the reference frame transformation system,thus contributing to our general understanding of the transformation system.

  12. WNT7B in fibroblastic foci of idiopathic pulmonary fibrosis

    Meuten Travis

    2012-07-01

    Full Text Available Abstract Background Idiopathic pulmonary fibrosis (IPF is a devastating interstitial pneumonia causing a loss of respiratory surface area due to a proliferative fibrotic response involving hyperplastic, hypertrophic, and metaplastic epithelium, cystic honeycomb change, septal expansion, and variable inflammation. Wnt (wingless signaling glycoproteins are known to be involved in lung development and tissue repair, and are up-regulated in patients with IPF. Based on previous qRT-PCR data showing increased Wnt7B in lungs of IPF patients, a systematic, quantitative examination of its tissue site distribution was undertaken. Methods Tissue samples from the Lung Tissue Research Consortium (LTRC of 39 patients diagnosed with mild to severe IPF/usual interstitial pneumonia (UIP and 19 normal patients were examined for the immunolocalization of Wnt7B. Results In normal lung, moderate Wnt7B reactivity was confined to airway epithelium, smooth muscle of airways and vasculature, and macrophages. IPF lung showed strong Wnt7B reactivity in fibroblastic foci, dysplastic airway and alveolar epithelium, and in highly discrete subepithelial, basement membrane-associated regions. All reactive sites were sized and counted relative to specific microscopic regions. Those in the subepithelial sites were found in significantly greater numbers and larger relative area compared with the others. No reactive sites were present in normal patient controls. Conclusions The results demonstrate Wnt7B to be expressed at high concentrations in regions of active hyperplasia, metaplasia, and fibrotic change in IPF patients. In this context and its previously established biologic activities, Wnt7B would be expected to be of potential importance in the pathogenesis of IPF.

  13. Auxin-induced regulation of protein synthesis in tobacco mesophyll protoplasts cultivated in vitro: I. Characteristics of auxin-sensitive proteins.

    Meyer, Y; Aspart, L; Chartier, Y

    1984-08-01

    The presence of auxin (2,4-D), in the culture medium of tobacco (Nicotiana tabacum var Maryland) mesophyll protoplasts is necessary both for cell wall regeneration and for passage of the cells from phase G(0) to phase G(1) of the cell cycle. Among about 250 proteins synthesized by protoplasts and characterized by their migration in a two-dimensional electrophoresis gel, 2,4-dichlorophenoxyacetic acid affects the synthesis of 11.Nine proteins are synthesized at a reduced level in the presence of the hormone, of which three are rapidly labeled and short-lived, while the others, which are long-lived, become detectable only after 2 hours of radioactive labeling, suggesting that they undergo slow posttranslational maturation. These nine proteins are proline-rich but the proline radicals are not strongly hydroxylated. The synthesis of these proteins is no longer inhibited by auxin if dichlorobenzonitril, a weed-killer which inhibits cell wall reformation of tobacco protoplasts, is added to the culture medium.Two proteins are only synthesized if protoplasts are cultivated in an auxin-containing medium. These polypeptides are rapidly labeled, and are long-lived. The inhibition of cell wall reformation by dichlorobenzonitril does not modify their synthesis.These results suggest that proteins whose synthesis is reduced by auxin are related to cell wall reformation and that they do not play a role in the induction of the cell cycle. In contrast, proteins whose synthesis is stimulated in the presence of auxin are good candidates for a role in the induction of the cell cycle. PMID:16663728

  14. Protein kinase C-mediated changes in synaptic efficacy at the neuromuscular junction in vitro: the role of postsynaptic acetylcholine receptors.

    Lanuza, M A; Li, M X; Jia, M; Kim, S; Davenport, R; Dunlap, V; Nelson, P G

    2000-09-15

    Activation of a mouse in vitro neuromuscular synapse produces a reduction in synaptic efficacy which is greater for nonactivated than for activated inputs to the myotubes. This has been shown to require thrombin and thrombin receptor activation and to involve a protein kinase C (PKC)-mediated step. We show in the present work that phorbol ester activation of PKC produces physiological loss of synapses in a time- and dose-related manner. We observe, using quantitative imaging methods, a parallel loss of acetylcholine receptors (AChR) from synaptically functional neurite-associated receptor aggregates in nerve-muscle cocultures. Biochemical measurements of total AChR show that PKC activation reduces both AChR stability (increases receptor loss) and receptor insertion into the surface membrane. Taken together, the data suggest that PKC activation decreases the stability of AChR aggregates in the muscle surface membrane. We conclude that PKC plays a crucial role in activity-dependent synapse reduction and does so, at least in part, by altering AChR stability. PMID:10972958

  15. Kidney specific protein-positive cells derived from embryonic stem cells reproduce tubular structures in vitro and differentiate into renal tubular cells.

    Ryuji Morizane

    Full Text Available Embryonic stem cells and induced pluripotent stem cells have the ability to differentiate into various organs and tissues, and are regarded as new tools for the elucidation of disease mechanisms as well as sources for regenerative therapies. However, a method of inducing organ-specific cells from pluripotent stem cells is urgently needed. Although many scientists have been developing methods to induce various organ-specific cells from pluripotent stem cells, renal lineage cells have yet to be induced in vitro because of the complexity of kidney structures and the diversity of kidney-component cells. Here, we describe a method of inducing renal tubular cells from mouse embryonic stem cells via the cell purification of kidney specific protein (KSP-positive cells using an anti-KSP antibody. The global gene expression profiles of KSP-positive cells derived from ES cells exhibited characteristics similar to those of cells in the developing kidney, and KSP-positive cells had the capacity to form tubular structures resembling renal tubular cells when grown in a 3D culture in Matrigel. Moreover, our results indicated that KSP-positive cells acquired the characteristics of each segment of renal tubular cells through tubular formation when stimulated with Wnt4. This method is an important step toward kidney disease research using pluripotent stem cells, and the development of kidney regeneration therapies.

  16. Extracellular-signal regulated kinase (Erk1/2), mitogen-activated protein kinase-activated protein kinase 2 (MK2) and tristetraprolin (TTP) comprehensively regulate injury-induced immediate early gene (IEG) response in in vitro liver organ culture.

    Tran, Doan Duy Hai; Koch, Alexandra; Saran, Shashank; Armbrecht, Marcel; Ewald, Florian; Koch, Martina; Wahlicht, Tom; Wirth, Dagmar; Braun, Armin; Nashan, Björn; Gaestel, Matthias; Tamura, Teruko

    2016-05-01

    Differentiated hepatocytes are long-lived and normally do not undergo cell division, however they have the unique capacity to autonomously decide their replication fate after liver injury. In this context, the key players of liver regeneration immediately after injury have not been adequately studied. Using an in vitro liver culture system, we show that after liver injury, p38 mitogen-activated protein kinase (p38MAPK), mitogen-activated protein kinase-activated protein kinase 2 (MK2) and extracellular-signal regulated kinase (Erk)1/2 were activated within 15min and continued to be phosphorylated for more than 2h. Both p38MAPK and Erk1/2 were activated at the edge of the cut as well as on the liver surface where the mesothelial cell sheet expresses several cytokines. Notably, in human liver Erk1/2 was also activated under the mesothelial cell sheet shortly after liver resections. Furthermore, in in vitro liver slice culture immediate early genes (IEGs) were upregulated within 1-2h and the S phase marker proliferation-cell-nuclear-antigen (PCNA) appeared 24h after injury. Although Erk1/2 was activated after injury, in MK2 depleted liver a set of IEGs, such as Dusp1, Cox2, or c-Myc and proliferation marker gene Ki67 were not induced. In addition, in immortalized hepatocyte cells, THLE-2, the same subset of genes was upregulated upon stimulation with lipopolysaccharide (LPS), but not in the presence of MK2 inhibitor. The protein level of tristetraprolin (TTP), a substrate for MK2 that plays a role in mRNA degradation, was increased in the presence of MK2 inhibitor. In this context, the depletion of TTP gene rescued Dusp1, Cox2, or c-Myc upregulation in the presence of MK2 inhibitor. These data imply that MK2 pathway is positively involved in Erk1/2 induced IEG response after liver injury. These data also suggest that in vitro liver culture may be a useful tool for measuring the proliferation potential of hepatocytes in individual liver. PMID:26876787

  17. Yeast Ivy1p Is a Putative I-BAR-domain Protein with pH-sensitive Filament Forming Ability in vitro.

    Itoh, Yuzuru; Kida, Kazuki; Hanawa-Suetsugu, Kyoko; Suetsugu, Shiro

    2016-01-01

    Bin-Amphiphysin-Rvs161/167 (BAR) domains mold lipid bilayer membranes into tubules, by forming a spiral polymer on the membrane. Most BAR domains are thought to be involved in forming membrane invaginations through their concave membrane binding surfaces, whereas some members have convex membrane binding surfaces, and thereby mold membranes into protrusions. The BAR domains with a convex surface form a subtype called the inverse BAR (I-BAR) domain or IRSp53-MIM-homology domain (IMD). Although the mammalian I-BAR domains have been studied, those from other organisms remain elusive. Here, we found putative I-BAR domains in Fungi and animal-like unicellular organisms. The fungal protein containing the putative I-BAR-domain is known as Ivy1p in yeast, and is reportedly localized in the vacuole. The phylogenetic analysis of the I-BAR domains revealed that the fungal I-BAR-domain containing proteins comprise a distinct group from those containing IRSp53 or MIM. Importantly, Ivy1p formed a polymer with a diameter of approximately 20 nm in vitro, without a lipid membrane. The filaments were formed at neutral pH, but disassembled when pH was reverted to basic. Moreover, Ivy1p and the I-BAR domain expressed in mammalian HeLa cells was localized at a vacuole-like structure as filaments as revealed by super-resolved microscopy. These data indicate the pH-sensitive polymer forming ability and the functional conservation of Ivy1p in eukaryotic cells. PMID:26657738

  18. Comparison of copper and zinc in vitro bioaccessibility from cyanobacteria rich in proteins and a synthetic supplement containing gluconate complexes: LC-MS mapping of bioaccessible copper complexes.

    Wojcieszek, Justyna; Witkoś, Katarzyna; Ruzik, Lena; Pawlak, Katarzyna

    2016-01-01

    An analytical procedure was proposed to estimate bioaccessibility of copper and zinc in Spirulina Pacifica tablets with respect to that of copper and zinc in gluconate complexes. Spirulina is the common name for diet supplements produced primarily from two species of cyanobacteria, namely Arthrospira platensis and Arthrospira maxima. Spirulina tablets are an excellent source of proteins, vitamins and minerals. To obtain information about the bioavailability of these elements, an in vitro bioaccessibility test was performed by application of a two-step protocol which simulated the gastric (pepsin) and intestinal (pancreatin) digestion. The species obtained were investigated by size exclusion chromatography on a chromatograph coupled to a mass spectrometer with inductively coupled plasma (SEC-ICP-MS) and an on-capillary liquid chromatograph coupled to an electrospray mass spectrometer (μ-HPLC-ESI-MS). Both copper and zinc were found to be highly bioaccessible in Spirulina tablets (90-111%) and those containing gluconate complexes (103% for Cu and 62% for Zn). In Spirulina tablets, copper was found to form two types of complex: (1) polar ones with glycine and aspartic acid and (2) more hydrophobic ones containing amino acids with cyclic hydrocarbons (phenylalanine, histidine, proline and tyrosine). Zinc and copper were also proved to form complexes during the digestion process with products of pepsin digestion, but the stability of these complexes is lower than that of the complexes formed in Spirulina. The results proving the involvement of proteins in the enhancement of copper and zinc bioaccessibility will be useful for the design of new copper and zinc supplements. PMID:26597916

  19. Osteomimicry of mammary adenocarcinoma cells in vitro; increased expression of bone matrix proteins and proliferation within a 3D collagen environment.

    Rachel F Cox

    Full Text Available Bone is the most common site of metastasis for breast cancer, however the reasons for this remain unclear. We hypothesise that under certain conditions mammary cells possess osteomimetic capabilities that may allow them to adapt to, and flourish within, the bone microenvironment. Mammary cells are known to calcify within breast tissue and we have recently reported a novel in vitro model of mammary mineralization using murine mammary adenocarcinoma 4T1 cells. In this study, the osteomimetic properties of the mammary adenocarcinoma cell line and the conditions required to induce mineralization were characterized extensively. It was found that exogenous organic phosphate and inorganic phosphate induce mineralization in a dose dependent manner in 4T1 cells. Ascorbic acid and dexamethasone alone have no effect. 4T1 cells also show enhanced mineralization in response to bone morphogenetic protein 2 in the presence of phosphate supplemented media. The expression of several bone matrix proteins were monitored throughout the process of mineralization and increased expression of collagen type 1 and bone sialoprotein were detected, as determined by real-time RT-PCR. In addition, we have shown for the first time that 3D collagen glycosaminoglycan scaffolds, bioengineered to represent the bone microenvironment, are capable of supporting the growth and mineralization of 4T1 adenocarcinoma cells. These 3D scaffolds represent a novel model system for the study of mammary mineralization and bone metastasis. This work demonstrates that mammary cells are capable of osteomimicry, which may ultimately contribute to their ability to preferentially metastasize to, survive within and colonize the bone microenvironment.

  20. Calcium and protein kinase C play an important role in Campylobacter jejuni-induced changes in Na+ and Cl- transport in rat ileum in vitro.

    Kanwar, R K; Ganguly, N K; Kumar, L; Rakesh, J; Panigrahi, D; Walia, B N

    1995-04-24

    The pathophysiological mechanism of Campylobacter jejuni (enterotoxigenic) induced secretory diarrhoea remains least understood. To investigate the mechanism(s) involved, the unidirectional fluxes of Na+ and Cl- were measured across the C. jejuni live culture infected and control (non infected) rat ileum (unstriped), in vitro by Ussing technique under short circuit conditions, in the presence or absence of: Ca2+ ionophore A23187 (5 microM), 1-verapamil (100 microM), calmodulin (CaM) antagonist W-7 (100 microM), dantrolene (25 microM), protein kinase C (PKC) activator PMA (100 ng/ml) and H-7 (60 microM), selective inhibitor of PKC. There was net absorption of Na+ and enhanced Cl- secretion in infected animals while in control animals there was net absorption of Na+ and marginal secretion Cl-.Ca2+ ionophore A23187 mimicked the effects of C. jejuni infection whereas 1-verapamil had significant antisecretory effect on Na+ and Cl- secretion in infected animals. In vitro measurement of undirectional 45Ca fluxes in Ussing chamber experiments revealed net absorption of Ca2+ in infected rat ileum as compared to net secretion of Ca2+ in control rat ileum. These observations clearly indicate that there is increased stimulation of Ca2+ uptake from extracellular milieu to the enterocytes during C. jejuni-induced diarrhoea. The intracellular calcium levels (Ca2+]i (as measured by fluorescent probe Fura-2AM) were found to be raised significantly (P < 0.0001) in enterocytes isolated from C. jejuni infected ileum as compared to the enterocytes from control ileum. The observed increase in [Ca2+]i in enterocytes isolated from C. jejuni live culture supernatant treated rat ileum further shows the involvement of enterotoxin in diarrhoeal process. Dantrolene decreased significantly C. jejuni-induced net Na+ and Cl- secretion but it could not reverse it to absorption suggesting the partial involvement of Ca2+ mobilised from intracellular stores in mediating secretion. W-7 failed to

  1. Short-hairpin RNA-mediated Heat shock protein 90 gene silencing inhibits human breast cancer cell growth in vitro and in vivo

    Highlights: ► Hsp90 is over-expressed in human breast cancer. ► The shRNA-mediated gene silencing of Hsp90 resulted in inhibition of cell growth. ► Akt and NF-kB were down-regulation after transfection due to Hsp90 silencing. ► The tumor growth ratio was decline due to Hsp90 silencing. ► The PCNA expression was down-regulation due to Hsp90 silencing. -- Abstract: Hsp90 interacts with proteins that mediate signaling pathways involved in the regulation of essential processes such as proliferation, cell cycle control, angiogenesis and apoptosis. Hsp90 inhibition is therefore an attractive strategy for blocking abnormal pathways that are crucial for cancer cell growth. In the present study, the role of Hsp90 in human breast cancer MCF-7 cells was examined by stably silencing Hsp90 gene expression with an Hsp90-silencing vector (Hsp90-shRNA). RT-PCR and Western blot analyses showed that Hsp90-shRNA specifically and markedly down-regulated Hsp90 mRNA and protein expression. NF-kB and Akt protein levels were down-regulated in Hsp90-shRNA transfected cells, indicating that Hsp90 knockout caused a reduction of survival factors and induced apoptosis. Treatment with Hsp90-shRNA significantly increased apoptotic cell death and caused cell cycle arrest in the G1/S phase in MCF-7 cells, as shown by flow cytometry. Silencing of Hsp90 also reduced cell viability, as determined by MTT assay. In vivo experiments showed that MCF-7 cells stably transfected with Hsp90-shRNA grew slowly in nude mice as compared with control groups. In summary, the Hsp90-shRNA specifically silenced the Hsp90 gene, and inhibited MCF-7 cell growth in vitro and in vivo. Possible molecular mechanisms underlying the effects of Hsp90-shRNA include the degradation of Hsp90 breast cancer-related client proteins, the inhibition of survival signals and the upregulation of apoptotic pathways. shRNA-mediated interference may have potential therapeutic utility in human breast cancer.

  2. In vitro fertilization (IVF)

    ... page: //medlineplus.gov/ency/article/007279.htm In vitro fertilization (IVF) To use the sharing features on this page, please enable JavaScript. In vitro fertilization (IVF) is the joining of a woman's ...

  3. Dynamics of DNA in vitro evolution

    Xiaojing Yang; Xili Liu; Chunbo Lou; Qi Ouyang

    2009-01-01

    In vitro evolution has become a very important research area in recent years. From a practical point of view, it provides a powerful and reliable tool for engineering functional molecules (DNA, RNA or proteins) in reasonably short periods of time. From a theoretical point of view, since in vitro evolution is analogous to natural evolution in many respects, the study of the dynamic details of in vitro evolution may provide some instructive insights into the process of evolution. In this review, we summarize current theoretical and exper-imental studies, including several efforts made by our group, on the dynamics of DNA in vitro evolution.

  4. Effect of growth factors (BMP-4/7 & bFGF on proliferation & osteogenic differentiation of bone marrow stromal cells

    Shaohui Yuan

    2013-01-01

    Full Text Available Background & objectives: BMP (bone morphogenetic protein-4/7 and bFGF (basic fibroblast growth factor significantly promote the osteogenic activity and the proliferation of rabbit BMSCs (bone marrow stromal cells, respectively. However, their synergistic effects on the proliferation and the differentiation of BMSCs remain unclear. In the present study, the effects of bFGF and BMP-4/7 were investigated on the proliferation and the differentiation of rat BMSCs in vitro. Methods: BMSCs were isolated from New Zealand white rabbits and cultured to the third passage. The samples were divided into five groups according to the material implanted: (A 80 ng/ml BMP-4/7; (B 80 ng/ml bFGF; (C 30 ng/ml BMP-4/7 and 30 ng/ml bFGF; (D 50 ng/ml BMP-4/7 and 50 ng/ml bFGF; and (E 80 ng/ml BMP-4/7 and 80 ng/ml bFGF. Cell proliferation was analyzed using methyl thiazolyl tetrazolium (MTT assay. Alkaline phosphatase activity and osteocalcin (OC dynamics were also measured. Results: BMP-4/7 alone significantly (P<0.05 promoted the proliferation of BMSCs. At the same time, it also promoted or inhibited the osteogenic differentiation of BMSCs. The synergistic effects of BMP-4/7 and bFGF significantly promoted both the proliferation and the osteogenic differentiation of BMSCs. The treatment of the synergistic effects was dose and time dependent. Interpretation & conclusions: A rational combination of BMP-4/7 and bFGF can promote the proliferation and the osteogenic differentiation of BMSCs. In addition, the synergistic functions are effective.

  5. In Vitro and In Vivo Relevance of Infectious Laryngotracheitis Virus gJ Proteins That Are Expressed from Spliced and Nonspliced mRNAs

    Fuchs, Walter; Wiesner, Dorothee; Veits, Jutta; Teifke, Jens P.; Mettenleiter, Thomas C.

    2005-01-01

    The positional homologue in the infectious laryngotracheitis virus (ILTV) genome of the glycoprotein gJ gene of herpes simplex virus and the gp2 gene of equine herpesvirus 1 is expressed into four proteins of 85, 115, 160, and 200 kDa (J. Veits, B. Köllner, J. P. Teifke, H. Granzow, T. C. Mettenleiter, and W. Fuchs, Avian Dis. 47:330-342, 2003). RNA analyses revealed that these proteins are expressed from two different late (γ2) transcripts, an unspliced 5.5-kb and a spliced 4.3-kb mRNA that are translated into proteins of 985 and 611 amino acids, respectively. ILTV gJ is incorporated into virions and is modified by N- and O-linked glycosylation. After cotransfection of chicken cells with genomic DNA of a pathogenic ILTV strain and transfer plasmids, gJ-negative ILTV mutants could be isolated. In vitro growth studies demonstrated that deletion of the gJ gene has only minor effects on direct cell-to-cell spread as measured by plaque size. However, progeny virus titers of ILTV-ΔgJ were significantly reduced in comparison to those of the parental virus and a gJ rescue mutant. After experimental infection of chickens the gJ rescue mutant, like wild-type ILTV, caused severe disease and considerable mortality, whereas ILTV-ΔgJ was significantly attenuated. All immunized animals were protected against subsequent challenge infection with virulent ILTV. In sera collected after immunization with the gJ-rescue mutant or with wild-type ILTV, gJ-specific antibodies were detectable by immunofluorescence on cells that had been transfected with a gJ expression plasmid. As expected, no gJ-specific antibodies were found in sera obtained from chickens immunized with ILTV-ΔgJ. Thus, gJ deletion mutants of ILTV might be usable as attenuated live-virus vaccines. Furthermore, the gJ gene might constitute a reliable marker for serological discrimination between vaccinated and field virus-infected chickens. PMID:15613298

  6. Factors involved in the initiation of phage ø29 DNA replication in vitro: requirement of the gene 2 product for the formation of the protein p3-dAMP complex

    1983-01-01

    To study the requirements for the in vitro formation of the protein p3-dAMP complex, the first step in φ29 DNA replication, extracts from B. subtilis infected with ø29 mutants in genes 2, 3, 5, 6 and 17, involved in DNA synthesis, have been used. The formation of the initiation complex is completely dependent on the presence of a functional gene 2 product, in addition to protein p3 and ø29 DNA-protein p3 as template. ATP is also required, although it can be replaced by other nucleotides. The ...

  7. Human papillomavirus 16 E2 interacts with neuregulin receptor degradation protein 1 affecting ErbB-3 expression in vitro and in clinical samples of cervical lesions.

    Paolini, Francesca; Curzio, Gianfranca; Melucci, Elisa; Terrenato, Irene; Antoniani, Barbara; Carosi, Mariantonia; Mottolese, Marcella; Vici, Patrizia; Mariani, Luciano; Venuti, Aldo

    2016-05-01

    The ErbB tyrosine kinase receptors play a key role in regulating many cellular functions and human papillomaviruses (HPVs) may interact with transductional pathway of different growth factor receptors. Here, these interactions were analysed in W12 cell line carrying HPV 16 genome and in clinical samples. W12 cells, in which HPV16 becomes integrated during passages, were utilised to detect viral and ErbB family expression at early (W12E) and late passages (W12G). Interestingly, a strong reduction of ErbB-3 expression was observed in W12G. Loss of the E2 and E5 viral genes occurs in W12G and this may affect ErbB-3 receptor expression. E2 and E5 rescue experiments demonstrated that only E2 gene was able to restore ErbB-3 expression. E2 is a transcriptional factor but the expression levels of ErbB3 were unaffected and ErbB-3 promoter did not show any consensus sequence for E2, thus E2 may interact in another way with ErbB3. Indeed, HPV 16 E2 can modulate ErbB-3 by interacting with the ubiquitin ligase neuregulin receptor degradation protein 1 (Nrdp-1) that is involved in the regulation of this receptor, via ubiquitination and degradation. E2 co-immunoprecipitated in a complex with Nrdp-1 leading to hypothesise an involvement of this interaction in ErbB-3 regulation. In addition, 90% of the clinical samples with integrated virus and E2 loss showed no or low ErbB-3 positivity, confirming in vitro results. In conclusion, the new discovered interaction of HPV-16 E2 with Nrdp-1 may affect ErbB-3 expression. PMID:26963794

  8. Comparative study of osteogenic potential of a composite scaffold incorporating either endogenous bone morphogenetic protein-2 or exogenous phytomolecule icaritin: an in vitro efficacy study.

    Chen, S-H; Wang, X-L; Xie, X-H; Zheng, L-Z; Yao, D; Wang, D-P; Leng, Y; Zhang, G; Qin, L

    2012-08-01

    A local delivery system with sustained and efficient release of therapeutic agents from an appropriate carrier is desirable for orthopedic applications. Novel composite scaffolds made of poly (lactic-co-glycolic acid) with tricalcium phosphate (PLGA/TCP) were fabricated by an advanced low-temperature rapid prototyping technique, which incorporated either endogenous bone morphogenetic protein-2 (BMP-2) (PLGA/TCP/BMP-2) or phytomolecule icaritin (ICT) (PLGA/TCP/ICT) at low, middle and high doses. PLGA/TCP served as control. In vitro degradation, osteogenesis and release tests showed statistical differences among PLGA/TCP/ICT, PLGA/TCP and PLGA/TCP/BMP-2 groups, where PLGA/TCP/ICT had the desired slow release of bioactive icaritin in a dose-dependent manner, whereas there was almost no BMP-2 release from the PLGA/TCP/BMP-2 scaffolds. PLGA/TCP/ICT significantly increased more ALP activity, upregulated mRNA expression of osteogenic genes and enhanced calcium deposition and mineralization in rabbit bone marrow stem cells cultured on scaffolds compared with the other two groups. These results indicate the desired degradation rate, osteogenic capability and release property in PLGA/TCP/ICT composite scaffold, as icaritin preserved its bioactivity and structure after incorporation, while PLGA/TCP/BMP-2 did not show an initially expected osteogenic potential, owing to loss of the original bioactivity of BMP-2 during its incorporation and fabrication procedure. The results suggest that PLGA/TCP composite scaffolds incorporating osteogenic ICT might be a promising approach for bone tissue bioengineering and regeneration. PMID:22543006

  9. THE MASS OF CoRoT-7b

    Hatzes, Artie P.; Wuchterl, Guenther [Thueringer Landessternwarte, D-07778 Tautenburg (Germany); Fridlund, Malcolm; Gandolfi, Davide [European Space Agency, ESTEC, SRE-SA, P.O. Box 299, NL-2200AG, Noordwijk (Netherlands); Nachmani, Gil; Mazeh, Tsevi [School of Physics and Astronomy, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv (Israel); Valencia, Diana [Observatoire de la Cote d' Azur, BP 4229, F-06304 Nice Cedex 4 (France); Hebrard, Guillaume; Borde, Pascal [Institut d' Astrophysique de Paris, UMR 7095 CNRS, Universite Pierre and Marie Curie, 98bis boulevard Arago, F-75014 Paris (France); Carone, Ludmila; Paetzold, Martin [Rheinisches Institut fuer Umweltforschung, Universitaet zu Koeln, Abt. Planetenforschung, Aachener Str. 209, D-50931 Koeln (Germany); Udry, Stephane [Observatoire de l' Universite de Geneve, 51 chemin des Maillettes, 1290 Sauverny (Switzerland); Bouchy, Francois [Observatoire de Haute Provence, F-04670 Saint Michel l' Observatoire (France); Deleuil, Magali; Moutou, Claire; Barge, Pierre [Laboratoire d' Astrophysique de Marseille, CNRS and University of Provence, 38 rue Frederic Joliot-Curie, F-13388 Marseille Cedex 13 (France); Deeg, Hans; Tingley, Brandon [Instituto de Astrofisica de Canarias, E-38205 La Laguna, Tenerife (Spain); Dvorak, Rudolf [University of Vienna, Institute of Astronomy, Tuerkenschanzstr. 17, A-1180, Vienna (Austria); Ferraz-Mello, Sylvio, E-mail: artie@tls-tautenburg.de, E-mail: malcolm.fridlund@esa.int [IAG, University of Sao Paulo (Brazil); and others

    2011-12-10

    The mass of CoRoT-7b, the first transiting super-Earth exoplanet, is still a subject of debate. A wide range of masses have been reported in the literature ranging from as high as 8 M{sub Circled-Plus} to as low as 2.3 M{sub Circled-Plus }. This range in mass is largely due to the activity level of the star that contributes a significant amount of radial velocity (RV) 'jitter' and how the various methods correct this jitter. Although most mass determinations give a density consistent with a rocky planet, the lower value permits a bulk composition that can be up to 50% water. We present an analysis of the CoRoT-7b RV measurements that uses very few and simple assumptions in treating the activity signal. By analyzing those RV data for which multiple measurements were made in a given night, we remove the activity related RV contribution without any a priori model. We argue that the contribution of activity to the final RV curve is negligible and that the K-amplitude due to the planet is well constrained. This yields a mass of 7.42 {+-} 1.21 M{sub Circled-Plus} and a mean density of {rho} = 10.4 {+-} 1.8 gm cm{sup -3}. CoRoT-7b is similar in mass and radius to the second rocky planet to be discovered, Kepler-10b, and within the errors they have identical bulk densities-they are virtual twins. These bulk densities lie close to the density-radius relationship for terrestrial planets similar to what is seen for Mercury. CoRoT-7b and Kepler-10b may have an internal structure more like Mercury than the Earth.

  10. On the Mass of CoRoT-7b

    Hatzes, Artie P; Nachmani, Gil; Mazeh, Tsevi; Valencia, Diana; Hebrard, Guillaume; Carone, Ludmila; Paetzold, Martin; Udry, Stephane; Bouchy, Francois; Borde, Pascal; Deeg, Hans; Tingley, Brandon; Dvorak, Rudolf; Gandolfi, Davide; Ferraz-Mello, Sylvio; Wuchterl, Guenther; Guenther, Eike; Rauer, Heike; Erikson, Anders; Cabrera, Juan; Csizmadia, Szilard; Leger, Alain; Lammer, Helmut; Weingrill, Joerg; Queloz, Didier; Alonso, Roi; Schneider, Jean

    2011-01-01

    The mass of CoRoT-7b, the first transiting superearth exoplanet, is still a subject of debate. A wide range of masses have been reported in the literature ranging from as high as 8 M_Earth to as low as 2.3 M_Earth. Although most mass determinations give a density consistent with a rocky planet, the lower value permits a bulk composition that can be up to 50% water. We present an analysis of the CoRoT-7b radial velocity measurements that uses very few and simple assumptions in treating the activity signal. By only analyzing those radial velocity data for which multiple measurements were made in a given night we remove the activity related radial velocity contribution without any a priori model. We demonstrate that the contribution of activity to the final radial velocity curve is negligible and that the K-amplitude due to the planet is well constrained. This yields a mass of 7.42 +/- 1.21 M_Earth and a mean density of rho = 10.4 +/- 1.8 gm cm^-3. CoRoT-7b is similar in mass and radius to the second rocky plane...

  11. Tank 40 Final SB7b Chemical Characterization Results

    A sample of Sludge Batch 7b (SB7b) was taken from Tank 40 in order to obtain radionuclide inventory analyses necessary for compliance with the Waste Acceptance Product Specifications (WAPS). The SB7b WAPS sample was also analyzed for chemical composition including noble metals and fissile constituents. At the Savannah River National Laboratory (SRNL) the 3-L Tank 40 SB7b sample was transferred from the shipping container into a 4-L high density polyethylene bottle and solids were allowed to settle over the weekend. Supernate was then siphoned off and circulated through the shipping container to complete the transfer of the sample. Following thorough mixing of the 3-L sample, a 558 g sub-sample was removed. This sub-sample was then utilized for all subsequent analytical samples. Eight separate aliquots of the slurry were digested, four with HNO3/HCl (aqua regia) in sealed Teflon(R) vessels and four with NaOH/Na2O2 (alkali or peroxide fusion) using Zr crucibles. Two Analytical Reference Glass -- 1 (ARG-1) standards were digested along with a blank for each preparation. Each aqua regia digestion and blank was diluted to 1:100 mL with deionized water and submitted to Analytical Development (AD) for inductively coupled plasma -- atomic emission spectroscopy (ICP-AES) analysis, inductively coupled plasma -- mass spectrometry (ICP-MS) analysis, atomic absorption spectroscopy (AA) for As and Se, and cold vapor atomic absorption spectroscopy (CV-AA) for Hg. Equivalent dilutions of the alkali fusion digestions and blank were submitted to AD for ICP-AES analysis. Tank 40 SB7b supernate was collected from a mixed slurry sample in the SRNL Shielded Cells and submitted to AD for ICP-AES, ion chromatography (IC), total base/free OH-/other base, total inorganic carbon/total organic carbon (TIC/TOC) analyses, and Cs-137 gamma scan. Weighted dilutions of slurry were submitted for IC, TIC/TOC, and total base/free OH-/other base analyses. Activities for U-233, U-235, and Pu-239 were

  12. Papain Degrades Tight Junction Proteins of Human Keratinocytes In Vitro and Sensitizes C57BL/6 Mice via the Skin Independent of its Enzymatic Activity or TLR4 Activation

    Stremnitzer, Caroline; Manzano-Szalai, Krisztina; Willensdorfer, Anna; Starkl, Philipp(*); Pieper, Mario; König, Peter; Mildner, Michael; Tschachler, Erwin; Reichart, Ursula; Jensen-Jarolim, Erika

    2015-01-01

    Papain is commonly used in food, pharmaceutical, textile, and cosmetic industries and is known to induce occupational allergic asthma. We have previously shown that the papain-like cysteine protease Dermatophagoides pteronyssinus 1 from house dust mite exhibits percutaneous sensitization potential. We aimed here to investigate the potential of papain itself in epicutaneous sensitization. The effects of papain on tight junction (TJ) proteins were tested in vitro in human primary keratinocytes....

  13. NasR, a novel RNA-binding protein, mediates nitrate-responsive transcription antitermination of the Klebsiella oxytoca M5al nasF operon leader in vitro.

    Chai, W; Stewart, V

    1998-10-23

    In Klebsiella oxytoca (pneumoniae), enzymes required for nitrate assimilation are encoded by the nasFEDCBA operon. Previous genetic studies led to the conclusion that nitrate and nitrite induction of nasF operon expression is determined by a transcriptional antitermination mechanism. In the presence of nitrate or nitrite, the nasR gene product is hypothesized to inhibit transcription termination at the factor-independent terminator site located in the nasF operon leader region. To test this model in vitro, we first purified NasR as both a maltose binding protein fusion form (MBP-NasR) and a His6-tagged form (His6-NasR). Templates for in vitro transcription contained the nasF operon leader region, with a substitution of the sigma70-dependent tac promoter for the native sigmaN-dependent promoter. We found that in vitro transcription of the leader template terminated at the terminator site, and that MBP-NasR and His6-NasR proteins both caused transcription readthrough of this site in response to nitrate or nitrite. Half-maximal antitermination required nitrate or nitrite at moderate (1 to 10 microM) concentrations, and several other anions tested, including chlorate, were without effect. Previous in vivo analysis of leader deletions identified regions required for both negative regulation (the terminator) and for positive regulation. Results from in vitro transcription of these deletion templates correlated fully with the in vivo analysis. Finally, electrophoresis mobility shift analysis revealed that His6-NasR bound specifically to nasF leader RNA. This binding was independent of nitrate in vitro. These results strongly support the conclusions drawn from previous in vivo analysis, and establish that NasR mediates ligand-responsive transcription antitermination through interaction with nasF leader RNA. PMID:9769209

  14. Sulfate Solubility Limit Verification For DWPF Sludge Batch 7B

    The objective of this study was to determine a sulfate solubility limit in glass for Sludge Batch 7b (SB7b). The SB7b composition projection provided by Savannah River Remediation (SRR) on May 25, 2011 was used as the basis for formulating glass compositions to determine the sulfate limit. Additions of Na2O to the projected sludge composition were made by the Savannah River National Laboratory (SRNL) due to uncertainty in the final concentration of Na2O for SB7b, which is dependent on washing effectiveness and the potential need to add NaOH to ensure an acceptable projected operating window. Additions of 4, 6, and 8 wt % Na2O were made to the nominal May 25, 2011 composition projection. An updated SB7b composition projection was received from SRR on August 4, 2011. Due to compositional similarities, no additional experimental work using the August 4, 2011 compositions was considered to be necessary for this study. Both Frit 418 and Frit 702 were included in this study. The targeted sulfate (SO42-) concentrations of the study glasses were selected within the range of 0.6 to 0.9 wt % in glass. A total of 52 glass compositions were selected based on the compositional variables of Na2O addition, Actinide Removal Process (ARP) stream addition, waste loading, frit composition, and sulfate concentration. The glasses were batched, melted, and characterized following SRNL procedures. Visual observations were recorded for each glass after it cooled and used as in indicator of sulfur retention. Representative samples of each of the glasses fabricated were subjected to chemical analysis to determine whether the targeted compositions were met, as well as to determine the quantity of sulfate that was retained after melting. In general, the measured composition data showed that there were only minor issues in meeting the targeted compositions for the study glasses, and the measured sulfate concentrations for each study glass were within 10% of the targeted values. The results for

  15. Tank 40 Final SB7b Chemical Characterization Results

    A sample of Sludge Batch 7b (SB7b) was taken from Tank 40 in order to obtain radionuclide inventory analyses necessary for compliance with the Waste Acceptance Product Specifications (WAPS). The SB7b WAPS sample was also analyzed for chemical composition including noble metals and fissile constituents, and these results are reported here. These analyses along with the WAPS radionuclide analyses will help define the composition of the sludge in Tank 40 that is currently being fed to the Defense Waste Processing Facility (DWPF) as SB7b. At the Savannah River National Laboratory (SRNL) the 3-L Tank 40 SB7b sample was transferred from the shipping container into a 4-L high density polyethylene bottle and solids were allowed to settle over the weekend. Supernate was then siphoned off and circulated through the shipping container to complete the transfer of the sample. Following thorough mixing of the 3-L sample, a 558 g sub-sample was removed. This sub-sample was then utilized for all subsequent analytical samples. Eight separate aliquots of the slurry were digested, four with HNO3/HCl (aqua regia) in sealed Teflon(regsign) vessels and four with NaOH/Na2O2 (alkali or peroxide fusion) using Zr crucibles. Two Analytical Reference Glass - 1 (ARG-1) standards were digested along with a blank for each preparation. Each aqua regia digestion and blank was diluted to 1:100 mL with deionized water and submitted to Analytical Development (AD) for inductively coupled plasma - atomic emission spectroscopy (ICP-AES) analysis, inductively coupled plasma - mass spectrometry (ICP-MS) analysis, atomic absorption spectroscopy (AA) for As and Se, and cold vapor atomic absorption spectroscopy (CV-AA) for Hg. Equivalent dilutions of the alkali fusion digestions and blank were submitted to AD for ICP-AES analysis. Tank 40 SB7b supernate was collected from a mixed slurry sample in the SRNL Shielded Cells and submitted to AD for ICP-AES, ion chromatography (IC), total base/free OH-/other base

  16. SULFATE SOLUBILITY LIMIT VERIFICATION FOR DWPF SLUDGE BATCH 7B

    Fox, K.

    2011-10-03

    The objective of this study was to determine a sulfate solubility limit in glass for Sludge Batch 7b (SB7b). The SB7b composition projection provided by Savannah River Remediation (SRR) on May 25, 2011 was used as the basis for formulating glass compositions to determine the sulfate limit. Additions of Na{sub 2}O to the projected sludge composition were made by the Savannah River National Laboratory (SRNL) due to uncertainty in the final concentration of Na{sub 2}O for SB7b, which is dependent on washing effectiveness and the potential need to add NaOH to ensure an acceptable projected operating window. Additions of 4, 6, and 8 wt % Na{sub 2}O were made to the nominal May 25, 2011 composition projection. An updated SB7b composition projection was received from SRR on August 4, 2011. Due to compositional similarities, no additional experimental work using the August 4, 2011 compositions was considered to be necessary for this study. Both Frit 418 and Frit 702 were included in this study. The targeted sulfate (SO{sub 4}{sup 2-}) concentrations of the study glasses were selected within the range of 0.6 to 0.9 wt % in glass. A total of 52 glass compositions were selected based on the compositional variables of Na{sub 2}O addition, Actinide Removal Process (ARP) stream addition, waste loading, frit composition, and sulfate concentration. The glasses were batched, melted, and characterized following SRNL procedures. Visual observations were recorded for each glass after it cooled and used as in indicator of sulfur retention. Representative samples of each of the glasses fabricated were subjected to chemical analysis to determine whether the targeted compositions were met, as well as to determine the quantity of sulfate that was retained after melting. In general, the measured composition data showed that there were only minor issues in meeting the targeted compositions for the study glasses, and the measured sulfate concentrations for each study glass were within 10% of

  17. Wnt7b is an important intrinsic regulator of hair follicle stem cell homeostasis and hair follicle cycling.

    Kandyba, Eve; Kobielak, Krzysztof

    2014-04-01

    The hair follicle (HF) is an exceptional mini-organ to study the mechanisms which regulate HF morphogenesis, cycling, hair follicle stem cell (hfSCs) homeostasis, and progeny differentiation. During morphogenesis, Wnt signaling is well-characterized in the initiation of HF patterning but less is known about which particular Wnt ligands are required and whether individual Wnt ligands act in an indispensable or redundant manner during postnatal hfSCs anagen onset and HF cycle progression. Previously, we described the function of the bone morphogenetic protein (BMP) signaling target gene WNT7a in intrinsic regulation of hfSCs homeostasis in vivo. Here, we investigated the role of Wnt7b, which was also intrinsically upregulated in hfSCs during physiological and precocious anagen after BMP inhibition in vivo. We demonstrated Wnt7b to be a direct target of canonical BMP signaling in hfSCs and using Wnt7b conditional gene targeting during HF morphogenesis revealed disrupted HF cycling including a shorter anagen, premature catagen onset with overall shorter hair production, and diminished HF differentiation marker expression. Additionally, we observed that postnatal ablation of Wnt7b resulted in delayed HF activation, affecting both the hair germ and bulge hfSCs but still maintaining a two-step sequence of HF stimulation. Interestingly, Wnt7b cKO hfSCs participated in reformation of the new HF bulge, but with slower self-renewal. These findings demonstrate the importance of intrinsic Wnt7b expression in hfSCs regulation and normal HF cycling and surprisingly reveal a nonredundant role for Wnt7b in the control of HF anagen length and catagen entry which was not compensated by other Wnt ligands. PMID:24222445

  18. Structural models of the human copper P-type ATPases ATP7A and ATP7B

    Gourdon, Pontus Emanuel; Sitsel, Oleg; Karlsen, Jesper Lykkegaard;

    2012-01-01

    The human copper exporters ATP7A and ATP7B contain domains common to all P-type ATPases as well as class-specific features such as six sequential heavy-metal binding domains (HMBD1-HMBD6) and a type-specific constellation of transmembrane helices. Despite the medical significance of ATP7A and ATP7B...... Legionella pneumophila. The models and sequence analyses show that the domains and residues involved in the catalytic phosphorylation events and copper transfer are highly conserved. In addition, there are only minor differences in the core structures of the two human proteins and the bacterial template...

  19. Ultrastructural changes and Heat Shock Proteins 70 induced by atmospheric pollution are similar to the effects observed under in vitro heavy metals stress in Conocephalum conicum (Marchantiales – Bryophyta)

    Changes in ultrastructure and induction of Heat Shock Proteins 70 have been studied in Conocephalum conicum (Marchantiales) collected in different urban and country sites in Italy. These results were compared to the effects in vitro of exposition to different heavy metals for several days. At urban sites, cellular ultrastructure was modified, and heavy metals could be observed accumulating in cell walls. Simultaneously, a strong increment in Hsp70 was detected, compared with results observed on control specimens. When C. conicum was exposed to heavy metals in vitro, comparable effects as in polluted sites were observed: Cd and Pb accumulated mostly within parenchyma and, within cells, were absorbed to cell walls or concentrated in vacuoles. Moreover, severe alterations were observed in organelles. Concomitantly, a progressive accumulation of Hsp70 was detected following heavy metals exposition. These effects are discussed in order to describe the dose and time-dependent response to heavy metal stress in C. conicum. -- Highlights: •Pollution induces ultrastructural changes and Hsp70 induction in C. conicum. •These changes can be mimicked by in vitro exposition to heavy metals. •The heavy metal induced changes, which are potential causes of toxitolerance. •C. conicum is a heavy metals tolerant liverwort that could be used as bioindicator. -- Pollution induces ultrastructural changes and Hsp70 induction in C. conicum, that can be mimicked in vitro. This liverworth could be used as bioindicator

  20. Expression of RIZ1 protein (Retinoblastoma-interacting zinc-finger protein 1) in prostate cancer epithelial cells changes with cancer grade progression and is modulated in vitro by DHT and E2.

    Rossi, Valentina; Staibano, Stefania; Abbondanza, Ciro; Pasquali, Daniela; De Rosa, Caterina; Mascolo, Massimo; Bellastella, Giuseppe; Visconti, Daniela; De Bellis, Annamaria; Moncharmont, Bruno; De Rosa, Gaetano; Puca, Giovanni Alfredo; Bellastella, Antonio; Sinisi, Antonio Agostino

    2009-12-01

    The nuclear protein methyl-transferase Retinoblastoma-interacting zinc-finger protein 1 (RIZ1) is considered to be a downstream effector of estrogen action in target tissues. Silencing of RIZ1 expression is common in many tumors. We analyzed RIZ1 expression in normal and malignant prostate tissue and evaluated whether estradiol (E2) or dihydrotestosterone (DHT) treatment modulated RIZ1 in cultured prostate epithelial cells (PEC). Moreover, we studied the possible involvement of RIZ1 in estrogen action on the EPN prostate cell line, constitutively expressing both estrogen receptor (ER)-alpha and beta. RIZ1 protein, found in the nucleus of normal PECs by immunohistochemistry, was progressively lost in cancer tissues as the Gleason score increased and was only detected in the cytoplasmic compartment. RIZ1 transcript levels, as assayed by semi-quantitative RT-PCR in primary PEC cultures, were significantly reduced in cancer cells (P < 0.05). In EPN DHT treatment significantly increased RIZ1 transcript and protein levels (P < 0.05); E2 induced a reduction of S phase without significant changes of RIZ1 expression. In E2-treated EPN cell extracts RIZ co-immunoprecipitated with ERbeta and ERalpha. Our data demonstrate that RIZ1 is expressed in normal PECs and down-regulated in cancer cells, with a switch of its sub-cellular localization from the nucleus to the cytoplasm upon cancer grade progression. RIZ1 expression levels in the PECs were modulated by DHT or E2 treatment in vitro. Furthermore, the E2 effects on ER-expressing prostate cells involve RIZ1, which confirms a possible role for ER-mediated pathways in a non-classic E(2)-target tissue. PMID:19746436

  1. Requirement of NifX and other nif proteins for in vitro biosynthesis of the iron-molybdenum cofactor of nitrogenase.

    Shah, V K; Rangaraj, P; Chatterjee, R; Allen, R M; Roll, J T; Roberts, G P; Ludden, P W

    1999-05-01

    The iron-molybdenum cofactor (FeMo-co) of nitrogenase contains molybdenum, iron, sulfur, and homocitrate in a ratio of 1:7:9:1. In vitro synthesis of FeMo-co has been established, and the reaction requires an ATP-regenerating system, dithionite, molybdate, homocitrate, and at least NifB-co (the metabolic product of NifB), NifNE, and dinitrogenase reductase (NifH). The typical in vitro FeMo-co synthesis reaction involves mixing extracts from two different mutant strains of Azotobacter vinelandii defective in the biosynthesis of cofactor or an extract of a mutant strain complemented with the purified missing component. Surprisingly, the in vitro synthesis of FeMo-co with only purified components failed to generate significant FeMo-co, suggesting the requirement for one or more other components. Complementation of these assays with extracts of various mutant strains demonstrated that NifX has a role in synthesis of FeMo-co. In vitro synthesis of FeMo-co with purified components is stimulated approximately threefold by purified NifX. Complementation of these assays with extracts of A. vinelandii DJ42. 48 (DeltanifENX DeltavnfE) results in a 12- to 15-fold stimulation of in vitro FeMo-co synthesis activity. These data also demonstrate that apart from the NifX some other component(s) is required for the cofactor synthesis. The in vitro synthesis of FeMo-co with purified components has allowed the detection, purification, and identification of an additional component(s) required for the synthesis of cofactor. PMID:10217770

  2. Requirement of NifX and Other nif Proteins for In Vitro Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase

    Shah, Vinod K.; Rangaraj, Priya; Chatterjee, Ranjini; Allen, Ronda M.; Roll, Jon T.; Roberts, Gary P.; Ludden, Paul W.

    1999-01-01

    The iron-molybdenum cofactor (FeMo-co) of nitrogenase contains molybdenum, iron, sulfur, and homocitrate in a ratio of 1:7:9:1. In vitro synthesis of FeMo-co has been established, and the reaction requires an ATP-regenerating system, dithionite, molybdate, homocitrate, and at least NifB-co (the metabolic product of NifB), NifNE, and dinitrogenase reductase (NifH). The typical in vitro FeMo-co synthesis reaction involves mixing extracts from two different mutant strains of Azotobacter vineland...

  3. Acute toxicity of phyto medicine Mulher Ativa and antioxidant properties on the labeling of blood cells and plasmatic proteins with 99mTc in vitro

    Full text: Medicinal plants originate natural products that are biologically active and widely employed as an alternative source in health care. Mulher Ativa is a phyto medicine used in several gynecological pathologies composed of eight medicinal plants which exhibits estrogen properties in the reproductive tract. The objective of this work was determining the acute toxicity studies investigated of Mulher Ativa (Ma) were performed in mice and antioxidant properties on the labeling of blood cells and plasmatic protein with 99mTc in vitro. For these studies, mice were divided in two groups, containing 05 animals each. The treated group received Ma in doses of 10, 100, 200, 300, 600, 1000, 2000, 3000 mg/kg of animal weight. Mice were carefully observed 0.5, 1, 2, 3, 4, 24, 48, and 72h after the treatment to assess possible clinical or toxicological symptoms. The second experiment was realized incubating heparin with blood carried out the experiments. Different concentrations of Mulher Ativa were chosen (200; 100; 50; 25; 12,5 mg/mL). A stannous chloride solution was also added and incubation was kept for 60 minutes. After this, 99mTc was added and the incubation was continued for 10 minutes. The mixture was centrifuged, precipitated with thichloroacetic acid 5% and soluble (SF) and insoluble fractions (IF) were separated. The radioactivities in the groups P, BC, IF-P, SF-P, IF- BC, SF-BC were determined in counter. The analysis of radioactivity in the samples of P and BC isolated from samples of whole blood treated with Mulher Ativa showed decrease significant (*p99mTc in the TCA-insoluble fraction of plasma. It is also concluded that presents antioxidant properties. As part of this pharmacological study, the acute toxicity of Ma in mice was first investigated. In these doses, the median lethal dose LD50 was determined to be higher than highest dose tested i.e 2.0 gkg -1 b.w. From this data, the estimated LD50 was 2060.1 mg/kg. The product was classified as slightly

  4. Synthesis and intracellular transport of proteins in the exocrine pancreas of the frog (Rana esculenta). II. An in vitro study of the transport process and the influence of temperature.

    Slot, J W; Geuze, J J

    1976-03-16

    Frog pancreatic tissue was pulse-labelled in vitro with 3H-leucine and protein transport was studied in exocrine cells by electron microscope autoradiography. The proteins appeared to be synthesized in the RER and transported to the secretory granules along a similar route and with the same velocity as previously described under in vitro conditions. Evidence was obtained for the involvement of the vesicular and tubular elements at the periphery of the Golgi system in transferring protein from the RER to the Golgi cisternae. Kinetics of the release of newly synthesized proteins from the RER and their appearance in the condensing vacuoles are discussed and related to results reported from other tissues. The transport velocity in this poikilothermic system was studied in relation to the incubation temperature and compared with results reported from its mammalian counterpart. At temperatures between 20 and 30 degrees C intracellular protein transport occurs faster in the frog than in the Guinea pig pancreas. At higher temperature the transport process was severely disturbed in the frog. PMID:1083293

  5. In vitro transcripts of wild-type and fluorescent protein-tagged triticum mosaic virus (family potyviridae) are biologically active in wheat

    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, and the progeny virus was efficiently transmitted by wheat curl m...

  6. IN VITRO COMPETITION OF NATURAL 'AUTOGRAPHA CALIFORNICA' NUCLEAR POLYHEDROSIS VIRUS AND A RECOMBINANT EXPRESSING A POLYHEDRIN-BETA GALACTOSIDASE FUSION PROTEIN

    The paper describes results of experiments conducted to investigate the kinetics of in vitro competition between natural progenitor Autographa californica (Acv E-2) and the recombinant Ac360-Bgal virus strains. The following conclusions can be drawn from the analysis: Selection p...

  7. Effects of bone morphogenic protein 4 (BMP4 and its inhibitor, Noggin, on in vitro maturation and culture of bovine preimplantation embryos

    Fernandez-Martin Rafael

    2011-02-01

    Full Text Available Abstract Background BMP4 is a member of the transforming growth factor beta (TGFbeta superfamily and Noggin is a potent BMP inhibitor that exerts its function by binding to BMPs preventing interactions with its receptors. The aim of this work was to investigate the role of BMP4 and Noggin, on oocytes in vitro maturation (m experiments and embryos in vitro development (c experiments of bovine. Methods For m experiments, COCs were collected from slaughterhouse ovaries and in vitro matured in TCM with 100 ng/ml of either BMP4 or Noggin. After 24 h, the nuclear stage of the oocytes was determined by staining with Hoechst 33342. In addition, RT-qPCR was performed on MII oocytes to study the relative concentration of ZAR1, GDF9, BAX, MATER and HSP70 transcripts. Treated oocytes were submitted to parthenogenic activation (PA or in vitro fertilization (IVF and cultured in CR2. For c experiments, non-treated matured oocytes were submitted to PA or IVF to generate embryos that were exposed to 100 ng/ml of BMP4 or Noggin in CR2 until day nine of culture. Cleavage, blastocyst and hatching rates, expression pattern of the transcription factor Oct-4 in blastocysts and embryo cell number at day two and nine post-activation or fertilization were evaluated. Results We found that Noggin, as BMP4, did not affect oocyte nuclear maturation. Noggin supplementation up-regulated the expression of HSP70 and MATER genes in matured oocytes. Moreover, BMP4 during maturation increased the proportion of Oct-4 positive cells in parthenogenic embryos. On the other hand, when Noggin was added to embryo culture medium, developmental rates of parthenogenic and in vitro fertilized embryos were reduced. However, BMP4 addition decreases the development only for in vitro fertilized embryos. BMP4 and Noggin during culture reduced the proportion of Oct-4-expressing cells. Conclusions Our results show that BMP4 is implicated in bovine oocytes maturation and embryo development. Moreover

  8. Base-pairing potential identified by in vitro selection predicts the kinked RNA backbone observed in the crystal structure of the alfalfa mosaic virus RNA-coat protein complex.

    Boyce, Michael; Scott, Felicia; Guogas, Laura M; Gehrke, Lee

    2006-01-01

    The three-dimensional structure of the 3' terminus of alfalfa mosaic virus RNA in complex with an amino-terminal coat protein peptide revealed an unusual RNA fold with inter-AUGC basepairing stabilized by key arginine residues (Guogas, et al., 2004). To probe viral RNA interactions with the full-length coat protein, we have used in vitro genetic selection to characterize potential folding patterns among RNAs isolated from a complex randomized pool. Nitrocellulose filter retention, electrophoretic mobility bandshift analysis, and hydroxyl radical footprinting techniques were used to define binding affinities and to localize the potential RNA-protein interaction sites. Minimized binding sites were identified that included both the randomized domain and a portion of the constant regions of the selected RNAs. The selected RNAs, identified by their ability to bind full-length coat protein, have the potential to form the same unusual inter-AUGC Watson-Crick base pairs observed in the crystal structure, although the primary sequences diverge from the wild-type RNA. A constant feature of both the wild-type RNA and the selected RNAs is a G ribonucleotide in the third position of an AUGC-like repeat. Competitive binding assays showed that substituting adenosine for the constant guanosine in either the wild-type or selected RNAs impaired coat protein binding. These data suggest that the interactions observed in the RNA-peptide structure are likely recapitulated when the full-length protein binds. Further, the results underscore the power of in vitro genetic selection for probing RNA-protein structure and function. PMID:16312015

  9. KEPLER-7b: A TRANSITING PLANET WITH UNUSUALLY LOW DENSITY

    We report on the discovery and confirmation of Kepler-7b, a transiting planet with unusually low density. The mass is less than half that of Jupiter, M P = 0.43 M J, but the radius is 50% larger, R P = 1.48 R J. The resulting density, ρP = 0.17 g cm-3, is the second lowest reported so far for an extrasolar planet. The orbital period is fairly long, P = 4.886 days, and the host star is not much hotter than the Sun, T eff = 6000 K. However, it is more massive and considerably larger than the Sun, M * = 1.35 M sun and R * = 1.84 R sun, and must be near the end of its life on the main sequence.

  10. A Single Amino Acid Substitution in the NS2A Protein of Japanese Encephalitis Virus Affects Virus Propagation In Vitro but Not In Vivo

    Takamatsu, Yuki; Morita, Kouichi; Hayasaka, Daisuke

    2015-01-01

    We identified a unique amino acid of NS2A113, phenylalanine, that affects the efficient propagation of two Japanese encephalitis virus strains, JaTH160 and JaOArS982, in neuroblastoma Neuro-2a cells but not in cell lines of extraneural origin. This amino acid did not affect viral loads in the brain or survival curves in mice. These findings suggest that virus propagation in vitro may not reflect the level of virus neuroinvasiveness in vivo.

  11. Total and free serum haloperidol levels in schizophrenic patients and the effect of age thioridazine and fatty acid on haloperidol-serum protein binding in vitro.

    Rowell, F. J.; Hui, S M; Fairbairn, A F; Eccleston, D

    1981-01-01

    1 Using radioimmunoassays, steady-state levels of total and free haloperidol (obtained by ultrafiltration and dialysis) have been determined in twenty-two patients on long term treatment receiving doses from 3 to 45 mg per day. 2 For the group, both total serum concentration and free drug concentration showed significant correlations (P less than 0.001) with daily dose. 3 No significant correlation was observed between age of the patient and percentage free haloperidol in serum. 4 In vitro ex...

  12. THE HIGH ALBEDO OF THE HOT JUPITER KEPLER-7 b

    Hot Jupiters are expected to be dark from both observations (albedo upper limits) and theory (alkali metals and/or TiO and VO absorption). However, only a handful of hot Jupiters have been observed with high enough photometric precision at visible wavelengths to investigate these expectations. The NASA Kepler mission provides a means to widen the sample and to assess the extent to which hot Jupiter albedos are low. We present a global analysis of Kepler-7 b based on Q0-Q4 data, published radial velocities, and asteroseismology constraints. We measure an occultation depth in the Kepler bandpass of 44 ± 5 ppm. If directly related to the albedo, this translates to a Kepler geometric albedo of 0.32 ± 0.03, the most precise value measured so far for an exoplanet. We also characterize the planetary orbital phase light curve with an amplitude of 42 ± 4 ppm. Using atmospheric models, we find it unlikely that the high albedo is due to a dominant thermal component and propose two solutions to explain the observed planetary flux. First, we interpret the Kepler-7 b albedo as resulting from an excess reflection over what can be explained solely by Rayleigh scattering, along with a nominal thermal component. This excess reflection might indicate the presence of a cloud or haze layer in the atmosphere, motivating new modeling and observational efforts. Alternatively, the albedo can be explained by Rayleigh scattering alone if Na and K are depleted in the atmosphere by a factor of 10-100 below solar abundances.

  13. DWPF SIMULANT CPC STUDIES FOR SB7B

    Koopman, D.

    2011-11-01

    Lab-scale DWPF simulations of Sludge Batch 7b (SB7b) processing were performed. Testing was performed at the Savannah River National Laboratory - Aiken County Technology Laboratory (SRNL-ACTL). The primary goal of the simulations was to define a likely operating window for acid stoichiometry for the DWPF Sludge Receipt and Adjustment Tank (SRAT). In addition, the testing established conditions for the SRNL Shielded Cells qualification simulation of SB7b-Tank 40 blend, supported validation of the current glass redox model, and validated the coupled process flowsheet at the nominal acid stoichiometry. An acid window of 105-140% by the Koopman minimum acid (KMA) equation (107-142% DWPF Hsu equation) worked for the sludge-only flowsheet. Nitrite was present in the SRAT product for the 105% KMA run at 366 mg/kg, while SME cycle hydrogen reached 94% of the DWPF Slurry Mix Evaporator (SME) cycle limit in the 140% KMA run. The window was determined for sludge with added caustic (0.28M additional base, or roughly 12,000 gallons 50% NaOH to 820,000 gallons waste slurry). A suitable processing window appears to be 107-130% DWPF acid equation for sludge-only processing allowing some conservatism for the mapping of lab-scale simulant data to full-scale real waste processing including potentially non-conservative noble metal and mercury concentrations. This window should be usable with or without the addition of up to 7,000 gallons of caustic to the batch. The window could potentially be wider if caustic is not added to SB7b. It is recommended that DWPF begin processing SB7b at 115% stoichiometry using the current DWPF equation. The factor could be increased if necessary, but changes should be made with caution and in small increments. DWPF should not concentrate past 48 wt.% total solids in the SME cycle if moderate hydrogen generation is occurring simultaneously. The coupled flowsheet simulation made more hydrogen in the SRAT and SME cycles than the sludge-only run with the

  14. In vitro fertilization (IVF)

    ... egg and a man's sperm in a laboratory dish. In vitro means outside the body. Fertilization means ... especially the woman's age. Unused embryos may be frozen and implanted or donated at a later date.

  15. 不同油脂对瘤胃原虫、细菌蛋白质及DNA影响的研究%Effects of Different Oils on the Protein and DNA of Ruminal Protozoa and Bacteria in Vitro

    拟豪杰; 钱金花; 李丹枫; 王梦芝

    2011-01-01

    The objectives of this study were to investigate the effects of different oils on the protein and DNA rf ruminal protozoa and bacteria in vitro. 3 Xuhuai White Goats with permanent cannulas were used to provide rumen liquor, in vitro culture were carried out by adding peanut oil, rapeseed oil, corn oil, and soybean oil respectively, with a set of control group (no oil). The results showed that, except for bacterial protein, no significant differences were found in the mean value of bacterial DNA, protozoal protein, protozoal DNA, microbial protein, microbial DNA, and protozoa to bacteria ratio (P>0.05), while significant differences were detected in all above indexes between sampling points according to culture time (P corn oil > peanut oil >control group > rapeseed oil; Whereas, protozoa to bacteria ratio of control group was the highest, followed by that of rapeseed oil, and protozoa to bacteria ratio in corn oil, or soybean oil, were relatively lower. Furthermore, correlation analysis showed that, good correlations existed between bacterial protein and DNA, also between microbial protein and DNA (2-tail0.05),但各项指标随培养时间延长在取样时点间都有显著或极显著的纵向差异(P玉米油>花生油>对照组>菜籽油;而原虫/细菌则以对照组最高,菜籽油组次之,玉米油、豆油组较低.相关分析表明,细菌蛋白与DNA间、微生物蛋白与DNA间皆相关显著(2-tail<0.05).

  16. Expression of Green Fluorescent Protein in Streptococcus gordonii DL1 and Its Use as a Species-Specific Marker in Coadhesion with Streptococcus oralis 34 in Saliva-Conditioned Biofilms In Vitro

    Aspiras, Marcelo B.; Kazmerzak, Karen M.; Kolenbrander, Paul E; McNab, Roderick; Hardegen, Neil; Jenkinson, Howard F.

    2000-01-01

    Streptococcus gordonii is one of the predominant streptococci in the biofilm ecology of the oral cavity. It interacts with other bacteria through receptor-adhesin complexes formed between cognate molecules on the surfaces of the partner cells. To study the spatial organization of S. gordonii DL1 in oral biofilms, we used green fluorescent protein (GFP) as a species-specific marker to identify S. gordonii in a two-species in vitro oral biofilm flowcell system. To drive expression of gfp, we is...

  17. Isolamento da globulina majoritária, digestibilidade in vivo e in vitro das proteínas do tremoço-doce (Lupinus albus L., var. Multolupa Isolation of major globulin, in vivo and in vitro digestibility of proteins from sweet lupin (Lupinus albus L. var. Multolupa

    Valdir Augusto Neves

    2006-12-01

    Full Text Available Os objetivos do trabalho foram isolar, purificar e estudar algumas propriedades da fração globulina majoritária de tremoço-doce, var. Multolupa; assim como avaliar as características de digestibilidade da farinha e frações isoladas. As frações protéicas foram separadas por fracionamento diferencial com uso de diferentes solventes. A globulina majoritária de tremoço-doce foi isolada, purificada por cromatografia em Q-Sepharose, revelando um único pico de proteína. Apresentou um peso molecular de 162,5 ± 10,0 kDa, determinado por cromatografia em Sephacryl S-300, e subunidades entre 20-70 kDa em PAGE-SDS. A solubilidade em função do pH e concentrações de NaCl revelaram curva típica dessa fração. A digestibilidade da proteína da farinha e das frações albumina, globulina e glutelina foi avaliada por experimentos in vitro e in vivo e se revelou alta para a fração globulina, seguida pela glutelina, albumina e farinha. A digestibilidade in vivo da fração globulina, tanto aparente quanto verdadeira, não diferiu significativamente daquela determinada para a caseína. Apesar da alta digestibilidade da fração majoritária e frações protéicas, a utilização como única fonte de proteína nas dietas revelou valores baixos de RPLR (razão protéica líquida relativa, indicando ser insuficiente para sustentar o crescimento dos animais, comparativamente à caseína.The aim of the present work was to isolate, purify and study some properties of the major globulin fraction from sweet lupin, var. Multolupa; as well as to evaluate the digestibility features of flour and isolated fractions. The protein fractions were separated by differential fractionation with different solvents. The lupin major globulin was isolated, purified by Q-Sepharose chromatography when it showed only one protein peak. The major protein presented a molecular weight of 162.5 ± 10.0 kDa, determined on Sephacryl S-300 chromatography; and subunits between 20

  18. Proanthocyanidins from the American Cranberry (Vaccinium macrocarpon Induce Cell Cycle Alterations in DU145 Human Prostate Cancer Cells in Vitro by Affecting the Expression of Cell Cycle-Associated Proteins

    Joseph Kim

    2014-04-01

    Full Text Available Background: Prostate cancer is one of the most common cancers in the world. There are genetic and environmental factors that can potentially impact the development and progression of many types of cancer, including prostate cancer. As a consequence of environmental factors, such as diet having a potential effect on the development of prostate cancer, considerable interest in the possible health benefits associated with the inclusion and consumption of certain foods in the diet exists. Context and purpose of this study: This study describes the effects of a proanthocyanidinenriched fraction (PACs isolated from the American cranberry (Vaccinium macrocarpon on the behaviour of androgen-refractory (insensitive DU145 human prostate cancer cells in vitro. Results: Following treatment of DU145 human prostate cancer cells with 25 µg/mL of PACs for six hours, PACs significantly decreased the cellular viability of DU145 cells. PACs treatment (25 µg/mL for 6 hours of DU145 cells increased the proportion of cells in the G2-M phase of the cell cycle and decreased the proportion of cells in the G1 phase of the cell cycle. These alterations were associated with changes in cell cycle regulatory proteins and other cell cycle associated proteins. PACs increased the expression of cyclin E, cyclin D1, CDK2 and CDK4, and decreased the expression of cyclin A and cyclin B1. The protein expression level of p27 increased, and the protein expression levels of p16INK4a, p21, and pRBp107 decreased in response to PACs treatment. The protein expression level of pRBp130 was unchanged in Functional Foods in Health and Disease 2014; 4(2:130- 146 Page 131 of 146 response to PACs treatment. Conclusions: These findings demonstrate that proanthocyanidins from the American cranberry can affect the behaviour of human prostate cancer cells in vitro and further support the potential health benefits associated with cranberries.

  19. Food protein-stabilized nanoemulsions as potential delivery systems for poorly water-soluble drugs: preparation, in vitro characterization, and pharmacokinetics in rats

    He, Wei; Tan, Yanan; Tian, Zhiqiang; Chen, Lingyun; Hu, Fuqiang; Wu, Wei

    2011-01-01

    Nanoemulsions stabilized by traditional emulsifiers raise toxicological concerns for long-term treatment. The present work investigates the potential of food proteins as safer stabilizers for nanoemulsions to deliver hydrophobic drugs. Nanoemulsions stabilized by food proteins (soybean protein isolate, whey protein isolate, β-lactoglobulin) were prepared by high-pressure homogenization. The toxicity of the nanoemulsions was tested in Caco-2 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-dip...

  20. A polygalacturonase-inhibiting protein from grapevine reduces the symptoms of the endopolygalacturonase BcPG2 from Botrytis cinerea in Nicotiana benthamiana leaves without any evidence for in vitro interaction.

    Joubert, Dirk A; Kars, Ilona; Wagemakers, Lia; Bergmann, Carl; Kemp, Gabré; Vivier, Melané A; van Kan, Jan A L

    2007-04-01

    Six endopolygalacturonases from Botrytis cinerea (BcPG1 to BcPG6) as well as mutated forms of BcPG1 and BcPG2 were expressed transiently in leaves of Nicotiana benthamiana using agroinfiltration. Expression of BcPG1, BcPG2, BcPG4, BcPG5, and mutant BcPG1-D203A caused symptoms, whereas BcPG3, BcPG6, and mutant BcPG2-D192A caused no symptoms. Expression of BcPG2 caused the most severe symptoms, including wilting and necrosis. BcPG2 previously has been shown to be essential for B. cinerea virulence. The in vivo effect of this enzyme and the inhibition by a polygalacturonase-inhibiting protein (PGIP) was examined by coexpressing Bcpg2 and the Vvpgipl gene from Vitis vinifera in N. benthamiana. Coinfiltration resulted in a substantial reduction of the symptoms inflicted by the activity of BcPG2 in planta, as evidenced by quantifying the variable chlorophyll fluorescence yield. In vitro, however, no interaction between pure VvPGIP1 and pure BcPG2 was detected. Specifically, VvPGIP1 neither inhibited BcPG2 activity nor altered the degradation profile of polygalacturonic acid by BcPG2. Furthermore, using surface plasmon resonance technology, no physical interaction between VvPGIP1 and BcPG2 was detected in vitro. The data suggest that the in planta environment provided a context to support the interaction between BcPG2 and VvPGIP1, leading to a reduction in symptom development, whereas neither of the in vitro assays detected any interaction between these proteins. PMID:17427809

  1. The high albedo of the hot Jupiter Kepler-7b

    Demory, Brice-Olivier; Madhusudhan, Nikku; Kjeldsen, Hans; Christensen-Dalsgaard, Joergen; Gillon, Michael; Rowe, Jason F; Welsh, William F; Adams, Elisabeth R; Dupree, Andrea; McCarthy, Don; Kulesa, Craig; Borucki, William J; Koch, David G

    2011-01-01

    Hot Jupiters are expected to be dark from both observations (albedo upper limits) and theory (alkali metals and/or TiO and VO absorption). However, only a handful of hot Jupiters have been observed with high enough photometric precision at visible wavelengths to investigate these expectations. The NASA Kepler mission provides a means to widen the sample and to assess the extent to which hot Jupiter albedos are low. We present a global analysis of Kepler-7b based on Q0-Q4 data, published radial velocities, and asteroseismology constraints. We measure an occultation depth in the Kepler bandpass of 44+-5 ppm. If directly related to the albedo, this translates to a Kepler geometric albedo of 0.32+-0.03, the most precise value measured so far for an exoplanet. We also characterize the planetary orbital phase lightcurve with an amplitude of 42+-4 ppm. Using atmospheric models, we find it unlikely that the high albedo is due to a dominant thermal component and propose two solutions to explain the observed planetary ...

  2. A C-terminal truncated hepatitis C virus core protein variant assembles in vitro into virus-like particles in the absence of structured nucleic acids

    Little is known about the assembly pathway or structure of the hepatitis C virus (HCV). In this work a truncated HCcAg variant covering the first 120 aa (HCcAg.120) with a 32 aa N-terminal fusion peptide (6x Histag-Xpress epitope) was purified as a monomer under strong denaturing conditions. In addition, minor HCcAg.120 peaks exhibiting little different molecular mass by SDS-PAGE which possibly represents alternative forms harboring the N-termini of HCcAg.120 were detected. Analysis using gel filtration chromatography showed that HCcAg.120 assembled into high molecular weight structures in vitro in the absence of structured nucleic acids. The negative-stain electron microscopy analysis revealed that these structures correspond with spherical VLPs of uniform morphology and size distribution. The diameters of these particles ranged from 20 to 43 nm with an average diameter of approximately 30 nm and were specifically immunolabelled with a mouse monoclonal antibody against the residues 5-35 of HCcAg. Results presented in this work showed that HCcAg.120 assembled in vitro into VLPs in the absence of structured nucleic acids with similar morphology and size distribution to those found in sera and hepatocytes from HCV-infected patients. Therefore, these VLPs would be important to elucidate the mechanisms behind the ability of HCcAg to assemble into a nucleocapsid structure

  3. In vitro protein-primed initiation of pneumococcal phage Cp-1 DNA replication occurs at the third 3' nucleotide of the linear template: a stepwise sliding-back mechanism.

    Martín, A C; Blanco, L; García, P; Salas, M; Méndez, J

    1996-07-19

    Phage Cp-1 from Streptoccocus pneumoniae makes use of a protein-priming mechanism to start replication of its linear DNA: the first reaction consists of the addition of 5' dAMP to a molecule of the primer protein, an initiation event occurring at both DNA ends. After elongation of the initiation complex, the primer protein remains linked to the 5' end of the nascent DNA chain, and is subsequently referred to as terminal protein (TP). In this paper, using DNA-free extracts from Cp-1-infected S. pneumoniae, we provide evidence that the formation of the covalent complex TP-dAMP is a template-instructed reaction and that ssDNA molecules can serve as templates for TP-primed replication. A mutational analysis of the 3' terminal nucleotides of Cp-1 DNA reveals that a precise DNA sequence is required for efficient template recognition, and that in vitro initiation of Cp-1 DNA replication is directed by the third nucleotide of the template. However, the two terminal nucleotides are recovered during the first steps of elongation. A new variant of the sliding-back mechanism for protein-primed initiation, firstly described for Bacillus subtilis phage phi29, is proposed to account for the maintenance of Cp-1 DNA ends. The results presented here reinforce the hypothesis that sliding-back must be a common feature in all genomes that use protein-priming to initiate replication. PMID:8757800

  4. Ruthenium(III) S-methylisothiosemicarbazone Schiff base complexes bearing PPh3/AsPh3 coligand: synthesis, structure and biological investigations, including antioxidant, DNA and protein interaction, and in vitro anticancer activities.

    Prakash, Govindan; Manikandan, Rajendran; Viswanathamurthi, Periasamy; Velmurugan, Krishnaswamy; Nandhakumar, Raju

    2014-09-01

    New Ru(III) isothiosemicarbazone complexes [RuCl(EPh3)L(1-4)] (E=P or As) were obtained from the reactions between [RuCl3(EPh3)3] and bis(salicylaldehyde)-S-methylisothiosemicarbazone (H2L(1-3))/bis(2-hydroxy-naphthaldehyde)-S-methylisothiosemicarbazone (H2L(4)) ligands. The new complexes were characterized by using elemental analyses and various spectral (UV-Vis, IR, (1)H NMR, FAB-Mass and EPR) methods. The redox properties of the complexes were studied by using cyclic voltammetric method. The new complexes were subjected to various biological investigations such as antioxidant assays involving DPPH radical, hydroxyl radical, nitric oxide radical and hydrogen peroxide, DNA/protein interaction studies and in vitro cytotoxic studies against human breast cancer cell line (MCF-7). New complexes showed excellent free radicals scavenging ability and could bind with DNA via intercalation. Protein binding studies using fluorescence spectroscopy showed that the new complexes could bind strongly with bovine serum albumin (BSA). Photo cleavage experiments using DNA of E-coli bacterium exhibited the DNA cleavage ability of the complexes. Further, the in vitro anticancer activity studies on the new complexes against MCF-7 cell line exhibited the ability of Ru(III) isothiosemicarbazone complexes to suppress the development of malignant neoplastic disease cells. PMID:24911273

  5. Inhibition of Nonsmall Cell Lung Cancer Cell Migration by Protein Arginine Methyltransferase 1-small Hairpin RNA Through Inhibiting Epithelial-mesenchymal Transition, Extracellular Matrix Degradation, and Src Phosphorylation In Vitro

    Ting Zhang

    2015-01-01

    Full Text Available Background: Protein arginine methyltransferases 1 (PRMT1 is over-expressed in a variety of cancers, including lung cancer, and is correlated with a poor prognosis of tumor development. This study aimed to investigate the role of PRMT1 in nonsmall cell lung cancer (NSCLC migration in vitro. Methods: In this study, PRMT1 expression in the NSCLC cell line A549 was silenced using lentiviral vector-mediated short hairpin RNAs. Cell migration was measured using both scratch wound healing and transwell cell migration assays. The mRNA expression levels of matrix metalloproteinase 2 (MMP-2 and tissue inhibitor of metalloproteinase 1, 2 (TIMP1, 2 were measured using quantitative real-time reverse transcription-polymerase chain reaction. The expression levels of protein markers for epithelial-mesenchymal transition (EMT (E-cadherin, N-cadherin, focal adhesion kinase (FAK, Src, AKT, and their corresponding phosphorylated states were detected by Western blot. Results: Cell migration was significantly inhibited in the PRMT1 silenced group compared to the control group. The mRNA expression of MMP-2 decreased while TIMP1 and TIMP2 increased significantly. E-cadherin mRNA expression also increased while N-cadherin decreased. Only phosphorylated Src levels decreased in the silenced group while FAK or AKT remained unchanged. Conclusions: PRMT1-small hairpin RNA inhibits the migration abilities of NSCLC A549 cells by inhibiting EMT, extracellular matrix degradation, and Src phosphorylation in vitro.

  6. Mutations in the fusion protein cleavage site of avian paramyxovirus serotype 4 confer increased replication and syncytium formation in vitro but not increased replication and pathogenicity in chickens and ducks.

    Kim, Shin-Hee; Xiao, Sa; Shive, Heather; Collins, Peter L; Samal, Siba K

    2013-01-01

    To evaluate the role of the F protein cleavage site in the replication and pathogenicity of avian paramyxoviruses (APMVs), we constructed a reverse genetics system for recovery of infectious recombinant APMV-4 from cloned cDNA. The recovered recombinant APMV-4 resembled the biological virus in growth characteristics in vitro and in pathogenicity in vivo. The F cleavage site sequence of APMV-4 (DIQPR↓F) contains a single basic amino acid, at the -1 position. Six mutant APMV-4 viruses were recovered in which the F protein cleavage site was mutated to contain increased numbers of basic amino acids or to mimic the naturally occurring cleavage sites of several paramyxoviruses, including neurovirulent and avirulent strains of NDV. The presence of a glutamine residue at the -3 position was found to be important for mutant virus recovery. In addition, cleavage sites containing the furin protease motif conferred increased replication and syncytium formation in vitro. However, analysis of viral pathogenicity in 9-day-old embryonated chicken eggs, 1-day-old and 2-week-old chickens, and 3-week-old ducks showed that none the F protein cleavage site mutations altered the replication, tropism, and pathogenicity of APMV-4, and no significant differences were observed among the parental and mutant APMV-4 viruses in vivo. Although parental and mutant viruses replicated somewhat better in ducks than in chickens, they all were highly restricted and avirulent in both species. These results suggested that the cleavage site sequence of the F protein is not a limiting determinant of APMV-4 pathogenicity in chickens and ducks. PMID:23341874

  7. Study on the In Vitro Simulated Digestion Process of Whey Protein and Effects of Heat Treatment%乳清蛋白的体外模拟消化过程及热处理的影响

    何光华; 黄俊; 尤玉如; 肖功年; 刘士旺; 储小军; 魏培莲; 王仁风

    2012-01-01

    The in vitro pepsin and trypsin digestion process of whey protein and the effects of heat treatments were investigated in this paper. SDS-PAGE analytical results indicated that the effects of pepsin and trypsin on the digestion of whey protein was not very obvious. The dry heat-treatment (75-100 ℃, 1 h) did nearly not affect the in vitro digestion process by trypsin, while undcr the same conditions, the wet heat-treatment could remarkably improve the digestive effect of trypsin on the whey protein. After wet heat-treatment(75 ℃ and 100 ℃, 30 min) and trypsin digestion (8 h) of whey protein, its nitrogen releasing rate reached to 82.1% and 91.3%, respectively. However, the nitrogen releasing rate without wet heat-treatment was only 66.7%. This result suggested that the in vitro digestion process of whey protein was affected by different heat treatments. Wet heat treatment can effectively improve the digestion rate of whey protein, the higher the temperature of wet heat treatment, the faster the in vitro digestion rate of whey protein.%探讨了乳清蛋白体外胃蛋白酶和胰蛋白酶的消化过程,以及热处理对该消化过程的影响.SDS-PAGE分析结果表明,胃蛋白酶和胰蛋白酶对乳清蛋白的体外消化作用的影响不明显.干热处理(75℃和100C,1h)几乎不影响乳清蛋白的体外胰蛋白酶消化过程,而在同样条件下,湿热处理能显著提高胰蛋白酶对乳清蛋白的消化作用.乳清蛋白分别经75℃和100℃湿热处理30 min后,再由胰蛋白酶消化8h,其氮释放率分别达到82.1%和91.3%.而未湿热处理的氮释放率仅为66.7%,表明不同热处理方式对乳清蛋白的体外消化过程产生不同的影响.湿热处理能有效地提高乳清蛋白的消化速率,且湿热处理温度越高,乳清蛋白的体外消化速率越快.

  8. Identification and purification of a human immunoglobulin-enhancer-binding protein (NF-. kappa. B) that activates transcription from a human immunodeficiency virus type 1 promoter in vitro

    Kawakami, K.; Scheidereit, C.; Roeder, R.G. (Rockefeller Univ., New York, NY (USA))

    1988-07-01

    The enhancer-binding factor NF-{kappa}B, which is found only in cells that transcribe immunoglobulin light chain genes, has been purified from nuclear extracts of Namalwa cells (human Burkitt lymphoma cells) by sequence-specific DNA affinity chromatography. The purified NF-{kappa}B has been identified as a 51-kDa polypeptide by UV-crosslinking analysis. Footprint and methylation-interference analyses have shown that purified NF-{kappa}B has a binding activity specific for the {kappa} light chain enhancer sequence. The purified factor activated in vitro transcription of the human immunodeficiency virus type I promoter by binding to an upstream NF-{kappa}B-binding site.

  9. Solid lipid particles for oral delivery of peptide and protein drugs III - the effect of fed state conditions on the in vitro release and degradation of desmopressin

    Christophersen, Philip C; Vaghela, Dimple; Müllertz, Anette;

    2014-01-01

    The effect of food intake on the release and degradation of peptide drugs from solid lipid particles is unknown and was therefore investigated in vitro using different fed state media in a lipolysis model. Desmopressin was used as a model peptide and incorporated into solid lipid particles...... of oleic acid glycerides accelerated the release of desmopressin significantly from all solid lipid particles both in the presence and absence of lipase. The presence of oleic acid glycerides also reduced the degradation rate of desmopressin, probably due to the interactions between the lipids...... and the protease or desmopressin. Addition of a medium chain triglyceride, trilaurin, in combination with drug-loaded lipid particles diminished the food effect on the TG18 particles, and trilaurin is therefore proposed to be a suitable excipient for reduction of the food effect. Overall, the present study shows...

  10. Food protein-stabilized nanoemulsions as potential delivery systems for poorly water-soluble drugs: preparation, in vitro characterization, and pharmacokinetics in rats

    Zhiqiang Tian

    2011-03-01

    Full Text Available Wei He1, Yanan Tan1, Zhiqiang Tian1, Lingyun Chen2, Fuqiang Hu3, Wei Wu11Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai, People's Republic of China; 2Department of Agricultural, Food and Nutritional Sciences, University of Alberta, Alberta, Canada; 3Department of Pharmaceutics, School of Pharmacy, Zhejiang University, Hangzhou, Zhejiang, People's Republic of ChinaAbstract: Nanoemulsions stabilized by traditional emulsifiers raise toxicological concerns for long-term treatment. The present work investigates the potential of food proteins as safer stabilizers for nanoemulsions to deliver hydrophobic drugs. Nanoemulsions stabilized by food proteins (soybean protein isolate, whey protein isolate, ß-lactoglobulin were prepared by high-pressure homogenization. The toxicity of the nanoemulsions was tested in Caco-2 cells using the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium-bromide viability assay. In vivo absorption in rats was also evaluated. Food protein-stabilized nanoemulsions, with small particle size and good size distribution, exhibited better stability and biocompatibility compared with nanoemulsions stabilized by traditional emulsifiers. Moreover, ß-lactoglobulin had a better emulsifying capacity and biocompatibility than the other two food proteins. The pancreatic degradation of the proteins accelerated drug release. It is concluded that an oil/water nanoemulsion system with good biocompatibility can be prepared by using food proteins as emulsifiers, allowing better and more rapid absorption of lipophilic drugs.Keywords: oil in water nanoemulsions, food proteins, poorly water-soluble drugs, biocompatibility, in vivo absorption

  11. In vitro uptake of 140 kDa Bacillus thuringiensis nematicidal crystal proteins by the second stage juvenile of Meloidogyne hapla.

    Fengjuan Zhang

    Full Text Available Plant-parasitic nematodes (PPNs are piercing/sucking pests, which cause severe damage to crops worldwide, and are difficult to control. The cyst and root-knot nematodes (RKN are sedentary endoparasites that develop specialized multinucleate feeding structures from the plant cells called syncytia or giant cells respectively. Within these structures the nematodes produce feeding tubes, which act as molecular sieves with exclusion limits. For example, Heterodera schachtii is reportedly unable to ingest proteins larger than 28 kDa. However, it is unknown yet what is the molecular exclusion limit of the Meloidogyne hapla. Several types of Bacillus thuringiensis crystal proteins showed toxicity to M. hapla. To monitor the entry pathway of crystal proteins into M. hapla, second-stage juveniles (J2 were treated with NHS-rhodamine labeled nematicidal crystal proteins (Cry55Aa, Cry6Aa, and Cry5Ba. Confocal microscopic observation showed that these crystal proteins were initially detected in the stylet and esophageal lumen, and subsequently in the gut. Western blot analysis revealed that these crystal proteins were modified to different molecular sizes after being ingested. The uptake efficiency of the crystal proteins by the M. hapla J2 decreased with increasing of protein molecular mass, based on enzyme-linked immunosorbent assay analysis. Our discovery revealed 140 kDa nematicidal crystal proteins entered M. hapla J2 via the stylet, and it has important implications in designing a transgenic resistance approach to control RKN.

  12. In Vitro Interaction of the Housekeeping SecA1 with the Accessory SecA2 Protein of Mycobacterium tuberculosis.

    Irfan Prabudiansyah

    Full Text Available The majority of proteins that are secreted across the bacterial cytoplasmic membrane leave the cell via the Sec pathway, which in its minimal form consists of the dimeric ATP-driven motor protein SecA that associates with the protein-conducting membrane pore SecYEG. Some Gram-positive bacteria contain two homologues of SecA, termed SecA1 and SecA2. SecA1 is the essential housekeeping protein, whereas SecA2 is not essential but is involved in the translocation of a subset of proteins, including various virulence factors. Some SecA2 containing bacteria also harbor a homologous SecY2 protein that may form a separate translocase. Interestingly, mycobacteria contain only one SecY protein and thus both SecA1 and SecA2 are required to interact with SecYEG, either individually or together as a heterodimer. In order to address whether SecA1 and SecA2 cooperate during secretion of SecA2 dependent proteins, we examined the oligomeric state of SecA1 and SecA2 of Mycobacterium tuberculosis and their interactions with SecA2 and the cognate SecA1, respectively. We conclude that both SecA1 and SecA2 individually form homodimers in solution but when both proteins are present simultaneously, they form dissociable heterodimers.

  13. Copper directs ATP7B to the apical domain of hepatic cells via basolateral endosomes.

    Nyasae, Lydia K; Schell, Michael J; Hubbard, Ann L

    2014-12-01

    Physiologic Cu levels regulate the intracellular location of the Cu ATPase ATP7B. Here, we determined the routes of Cu-directed trafficking of endogenous ATP7B in the polarized hepatic cell line WIF-B and in the liver in vivo. Copper (10 µm) caused ATP7B to exit the trans-Golgi network (TGN) in vesicles, which trafficked via large basolateral endosomes to the apical domain within 1 h. Although perturbants of luminal acidification had little effect on the TGN localization of ATP7B in low Cu, they blocked delivery to the apical membrane in elevated Cu. If the vesicular proton-pump inhibitor bafilomycin-A1 (Baf) was present with Cu, ATP7B still exited the TGN, but accumulated in large endosomes located near the coverslip, in the basolateral region. Baf washout restored ATP7B trafficking to the apical domain. If ATP7B was staged apically in high Cu, Baf addition promoted the accumulation of ATP7B in subapical endosomes, indicating a blockade of apical recycling, with concomitant loss of ATP7B at the apical membrane. The retrograde pathway to the TGN, induced by Cu removal, was far less affected by Baf than the anterograde (Cu-stimulated) case. Overall, loss of acidification-impaired Cu-regulated trafficking of ATP7B at two main sites: (i) sorting and exit from large basolateral endosomes and (ii) recycling via endosomes near the apical membrane. PMID:25243755

  14. In-vitro diagnosis

    Poulsen, Lars K.

    2001-01-01

    This review on in-vitro diagnostic methods focuses on the use of methods to perform risk assessment on foods. Based on the International Life Science Institute (ILSI) risk decision tree, the methods are discussed and three scenarios are suggested: (i) testing for a well-known allergen; (ii) testing...

  15. The potential of chitosan in enhancing peptide and protein absorption across the TR146 cell culture model-an in vitro model of the buccal epithelium

    Portero, Ana; Remuñán-López, Carmen; Nielsen, Hanne Mørck

    2002-01-01

    To investigate the potential of chitosan (CS) to enhance buccal peptide and protein absorption, the TR146 cell culture model, a model of the buccal epithelium, was used.......To investigate the potential of chitosan (CS) to enhance buccal peptide and protein absorption, the TR146 cell culture model, a model of the buccal epithelium, was used....

  16. Antioxidant activity of Cod (Gadus morhua) protein hydrolysates: In vitro assays and evaluation in 5% fish oil-in-water emulsion

    Farvin, Sabeena; Andersen, Lisa Lystbæk; Nielsen, Henrik Hauch;

    2014-01-01

    Cod protein hydrolysates were fractionated according to the molecular mass into three fractions of >5kDa, 3–5kDa and......Cod protein hydrolysates were fractionated according to the molecular mass into three fractions of >5kDa, 3–5kDa and...

  17. Expression of Chlamydomonas actin-gfp fusion gene in to-bacco suspension cell and polymerization of the actin-gfp protein in vitro

    2001-01-01

    The fusion gene of actin (cDNA of Chlamydo- monas reinhardtii) and green fluorescence protein (gfp) had been constructed into two expression vectors which could be expressed in E. coli and tobacco suspension cells BY2. The correct expression was observed in E. coli and BY2 with a fluorescence microscopy. The fusion protein, which took part in the membrane skeleton, was mainly located peripherally along the membrane, specially the fusion protein was dis-tributed around nucleus and cell plate, while the fusion pro-tein also forms F-actin in the cell. The fusion protein was purified from Bl21plus by ammonium sulfate fractionation, ion exchange chromatography and hydrophobic interaction chromatography. The purified production could polymerize into F-actin when the actin polymerizing buffer was added. It was demonstrated that the characteristics and function of actin in Chlamydomonas was similar with those of animals and higher plants.

  18. Host-range restriction of vaccinia virus E3L deletion mutant can be overcome in vitro, but not in vivo, by expression of the influenza virus NS1 protein.

    Susana Guerra

    Full Text Available During the last decades, research focused on vaccinia virus (VACV pathogenesis has been intensified prompted by its potential beneficial application as a vector for vaccine development and anti-cancer therapies, but also due to the fear of its potential use as a bio-terrorism threat. Recombinant viruses lacking a type I interferon (IFN antagonist are attenuated and hence good vaccine candidates. However, vaccine virus growth requires production in IFN-deficient systems, and thus viral IFN antagonists that are active in vitro, yet not in vivo, are of great value. The VACV E3 and influenza virus NS1 proteins are distinct double-stranded RNA-binding proteins that play an important role in pathogenesis by inhibiting the mammalian IFN-regulated innate antiviral response. Based on the functional similarities between E3 and NS1, we investigated the ability of NS1 to replace the biological functions of E3 of VACV in both in vitro and in vivo systems. For this, we generated a VACV recombinant virus lacking the E3L gene, yet expressing NS1 (VVΔE3L/NS1. Our study revealed that NS1 can functionally replace E3 in cultured cells, rescuing the protein synthesis blockade, and preventing apoptosis and RNA breakdown. In contrast, in vivo the VVΔE3L/NS1 virus was highly attenuated after intranasal inoculation, as it was unable to spread to the lungs and other organs. These results indicate that there are commonalities but also functional differences in the roles of NS1 and E3 as inhibitors of the innate antiviral response, which could potentially be utilized for vaccine production purposes in the future.

  19. In vitro binding of the Salmonella dublin virulence plasmid regulatory protein SpvR to the promoter regions of spvA and spvR.

    Grob, P; Guiney, D G

    1996-01-01

    The spv regulon of Salmonella dublin is essential for virulence in mice. SpvR, a LysR-type regulator, induces the expression of the spvABCD operon and its own expression in the stationary phase of bacterial growth and in macrophages. We constructed fusion proteins to the maltose-binding protein (MBP) and a His tag peptide (His) to overcome the insolubility and to facilitate purification of SpvR. We demonstrated that both fusion proteins, MBP-SpvR and His-SpvR, were able to induce spvA express...

  20. Evidence for in vitro and in vivo expression of the conserved VAR3 (type 3) plasmodium falciparum erythrocyte membrane protein 1

    Wang, Christian W; Lavstsen, Thomas; Bengtsson, Dominique C;

    2012-01-01

    antibodies and the var transcript and PfEMP1 expression profiles of the generated parasites were investigated. The IgG reactivity by plasma from children living in malaria-endemic Tanzania was tested to parasites and recombinant VAR3 protein. Parasites from hospitalized children were isolated and the...... recognition of parasites expressing VAR3 and to the recombinant VAR3 protein after a malaria episode and the isolated parasites showed high levels of var3 transcripts. CONCLUSIONS: Collectively, the presented data suggest that var3 is transcribed and its protein product expressed on the surface of infected...... transcript level of var3 was investigated. RESULTS: Var3 is transcribed and its protein product expressed on the surface of infected erythrocytes. The VAR3-expressing parasites were better recognized by children's IgG than a parasite line expressing a Group B var gene. Two in 130 children showed increased...