Tumor promoter induced membrane-bound protein kinase C - its influence on hematogenous metastasis

International Nuclear Information System (INIS)

Gopalakrishna, R.; Barsky, S.H.

1987-01-01

A correlation between the amount of membrane-bound detergent-extractable protein kinase C activity in various B16 melanoma sublines (F10, F1, BL6) and their lung metastasizing abilities following intravenous injection was found. The F10 subline which exhibits higher metastasizing ability was found to have higher membrane-bound protein kinase C compared to the lower metastasizing subline, F1. Treatment of F1 cells with 100 nM 12-0 tetradecanoylphorbol-13-acetate (TPA) for 1h resulted in 90% decrease in protein kinase C activity in the cytosol with a concommitent increase in membrane-bound activity. These TPA-treated cells when injected intravenously in C57BL/6 mice produced 6-fold increase in pulmonary metastases compared to untreated F1 cells. However, biologically inactive analogues 4 α-phorbol 12,13-didecanoate and phorbol 13-acetate had no effect on either membrane-bound protein kinase C activity or pulmonary metastases. Treating F1 cells with the second-stage tumor promoter, mezerin, resulted in increase in both membrane association of protein kinase C and also lung metastases. Thus, these results strongly suggests that membrane associated protein kinase C activity influences hematogenous metastasis of these melanoma cells

TUNABLE TENSOR VOTING FOR REGULARIZING PUNCTATE PATTERNS OF MEMBRANE-BOUND PROTEIN SIGNALS

OpenAIRE

Loss, Leandro

2009-01-01

Membrane-bound protein, expressed in the basal-lateral region, is heterogeneous and an important endpoint for understanding biological processes. At the optical resolution, membrane-bound protein can be visualized as being diffused (e.g., E-cadherin), punctate (e.g., connexin), or simultaneously diffused and punctate as a result of sample preparation or conditioning. Furthermore, there is a significant amount of heterogeneity as a result of technical and biological variations. This paper aims...

Speciation of protein-bound trace elements by gel electrophoresis and atomic spectrometry.

Science.gov (United States)

Ma, Renli; McLeod, Cameron W; Tomlinson, Kerry; Poole, Robert K

2004-08-01

The metabolism of trace elements, in particular their binding to proteins in biological systems is of great importance in biochemical, toxicological, and pharmacological studies. As a result there has been a sustained interest over the last two decades in the speciation of protein-bound metals. Various analytical approaches have been employed, combining efficient separation of metalloproteins by liquid chromatography or electrophoresis with high-sensitivity elemental detection. Slab-gel electrophoresis (GE) is a key platform for high-resolution protein separation, and has been combined with autoradiography and various atomic spectrometric techniques for in-gel determination of protein-bound metals. Recently, the combination of GE with state-of-the-art inductively coupled plasma-mass spectrometry (ICP-MS), particularly when linked to laser ablation (LA) for direct gel interrogation, has opened up new opportunities for rapid characterization of metalloproteins. The use of GE and atomic spectrometry for the speciation of protein-bound trace elements is reviewed in this paper. Technical requirements for gel electrophoresis/atomic spectrometric measurement are considered in terms of method compatibilities, detection capability and potential usefulness. The literature is also surveyed to illustrate current status and future trends. Copyright 2004 Wiley-VCH Verlag GmbH and Co.

NF-kappa B activity in T cells stably expressing the Tax protein of human T cell lymphotropic virus type I

International Nuclear Information System (INIS)

Lacoste, J.; Cohen, L.; Hiscott, J.

1991-01-01

The effect of constitutive Tax expression on the interaction of NF-κ B with its recognition sequence and on NF-κ B-dependent gene expression was examined in T lymphoid Jurkat cell lines (19D and 9J) stably transformed with a Tax expression vector. Tax expressing T cell lines contained a constitutive level of NF-κ B binding activity, detectable by mobility shift assay and uv cross-linking using a palindromic NF-κ B probe homologous to the interferon beta PRDII site. In Jurkat and NC2.10 induction with phorbol esters resulted in the appearance of new DNA binding proteins of 85, 75, and 54 kDa, whereas in Tax expressing cells the 85-kDa protein and a 92-kDa DNA binding protein were constitutively induced. Expression of Tax protein in 19D and 9J resulted in transcription of the endogenous NF-kappa B-dependent granulocyte-macrophage colony stimulating factor gene and increased basal level expression of transfected NF-kappa B-regulated promoters. Nonetheless transcription of both the endogenous and the transfected gene was inducible by PMA treatment. Tax expression in Jurkat T cells may alter the stoichiometry of NF-kappa B DNA binding proteins and thus change the expression of NF-kappa B-regulated promoters

Spermidine is bound to a unique protein in early sea urchin embryos

International Nuclear Information System (INIS)

Canellakis, Z.N.; Bondy, P.K.; Infante, A.A.

1985-01-01

Spermidine is rapidly taken up and becomes bound to protein during the very early hours of sea urchin embryogenesis. During the first 6 hr after fertilization of freshly obtained sea urchin eggs (Strongylocentrotus purpuratus), which are incubated in the presence of exogenous [ 3 H]-spermidine, up to 7% of the total cell-associated spermidine appears uniquely as spermidine bound in macromolecular form. This unique protein containing spermidine migrates as a single radioactive band in gel electrophoresis. It has a Mr of approximately equal to 30,000 and is readily distinguishable from the protein initiation factor eIF-4D, which has a Mr of 18,000, the only other identifiable protein known to date to be posttranslationally modified by polyamines

Oxidation of DNA, proteins and lipids by DOPA, protein-bound DOPA, and related catechol(amine)s

DEFF Research Database (Denmark)

Pattison, David I; Dean, Roger T; Davies, Michael Jonathan

2002-01-01

Incubation of free 3,4-dihydroxyphenylalanine (DOPA), protein-bound DOPA (PB-DOPA) and related catechols with DNA, proteins and lipids has been shown to result in oxidative damage to the target molecule. This article reviews these reactions with particular emphasis on those that occur in the pres......Incubation of free 3,4-dihydroxyphenylalanine (DOPA), protein-bound DOPA (PB-DOPA) and related catechols with DNA, proteins and lipids has been shown to result in oxidative damage to the target molecule. This article reviews these reactions with particular emphasis on those that occur...... in the presence of molecular O(2) and redox-active metal ions (e.g. Fe(3+), Cu(2+), Cr(6+)), which are known to increase the rate of DOPA oxidation. The majority of oxidative damage appears to be mediated by reactive oxygen species (ROS) such as superoxide and HO(.) radicals, though other DOPA oxidation products...

Large eddy simulation of stably stratified turbulence

International Nuclear Information System (INIS)

Shen Zhi; Zhang Zhaoshun; Cui Guixiang; Xu Chunxiao

2011-01-01

Stably stratified turbulence is a common phenomenon in atmosphere and ocean. In this paper the large eddy simulation is utilized for investigating homogeneous stably stratified turbulence numerically at Reynolds number Re = uL/v = 10 2 ∼10 3 and Froude number Fr = u/NL = 10 −2 ∼10 0 in which u is root mean square of velocity fluctuations, L is integral scale and N is Brunt-Vaïsälä frequency. Three sets of computation cases are designed with different initial conditions, namely isotropic turbulence, Taylor Green vortex and internal waves, to investigate the statistical properties from different origins. The computed horizontal and vertical energy spectra are consistent with observation in atmosphere and ocean when the composite parameter ReFr 2 is greater than O(1). It has also been found in this paper that the stratification turbulence can be developed under different initial velocity conditions and the internal wave energy is dominated in the developed stably stratified turbulence.

Protein-Bound Uremic Toxin Profiling as a Tool to Optimize Hemodialysis.

Directory of Open Access Journals (Sweden)

Sunny Eloot

Full Text Available We studied various hemodialysis strategies for the removal of protein-bound solutes, which are associated with cardiovascular damage.This study included 10 patients on standard (3 x 4 h/week high-flux hemodialysis. Blood was collected at the dialyzer inlet and outlet at several time points during a midweek session. Total and free concentration of several protein-bound solutes was determined as well as urea concentration. Per solute, a two-compartment kinetic model was fitted to the measured concentrations, estimating plasmatic volume (V1, total distribution volume (V tot and intercompartment clearance (K21. This calibrated model was then used to calculate which hemodialysis strategy offers optimal removal. Our own in vivo data, with the strategy variables entered into the mathematical simulations, was then validated against independent data from two other clinical studies.Dialyzer clearance K, V1 and V tot correlated inversely with percentage of protein binding. All Ks were different from each other. Of all protein-bound solutes, K21 was 2.7-5.3 times lower than that of urea. Longer and/or more frequent dialysis that processed the same amount of blood per week as standard 3 x 4 h dialysis at 300 mL/min blood flow showed no difference in removal of strongly bound solutes. However, longer and/or more frequent dialysis strategies that processed more blood per week than standard dialysis were markedly more adequate. These conclusions were successfully validated.When blood and dialysate flow per unit of time and type of hemodialyzer are kept the same, increasing the amount of processed blood per week by increasing frequency and/or duration of the sessions distinctly increases removal.

Protein-Bound Uremic Toxin Profiling as a Tool to Optimize Hemodialysis.

Science.gov (United States)

Eloot, Sunny; Schneditz, Daniel; Cornelis, Tom; Van Biesen, Wim; Glorieux, Griet; Dhondt, Annemie; Kooman, Jeroen; Vanholder, Raymond

2016-01-01

We studied various hemodialysis strategies for the removal of protein-bound solutes, which are associated with cardiovascular damage. This study included 10 patients on standard (3 x 4 h/week) high-flux hemodialysis. Blood was collected at the dialyzer inlet and outlet at several time points during a midweek session. Total and free concentration of several protein-bound solutes was determined as well as urea concentration. Per solute, a two-compartment kinetic model was fitted to the measured concentrations, estimating plasmatic volume (V1), total distribution volume (V tot) and intercompartment clearance (K21). This calibrated model was then used to calculate which hemodialysis strategy offers optimal removal. Our own in vivo data, with the strategy variables entered into the mathematical simulations, was then validated against independent data from two other clinical studies. Dialyzer clearance K, V1 and V tot correlated inversely with percentage of protein binding. All Ks were different from each other. Of all protein-bound solutes, K21 was 2.7-5.3 times lower than that of urea. Longer and/or more frequent dialysis that processed the same amount of blood per week as standard 3 x 4 h dialysis at 300 mL/min blood flow showed no difference in removal of strongly bound solutes. However, longer and/or more frequent dialysis strategies that processed more blood per week than standard dialysis were markedly more adequate. These conclusions were successfully validated. When blood and dialysate flow per unit of time and type of hemodialyzer are kept the same, increasing the amount of processed blood per week by increasing frequency and/or duration of the sessions distinctly increases removal.

Magnetic cell sorting purification of differentiated embryonic stem cells stably expressing truncated human CD4 as surface marker.

Science.gov (United States)

David, Robert; Groebner, Michael; Franz, Wolfgang-Michael

2005-04-01

Embryonic stem (ES) cells offer great potential in regenerative medicine and tissue engineering. Clinical applications are still hampered by the lack of protocols for gentle, high-yield isolation of specific cell types for transplantation expressing no immunogenic markers. We describe labeling of stably transfected ES cells expressing a human CD4 molecule lacking its intracellular domain (DeltaCD4) under control of the phosphoglycerate kinase promoter for magnetic cell sorting (MACS). To track the labeled ES cells, we fused DeltaCD4 to an intracellular enhanced green fluorescent protein domain (DeltaCD4EGFP). We showed functionality of the membrane-bound fluorescent fusion protein and its suitability for MACS leading to purities greater than 97%. Likewise, expression of DeltaCD4 yielded up to 98.5% positive cells independently of their differentiation state. Purities were not limited by the initial percentage of DeltaCD4(+) cells, ranging from 0.6%-16%. The viability of MACS-selected cells was demonstrated by reaggregation and de novo formation of embryoid bodies developing all three germ layers. Thus, expression of DeltaCD4 in differentiated ES cells may enable rapid, high-yield purification of a desired cell type for tissue engineering and transplantation studies.

Generation of a Homozygous Transgenic Rat Strain Stably Expressing a Calcium Sensor Protein for Direct Examination of Calcium Signaling.

Science.gov (United States)

Szebényi, Kornélia; Füredi, András; Kolacsek, Orsolya; Pergel, Enikő; Bősze, Zsuzsanna; Bender, Balázs; Vajdovich, Péter; Tóvári, József; Homolya, László; Szakács, Gergely; Héja, László; Enyedi, Ágnes; Sarkadi, Balázs; Apáti, Ágota; Orbán, Tamás I

2015-08-03

In drug discovery, prediction of selectivity and toxicity require the evaluation of cellular calcium homeostasis. The rat is a preferred laboratory animal for pharmacology and toxicology studies, while currently no calcium indicator protein expressing rat model is available. We established a transgenic rat strain stably expressing the GCaMP2 fluorescent calcium sensor by a transposon-based methodology. Zygotes were co-injected with mRNA of transposase and a CAG-GCaMP2 expressing construct, and animals with one transgene copy were pre-selected by measuring fluorescence in blood cells. A homozygous rat strain was generated with high sensor protein expression in the heart, kidney, liver, and blood cells. No pathological alterations were found in these animals, and fluorescence measurements in cardiac tissue slices and primary cultures demonstrated the applicability of this system for studying calcium signaling. We show here that the GCaMP2 expressing rat cardiomyocytes allow the prediction of cardiotoxic drug side-effects, and provide evidence for the role of Na(+)/Ca(2+) exchanger and its beneficial pharmacological modulation in cardiac reperfusion. Our data indicate that drug-induced alterations and pathological processes can be followed by using this rat model, suggesting that transgenic rats expressing a calcium-sensitive protein provide a valuable system for pharmacological and toxicological studies.

Production and characterization of active recombinant interleukin-12/eGFP fusion protein in stably-transfected DF1 chicken cells.

Science.gov (United States)

Wu, Hsing Chieh; Chen, Yu San; Shen, Pin Chun; Shien, Jui Hung; Lee, Long Huw; Chiu, Hua Hsien

2015-01-01

The adjuvant activity of chicken interleukin-12 (chIL-12) protein has been described as similar to that of mammalian IL-12. Recombinant chIL-12 can be produced using several methods, but chIL-12 production in eukaryotic cells is lower than that in prokaryotic cells. Stimulating compounds, such as dimethyl sulfoxide (DMSO), can be added to animal cell cultures to overcome this drawback. In this study, we constructed a cell line, DF1/chIL-12 which stably expressed a fusion protein, chIL-12 and enhanced green fluorescent protein (eGFP) connected by a (G4 S)3 linker sequence. Fusion protein production was increased when cells were cultured in the presence of DMSO. When 1 × 10(6) DF1/chIL-12 cells were inoculated in a T-175 flask containing 30 mL of media, incubated for 15 h, and further cultivated in the presence of 4% DMSO for 48 h, the production of total fusion protein was mostly enhanced compared with the production of total fusion protein by using cell lysates induced with DMSO at other concentrations. The concentrations of the unpurified and purified total fusion proteins in cell lysates were 2,781 ± 2.72 ng mL(-1) and 2,207 ± 3.28 ng mL(-1) , respectively. The recovery rate was 79%. The fusion protein stimulated chicken splenocytes to produce IFN-γ, which was measured using an enzyme-linked immunosorbent assay, in the culture supernatant, indicating that treating DF1/chIL-12 cells with DMSO or producing chIL-12 in a fusion protein form does not have adverse effects on the bioactivity of chIL-12. © 2015 American Institute of Chemical Engineers.

Dynamic nuclear polarization of membrane proteins: covalently bound spin-labels at protein–protein interfaces

International Nuclear Information System (INIS)

Wylie, Benjamin J.; Dzikovski, Boris G.; Pawsey, Shane; Caporini, Marc; Rosay, Melanie; Freed, Jack H.; McDermott, Ann E.

2015-01-01

We demonstrate that dynamic nuclear polarization of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of sixfold for the dimeric protein. The enhancement effect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces

Protein-bound homocyst(e)ine. A possible risk factor for coronary artery disease.

Science.gov (United States)

Kang, S S; Wong, P W; Cook, H Y; Norusis, M; Messer, J V

1986-01-01

The development of atherosclerotic changes and thromboembolism are common features in homocystinurics. Hence, we postulate a positive correlation between the level of homocyst(e)ine in the blood and the occurrence of coronary artery disease. Homocysteine is found either as free homocystine, cysteine-homocysteine mixed disulfide, or protein-bound homocyst(e)ine. In nonhomocystinuric subjects, most homocysteine molecules are detectable in the protein-bound form. Thus, protein-bound homocyst(e)ine in stored plasma which reflected total plasma homocyst(e)ine was determined in 241 patients with coronary artery disease (173 males and 68 females). The mean +/- SD total plasma homocyst(e)ine was 5.41 +/- 1.62 nmol/ml in male patients, 4.37 +/- 1.09 nmol/ml in male controls, 5.66 +/- 1.93 nmol/ml in female patients, and 4.16 +/- 1.62 nmol/ml in female controls. The differences between the patients with coronary artery disease and the controls were statistically significant (P less than 0.0005). PMID:3700650

Thermal response of rat fibroblasts stably transfected with the human 70-kDa heat shock protein-encoding gene

International Nuclear Information System (INIS)

Li, G.C.; Li, Ligeng; Liu, Yunkang; Mak, J.Y.; Chen, Lili; Lee, W.M.F.

1991-01-01

The major heat shock protein hsp70 is synthesized by cells of a wide variety of organisms in response to heat shock or other environmental stresses and is assumed to play an important role in protecting cells from thermal stress. The authors have tested this hypothesis directly by transfecting a constitutively expressed recombinant human hsp70-encoding gene into rat fibroblasts and examining the relationship between the levels of human hsp70 expressed and thermal resistance of the stably transfected rat cells. Successful transfection and expression of the gene for human hsp70 were characterized by RNA hybridization analysis, low-dimensional gel electrophoresis, and immunoblot analysis. When individual cloned cell lines were exposed to 45C and their thermal survivals were determined by colony-formation assay, they found that the expression of human hsp70 conferred heat resistance to the rat cells. These results reinforce the hypothesis that hsp70 has a protective function against thermal stress

Casein kinase II protein kinase is bound to lamina-matrix and phosphorylates lamin-like protein in isolated pea nuclei

Science.gov (United States)

Li, H.; Roux, S. J.

1992-01-01

A casein kinase II (CK II)-like protein kinase was identified and partially isolated from a purified envelope-matrix fraction of pea (Pisum sativum L.) nuclei. When [gamma-32P]ATP was directly added to the envelope-matrix preparation, the three most heavily labeled protein bands had molecular masses near 71, 48, and 46 kDa. Protein kinases were removed from the preparation by sequential extraction with Triton X-100, EGTA, 0.3 M NaCl, and a pH 10.5 buffer, but an active kinase still remained bound to the remaining lamina-matrix fraction after these treatments. This kinase had properties resembling CK II kinases previously characterized from animal and plant sources: it preferred casein as an artificial substrate, could use GTP as efficiently as ATP as the phosphoryl donor, was stimulated by spermine, was calcium independent, and had a catalytic subunit of 36 kDa. Some animal and plant CK II kinases have regulatory subunits near 29 kDa, and a lamina-matrix-bound protein of this molecular mass was recognized on immunoblot by anti-Drosophila CK II polyclonal antibodies. Also found associated with the envelope-matrix fraction of pea nuclei were p34cdc2-like and Ca(2+)-dependent protein kinases, but their properties could not account for the protein kinase activity bound to the lamina. The 71-kDa substrate of the CK II-like kinase was lamin A-like, both in its molecular mass and in its cross-reactivity with anti-intermediate filament antibodies. Lamin phosphorylation is considered a crucial early step in the entry of cells into mitosis, so lamina-bound CK II kinases may be important control points for cellular proliferation.

Unique Pattern of Protein-Bound Maillard Reaction Products in Manuka (Leptospermum scoparium) Honey.

Science.gov (United States)

Hellwig, Michael; Rückriemen, Jana; Sandner, Daniel; Henle, Thomas

2017-05-03

As a unique feature, honey from the New Zealand manuka tree (Leptospermum scoparium) contains substantial amounts of dihydroxyacetone (DHA) and methylglyoxal (MGO). Although MGO is a reactive intermediate in the Maillard reaction, very little is known about reactions of MGO with honey proteins. We hypothesized that the abundance of MGO should result in a particular pattern of protein-bound Maillard reaction products (MRPs) in manuka honey. A protein-rich high-molecular-weight fraction was isolated from 12 manuka and 8 non-manuka honeys and hydrolyzed enzymatically. By HPLC-MS/MS, 8 MRPs, namely, N-ε-fructosyllysine, N-ε-maltulosyllysine, carboxymethyllysine, carboxyethyllysine (CEL), pyrraline, formyline, maltosine, and methylglyoxal-derived hydroimidazolone 1 (MG-H1), were quantitated. Compared to non-manuka honeys, the manuka honeys were characterized by high concentrations of CEL and MG-H1, whereas the formation of N-ε-fructosyllysine was suppressed, indicating concurrence reactions of glucose and MGO at the ε-amino group of protein-bound lysine. Up to 31% of the lysine and 8% of the arginine residues, respectively, in the manuka honey protein can be modified to CEL and MG-H1, respectively. CEL and MG-H1 concentrations correlated strongly with the MGO concentration of the honeys. Manuka honey possesses a special pattern of protein-bound MRPs, which might be used to prove the reliability of labeled MGO levels in honeys and possibly enable the detection of fraudulent MGO or DHA addition to honey.

Characterization of a major 31-kilodalton peptidoglycan-bound protein of Legionella pneumophila

International Nuclear Information System (INIS)

Butler, C.A.; Hoffman, P.S.

1990-01-01

A 31-kilodalton (kDa) protein was solubilized from the peptidoglycan (PG) fraction of Legionella pneumophila after treatment with either N-acetylmuramidase from the fungus Chalaropsis sp. or with mutanolysin from Streptomyces globisporus. The protein exhibited a ladderlike banding pattern by autoradiography when radiolabeled [(35S]cysteine or [35S]methionine) PG material was extensively treated with hen lysozyme. The banding patterns ranging between 31 and 45 kDa and between 55 and 60 kDa resolved as a single 31-kDa protein when the material was subsequently treated with N-acetylmuramidase. Analysis of the purified 31-kDa protein for diaminopimelic acid by gas chromatography revealed 1 mol of diaminopimelic acid per mol of protein. When outer membrane PG material containing the major outer membrane porin protein was treated with N-acetylmuramidase or mutanolysin, both the 28.5-kDa major outer membrane protein and the 31-kDa protein were solubilized from the PG material under reducing conditions. In the absence of 2-mercaptoethanol, a high-molecular-mass complex (100 kDa) was resolved. The results of this study indicate that a 31-kDa PG-bound protein is a major component of the cell wall of L. pneumophila whose function may be to anchor the major outer membrane protein to PG. Finally, a survey of other Legionella species and other serogroups of L. pneumophila suggested that PG-bound proteins may be a common feature of this genus

Fragment-based modelling of single stranded RNA bound to RNA recognition motif containing proteins

Science.gov (United States)

de Beauchene, Isaure Chauvot; de Vries, Sjoerd J.; Zacharias, Martin

2016-01-01

Abstract Protein-RNA complexes are important for many biological processes. However, structural modeling of such complexes is hampered by the high flexibility of RNA. Particularly challenging is the docking of single-stranded RNA (ssRNA). We have developed a fragment-based approach to model the structure of ssRNA bound to a protein, based on only the protein structure, the RNA sequence and conserved contacts. The conformational diversity of each RNA fragment is sampled by an exhaustive library of trinucleotides extracted from all known experimental protein–RNA complexes. The method was applied to ssRNA with up to 12 nucleotides which bind to dimers of the RNA recognition motifs (RRMs), a highly abundant eukaryotic RNA-binding domain. The fragment based docking allows a precise de novo atomic modeling of protein-bound ssRNA chains. On a benchmark of seven experimental ssRNA–RRM complexes, near-native models (with a mean heavy-atom deviation of <3 Å from experiment) were generated for six out of seven bound RNA chains, and even more precise models (deviation < 2 Å) were obtained for five out of seven cases, a significant improvement compared to the state of the art. The method is not restricted to RRMs but was also successfully applied to Pumilio RNA binding proteins. PMID:27131381

Incorporation of membrane-bound, mammalian-derived immunomodulatory proteins into influenza whole virus vaccines boosts immunogenicity and protection against lethal challenge

Directory of Open Access Journals (Sweden)

Roberts Paul C

2009-04-01

Full Text Available Abstract Background Influenza epidemics continue to cause morbidity and mortality within the human population despite widespread vaccination efforts. This, along with the ominous threat of an avian influenza pandemic (H5N1, demonstrates the need for a much improved, more sophisticated influenza vaccine. We have developed an in vitro model system for producing a membrane-bound Cytokine-bearing Influenza Vaccine (CYT-IVAC. Numerous cytokines are involved in directing both innate and adaptive immunity and it is our goal to utilize the properties of individual cytokines and other immunomodulatory proteins to create a more immunogenic vaccine. Results We have evaluated the immunogenicity of inactivated cytokine-bearing influenza vaccines using a mouse model of lethal influenza virus challenge. CYT-IVACs were produced by stably transfecting MDCK cell lines with mouse-derived cytokines (GM-CSF, IL-2 and IL-4 fused to the membrane-anchoring domain of the viral hemagglutinin. Influenza virus replication in these cell lines resulted in the uptake of the bioactive membrane-bound cytokines during virus budding and release. In vivo efficacy studies revealed that a single low dose of IL-2 or IL-4-bearing CYT-IVAC is superior at providing protection against lethal influenza challenge in a mouse model and provides a more balanced Th1/Th2 humoral immune response, similar to live virus infections. Conclusion We have validated the protective efficacy of CYT-IVACs in a mammalian model of influenza virus infection. This technology has broad applications in current influenza virus vaccine development and may prove particularly useful in boosting immune responses in the elderly, where current vaccines are minimally effective.

Causal boundary for stably causal space-times

International Nuclear Information System (INIS)

Racz, I.

1987-12-01

The usual boundary constructions for space-times often yield an unsatisfactory boundary set. This problem is reviewed and a new solution is proposed. An explicit identification rule is given on the set of the ideal points of the space-time. This construction leads to a satisfactory boundary point set structure for stably causal space-times. The topological properties of the resulting causal boundary construction are examined. For the stably causal space-times each causal curve has a unique endpoint on the boundary set according to the extended Alexandrov topology. The extension of the space-time through the boundary is discussed. To describe the singularities the defined boundary sets have to be separated into two disjoint sets. (D.Gy.) 8 refs

Nanoscale observation of local bound charges of patterned protein arrays by scanning force microscopy

International Nuclear Information System (INIS)

Oh, Y J; Jo, W; Kim, S; Park, S; Kim, Y S

2008-01-01

A protein patterned surface using micro-contact printing methods has been investigated by scanning force microscopy. Electrostatic force microscopy (EFM) was utilized for imaging the topography and detecting the electrical properties such as the local bound charge distribution of the patterned proteins. It was found that the patterned IgG proteins are arranged down to 1 μm, and the 90 deg. rotation of patterned anti-IgG proteins was successfully undertaken. Through the estimation of the effective areas, it was possible to determine the local bound charges of patterned proteins which have opposite electrostatic force behaviors. Moreover, we studied the binding probability between IgG and anti-IgG in a 1 μm 2 MIMIC system by topographic and electrostatic signals for applicable label-free detections. We showed that the patterned proteins can be used for immunoassay of proteins on the functional substrate, and that they can also be used for bioelectronics device application, indicating distinct advantages with regard to accuracy and a label-free detection

Visualization of mole fraction distribution of slow jet forming stably stratified field

International Nuclear Information System (INIS)

Fumizawa, Motoo; Hishida, Makoto

1990-01-01

An experimental study has been performed to investigate the behavior of flow and mass transfer in gaseous slow jet in which buoyancy force opposed the flow forming stably stratified field. The study has been performed to understand the basic features of air ingress phenomena at pipe rupture accident of the high temperature gas-cooled reactor. A displacement fringe technique was adopted in Mach-Zehnder interferometer to visualize the mole fraction distribution. As the result, the followings were obtained: (1) The stably stratified fields were formed in the vicinity of the outlet of the slow jet. The penetration distance of the stably stratified fields increased with Froude number. (2) Mass fraction distributions in the stably stratified fields were well correlated with the present model using the ramp mole velocity profile. (author)

Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload

Science.gov (United States)

Kuipers, Marjorie A.; Stasevich, Timothy J.; Sasaki, Takayo; Wilson, Korey A.; Hazelwood, Kristin L.; McNally, James G.; Davidson, Michael W.

2011-01-01

The heterohexameric minichromosome maintenance protein complex (Mcm2-7) functions as the eukaryotic helicase during DNA replication. Mcm2-7 loads onto chromatin during early G1 phase but is not converted into an active helicase until much later during S phase. Hence, inactive Mcm complexes are presumed to remain stably bound from early G1 through the completion of S phase. Here, we investigated Mcm protein dynamics in live mammalian cells. We demonstrate that Mcm proteins are irreversibly loaded onto chromatin cumulatively throughout G1 phase, showing no detectable exchange with a gradually diminishing soluble pool. Eviction of Mcm requires replication; during replication arrest, Mcm proteins remained bound indefinitely. Moreover, the density of immobile Mcms is reduced together with chromatin decondensation within sites of active replication, which provides an explanation for the lack of colocalization of Mcm with replication fork proteins. These results provide in vivo evidence for an exceptionally stable lockdown mechanism to retain all loaded Mcm proteins on chromatin throughout prolonged cell cycles. PMID:21220507

Convective transport of highly plasma protein bound drugs facilitates direct penetration into deep tissues after topical application

Science.gov (United States)

Dancik, Yuri; Anissimov, Yuri G; Jepps, Owen G; Roberts, Michael S

2012-01-01

AIMS To relate the varying dermal, subcutaneous and muscle microdialysate concentrations found in man after topical application to the nature of the drug applied and to the underlying physiology. METHODS We developed a physiologically based pharmacokinetic model in which transport to deeper tissues was determined by tissue diffusion, blood, lymphatic and intersitial flow transport and drug properties. The model was applied to interpret published human microdialysis data, estimated in vitro dermal diffusion and protein binding affinity of drugs that have been previously applied topically in vivo and measured in deep cutaneous tissues over time. RESULTS Deeper tissue microdialysis concentrations for various drugs in vivo vary widely. Here, we show that carriage by the blood to the deeper tissues below topical application sites facilitates the transport of highly plasma protein bound drugs that penetrate the skin, leading to rapid and significant concentrations in those tissues. Hence, the fractional concentration for the highly plasma protein bound diclofenac in deeper tissues is 0.79 times that in a probe 4.5 mm below a superficial probe whereas the corresponding fractional concentration for the poorly protein bound nicotine is 0.02. Their corresponding estimated in vivo lag times for appearance of the drugs in the deeper probes were 1.1 min for diclofenac and 30 min for nicotine. CONCLUSIONS Poorly plasma protein bound drugs are mainly transported to deeper tissues after topical application by tissue diffusion whereas the transport of highly plasma protein bound drugs is additionally facilitated by convective blood, lymphatic and interstitial transport to deep tissues. PMID:21999217

Measuring binding of protein to gel-bound ligands using magnetic levitation.

Science.gov (United States)

Shapiro, Nathan D; Mirica, Katherine A; Soh, Siowling; Phillips, Scott T; Taran, Olga; Mace, Charles R; Shevkoplyas, Sergey S; Whitesides, George M

2012-03-28

This paper describes the use of magnetic levitation (MagLev) to measure the association of proteins and ligands. The method starts with diamagnetic gel beads that are functionalized covalently with small molecules (putative ligands). Binding of protein to the ligands within the bead causes a change in the density of the bead. When these beads are suspended in a paramagnetic aqueous buffer and placed between the poles of two NbFeB magnets with like poles facing, the changes in the density of the bead on binding of protein result in changes in the levitation height of the bead that can be used to quantify the amount of protein bound. This paper uses a reaction-diffusion model to examine the physical principles that determine the values of rate and equilibrium constants measured by this system, using the well-defined model system of carbonic anhydrase and aryl sulfonamides. By tuning the experimental protocol, the method is capable of quantifying either the concentration of protein in a solution, or the binding affinities of a protein to several resin-bound small molecules simultaneously. Since this method requires no electricity and only a single piece of inexpensive equipment, it may find use in situations where portability and low cost are important, such as in bioanalysis in resource-limited settings, point-of-care diagnosis, veterinary medicine, and plant pathology. It still has several practical disadvantages. Most notably, the method requires relatively long assay times and cannot be applied to large proteins (>70 kDa), including antibodies. The design and synthesis of beads with improved characteristics (e.g., larger pore size) has the potential to resolve these problems.

Ligand-free, protein-bound technetium-99m. Evidence for tumour localisation

International Nuclear Information System (INIS)

Jakovljevic, A.C.; Pojer, P.M.

1984-11-01

An hypothesis that cations accumulate in tumours independent of ligand is tested. A preparation of technetium-99m known to be ligand-free (that is, the technetium is protein bound and no other ligand is injected) has been shown to accumulate in a T-cell lymphoma

Photochemistry of triarylmethane dyes bound to proteins

Science.gov (United States)

Indig, Guilherme L.

1996-04-01

Triarylmethanes represent a class of cationic dyes whose potential as photosensitizers for use in photodynamic therapy of neoplastic diseases has never been comprehensively evaluated. Here, the laser-induced photodecomposition of three triarylmethane dyes, crystal violet, ethyl violet, and malachite green, non-covalently bound to bovine serum albumin (a model biological target) was investigated. Upon laser excitation at 532 nm, the bleaching of the corresponding dye-protein molecular complexes follows spectroscopic patterns that suggest the formation of reduced forms of the dyes as major reaction photoproducts. That implies that an electron or hydrogen atom transfer from the protein to the dye's moiety within the guest-host complex is the first step of the photobleaching process. Since the availability of dissolved molecular oxygen was not identified as a limiting factor for the phototransformations to occur, these dyes can be seen as potential phototherapeutic agents for use in hypoxic areas of tumors. These triarylmethane dyes strongly absorb at relatively long wavelengths (absorption maximum around 600 nm; (epsilon) max approximately equals 105 M-1 cm-1), and only minor changes in their absorption characteristics are observed upon binding to the protein. However the binding event leads to a remarkable increase in their fluorescence quantum yield and photoreactivity.

Interaction of angiotensin-converting enzyme (ACE) with membrane-bound carboxypeptidase M (CPM) - a new function of ACE.

Science.gov (United States)

Sun, Xiaoou; Wiesner, Burkhard; Lorenz, Dorothea; Papsdorf, Gisela; Pankow, Kristin; Wang, Po; Dietrich, Nils; Siems, Wolf-Eberhard; Maul, Björn

2008-12-01

Angiotensin-converting enzyme (ACE) demonstrates, besides its typical dipeptidyl-carboxypeptidase activity, several unusual functions. Here, we demonstrate with molecular, biochemical, and cellular techniques that the somatic wild-type murine ACE (mACE), stably transfected in Chinese Hamster Ovary (CHO) or Madin-Darby Canine Kidney (MDCK) cells, interacts with endogenous membranal co-localized carboxypeptidase M (CPM). CPM belongs to the group of glycosylphosphatidylinositol (GPI)-anchored proteins. Here we report that ACE, completely independent of its known dipeptidase activities, has GPI-targeted properties. Our results indicate that the spatial proximity between mACE and the endogenous CPM enables an ACE-evoked release of CPM. These results are discussed with respect to the recently proposed GPI-ase activity and function of sperm-bound ACE.

Comparative LC-MS/MS profiling of free and protein-bound early and advanced glycation-induced lysine modifications in dairy products

International Nuclear Information System (INIS)

Hegele, Joerg; Buetler, Timo; Delatour, Thierry

2008-01-01

Free and protein-bound forms of early and advanced glycation-induced lysine (Lys) modifications were quantified in dairy products by LC-MS/MS using a stable isotope dilution assay. The glycation profiles for N ε -fructoselysine (FL), N ε -carboxymethyllysine (CML) and pyrraline (Pyr) were monitored in raw and processed cow milk to investigate whether free glycation products could serve as fast and simple markers to assess the extent of protein glycation in dairy products. In all milk samples, the fraction of free glycation adducts was predominantly composed of advanced modifications, e.g. 8.34 ± 3.81 nmol CML per μmol of free Lys (Lys free ) and 81.5 ± 87.8 nmol Pyr μmol -1 Lys free -1 vs. 3.72 ± 1.29 nmol FL μmol -1 Lys free -1 . In contrast, the protein-bound early glycation product FL considerably outweighed the content of CML and Pyr in milk proteins of raw and processed cow milk, whereas severely heat treated milk products, e.g. condensed milk, contained a higher amount of protein-bound advanced glycation adducts. Typical values recorded for milk samples processed under mild conditions were 0.47 ± 0.08 nmol FL μmol -1 of protein-bound Lys (Lys p-b ), 0.04 ± 0.03 nmol CML μmol -1 Lys p-b -1 and 0.06 ± 0.02 nmol Pyr μmol -1 Lys p-b -1 . It was particularly noticeable, however, that mild heat treatment of raw milk, i.e. pasteurization and UHT treatment, did not significantly increase the amount of both free and protein-bound Lys modifications. In conclusion, the profiles of free and protein-bound glycation-induced Lys modifications were found to be different and a screening of free glycation adducts does, therefore, not allow for a conclusion about the protein glycation status of dairy products

Structures of holo wild-type human cellular retinol-binding protein II (hCRBPII) bound to retinol and retinal.

Science.gov (United States)

Nossoni, Zahra; Assar, Zahra; Yapici, Ipek; Nosrati, Meisam; Wang, Wenjing; Berbasova, Tetyana; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James

2014-12-01

Cellular retinol-binding proteins (CRBPs) I and II, which are members of the intracellular lipid-binding protein (iLBP) family, are retinoid chaperones that are responsible for the intracellular transport and delivery of both retinol and retinal. Although structures of retinol-bound CRBPI and CRBPII are known, no structure of a retinal-bound CRBP has been reported. In addition, the retinol-bound human CRBPII (hCRBPII) structure shows partial occupancy of a noncanonical conformation of retinol in the binding pocket. Here, the structure of retinal-bound hCRBPII and the structure of retinol-bound hCRBPII with retinol fully occupying the binding pocket are reported. It is further shown that the retinoid derivative seen in both the zebrafish CRBP and the hCRBPII structures is likely to be the product of flux-dependent and wavelength-dependent X-ray damage during data collection. The structures of retinoid-bound CRBPs are compared and contrasted, and rationales for the differences in binding affinities for retinal and retinol are provided.

Evidence against the involvement of ionically bound cell wall proteins in pea epicotyl growth

Science.gov (United States)

Melan, M. A.; Cosgrove, D. J.

1988-01-01

Ionically bound cell wall proteins were extracted from 7 day old etiolated pea (Pisum sativum L. cv Alaska) epicotyls with 3 molar LiCl. Polyclonal antiserum was raised in rabbits against the cell wall proteins. Growth assays showed that treatment of growing region segments (5-7 millimeters) of peas with either dialyzed serum, serum globulin fraction, affinity purified immunoglobulin, or papain-cleaved antibody fragments had no effect on growth. Immunofluorescence microscopy confirmed antibody binding to cell walls and penetration of the antibodies into the tissues. Western blot analysis, immunoassay results, and affinity chromatography utilizing Sepharose-bound antibodies confirmed recognition of the protein preparation by the antibodies. Experiments employing in vitro extension as a screening measure indicated no effect upon extension by antibodies, by 50 millimolar LiCl perfusion of the apoplast or by 3 molar LiCl extraction. Addition of cell wall protein to protease pretreated segments did not restore extension nor did addition of cell wall protein to untreated segments increase extension. It is concluded that, although evidence suggests that protein is responsible for the process of extension, the class(es) of proteins which are extracted from pea cell walls with 3 molar LiCl are probably not involved in this process.

Proteomic Analysis to Identify Tightly-Bound Cell Wall Protein in Rice Calli

OpenAIRE

Cho, Won Kyong; Hyun, Tae Kyung; Kumar, Dhinesh; Rim, Yeonggil; Chen, Xiong Yan; Jo, Yeonhwa; Kim, Suwha; Lee, Keun Woo; Park, Zee-Yong; Lucas, William J.; Kim, Jae-Yean

2015-01-01

Rice is a model plant widely used for basic and applied research programs. Plant cell wall proteins play key roles in a broad range of biological processes. However, presently, knowledge on the rice cell wall proteome is rudimentary in nature. In the present study, the tightly-bound cell wall proteome of rice callus cultured cells using sequential extraction protocols was developed using mass spectrometry and bioinformatics methods, leading to the identification of 1568 candidate proteins. Ba...

Utilization of crystalline and protein-bound amino acids by growing-finishing pigs

DEFF Research Database (Denmark)

Nørgaard, Jan Værum; Buxant, L.; Clausen, D.

2016-01-01

It was hypothesized that diets containing crystalline AA (CAA) and protein-bound AA had a comparable nitrogen retention rate, even though the CAA-based diet is optimized as having a standardized ileal digestibility (SID) of 100% for the CAA. Two isoenergetic diets were formulated to provide ident...

Backbone resonance assignments for G protein α(i3) subunit in the GDP-bound state.

Science.gov (United States)

Mase, Yoko; Yokogawa, Mariko; Osawa, Masanori; Shimada, Ichio

2014-10-01

Guanine-nucleotide binding proteins (G proteins) serve as molecular switches in signaling pathways, by coupling the activation of G protein-coupled receptors (GPCRs) at the cell surface to intracellular responses. In the resting state, G protein forms a heterotrimer, consisting of the G protein α subunit with GDP (Gα·GDP) and the G protein βγ subunit (Gβγ). Ligand binding to GPCRs promotes the GDP-GTP exchange on Gα, leading to the dissociation of the GTP-bound form of Gα (Gα·GTP) and Gβγ. Then, Gα·GTP and Gβγ bind to their downstream effector enzymes or ion channels and regulate their activities, leading to a variety of cellular responses. Finally, Gα hydrolyzes the bound GTP to GDP and returns to the resting state by re-associating with Gβγ. The G proteins are classified with four major families based on the amino acid sequences of Gα: i/o, s, q/11, and 12/13. Here, we established the backbone resonance assignments of human Gαi3, a member of the i/o family with a molecular weight of 41 K, in complex with GDP. The chemical shifts were compared with those of Gα(i3) in complex with a GTP-analogue, GTPγS, which we recently reported, indicating that the residues with significant chemical shift differences are mostly consistent with the regions with the structural differences between the GDP- and GTPγS-bound states, as indicated in the crystal structures. The assignments of Gα(i3)·GDP would be useful for the analyses of the dynamics of Gα(i3) and its interactions with various target molecules.

Using isoelectric focussing and neutron activation analysis to study protein-bound tracer elements

International Nuclear Information System (INIS)

Schmelzer, W.

1976-01-01

A method to determine protein-bound tracer elements was determined by combining a protein separation method with neutron activation analysis. Gel filtration, disk electrophoresis, and isoelectric focussing were studied with regard to their suitability as separation methods. Using isoelectric focussing, human serum protein could be separated with good resolution on a preparative scale. The Se, Cr, Ag, Sc, Fe, Zn, Co, Br, Na, Rb, and Cs contents of the various protein fractions were determined by instrumental neutron activation analysis and by gamma-spectroscopic identification of their long-lived nuclides. Particular attention was paid to the main source of error with this method, i.e. contamination of the proteins in the course of the separation process. Information on the binding of the elements to protein was obtained by comparing the contents in the serum and in the protein separated by gel chromatography. For example, 75% of the Se and 30% of the Cs are bound to protein. Contamination of the protein fractions was studied by means of tracer elements with the element Se, errors due to contamination could be ruled out. The method was modified for the special imvestigation of Se-protein complexes in the serum. The Se content was determined by activation analysis via the short-lived radionuclide sup(77m)Se, this considerably reducing the duration of analysis. With regard to focussing, discrimination was improved in the pH region in which specific Se complexes were found. The activity distribution in fractionated serum protein labelled in vitro with 75 Se in the presence of erythrocytes showed that specific labelling is possible in this way. It is thus possible to study the distribution of Se carrier proteins with the aid of a radiotracer technique. (orig./RB) [de

Uniform Recovery Bounds for Structured Random Matrices in Corrupted Compressed Sensing

Science.gov (United States)

Zhang, Peng; Gan, Lu; Ling, Cong; Sun, Sumei

2018-04-01

We study the problem of recovering an $s$-sparse signal $\\mathbf{x}^{\\star}\\in\\mathbb{C}^n$ from corrupted measurements $\\mathbf{y} = \\mathbf{A}\\mathbf{x}^{\\star}+\\mathbf{z}^{\\star}+\\mathbf{w}$, where $\\mathbf{z}^{\\star}\\in\\mathbb{C}^m$ is a $k$-sparse corruption vector whose nonzero entries may be arbitrarily large and $\\mathbf{w}\\in\\mathbb{C}^m$ is a dense noise with bounded energy. The aim is to exactly and stably recover the sparse signal with tractable optimization programs. In this paper, we prove the uniform recovery guarantee of this problem for two classes of structured sensing matrices. The first class can be expressed as the product of a unit-norm tight frame (UTF), a random diagonal matrix and a bounded columnwise orthonormal matrix (e.g., partial random circulant matrix). When the UTF is bounded (i.e. $\\mu(\\mathbf{U})\\sim1/\\sqrt{m}$), we prove that with high probability, one can recover an $s$-sparse signal exactly and stably by $l_1$ minimization programs even if the measurements are corrupted by a sparse vector, provided $m = \\mathcal{O}(s \\log^2 s \\log^2 n)$ and the sparsity level $k$ of the corruption is a constant fraction of the total number of measurements. The second class considers randomly sub-sampled orthogonal matrix (e.g., random Fourier matrix). We prove the uniform recovery guarantee provided that the corruption is sparse on certain sparsifying domain. Numerous simulation results are also presented to verify and complement the theoretical results.

A method for analysing small samples of floral pollen for free and protein-bound amino acids.

Science.gov (United States)

Stabler, Daniel; Power, Eileen F; Borland, Anne M; Barnes, Jeremy D; Wright, Geraldine A

2018-02-01

Pollen provides floral visitors with essential nutrients including proteins, lipids, vitamins and minerals. As an important nutrient resource for pollinators, including honeybees and bumblebees, pollen quality is of growing interest in assessing available nutrition to foraging bees. To date, quantifying the protein-bound amino acids in pollen has been difficult and methods rely on large amounts of pollen, typically more than 1 g. More usual is to estimate a crude protein value based on the nitrogen content of pollen, however, such methods provide no information on the distribution of essential and non-essential amino acids constituting the proteins.Here, we describe a method of microwave-assisted acid hydrolysis using low amounts of pollen that allows exploration of amino acid composition, quantified using ultra high performance liquid chromatography (UHPLC), and a back calculation to estimate the crude protein content of pollen.Reliable analysis of protein-bound and free amino acids as well as an estimation of crude protein concentration was obtained from pollen samples as low as 1 mg. Greater variation in both protein-bound and free amino acids was found in pollen sample sizes amino acids in smaller sample sizes, we suggest a correction factor to apply to specific sample sizes of pollen in order to estimate total crude protein content.The method described in this paper will allow researchers to explore the composition of amino acids in pollen and will aid research assessing the available nutrition to pollinating animals. This method will be particularly useful in assaying the pollen of wild plants, from which it is difficult to obtain large sample weights.

Comparative LC-MS/MS profiling of free and protein-bound early and advanced glycation-induced lysine modifications in dairy products

Energy Technology Data Exchange (ETDEWEB)

Hegele, Joerg [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland)], E-mail: joerg.hegele@rdls.nestle.com; Buetler, Timo; Delatour, Thierry [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland)

2008-06-09

Free and protein-bound forms of early and advanced glycation-induced lysine (Lys) modifications were quantified in dairy products by LC-MS/MS using a stable isotope dilution assay. The glycation profiles for N{sup {epsilon}}-fructoselysine (FL), N{sup {epsilon}}-carboxymethyllysine (CML) and pyrraline (Pyr) were monitored in raw and processed cow milk to investigate whether free glycation products could serve as fast and simple markers to assess the extent of protein glycation in dairy products. In all milk samples, the fraction of free glycation adducts was predominantly composed of advanced modifications, e.g. 8.34 {+-} 3.81 nmol CML per {mu}mol of free Lys (Lys{sub free}) and 81.5 {+-} 87.8 nmol Pyr {mu}mol{sup -1} Lys{sub free}{sup -1} vs. 3.72 {+-} 1.29 nmol FL {mu}mol{sup -1} Lys{sub free}{sup -1}. In contrast, the protein-bound early glycation product FL considerably outweighed the content of CML and Pyr in milk proteins of raw and processed cow milk, whereas severely heat treated milk products, e.g. condensed milk, contained a higher amount of protein-bound advanced glycation adducts. Typical values recorded for milk samples processed under mild conditions were 0.47 {+-} 0.08 nmol FL {mu}mol{sup -1} of protein-bound Lys (Lys{sub p-b}), 0.04 {+-} 0.03 nmol CML {mu}mol{sup -1} Lys{sub p-b}{sup -1} and 0.06 {+-} 0.02 nmol Pyr {mu}mol{sup -1} Lys{sub p-b}{sup -1}. It was particularly noticeable, however, that mild heat treatment of raw milk, i.e. pasteurization and UHT treatment, did not significantly increase the amount of both free and protein-bound Lys modifications. In conclusion, the profiles of free and protein-bound glycation-induced Lys modifications were found to be different and a screening of free glycation adducts does, therefore, not allow for a conclusion about the protein glycation status of dairy products.

Comparison of methods for determination of testosterone and non-protein bound testosterone in men with alcoholic liver disease

DEFF Research Database (Denmark)

Gluud, C; Bennett, Patrick

1986-01-01

The serum concentrations of testosterone and of non-protein bound testosterone were determined in 28 men with alcoholic liver disease having normal to decreased serum albumin concentrations and normal to raised SHBG concentrations. Serum testosterone concentrations determined with two...... radioimmunoassays using different purification procedures and antibody batches did not differ significantly and correlated significantly (r=0.91; p less than 0.001). The median serum concentration of non-protein bound testosterone was 0.265 nmol/l (range 0.068-0.495 nmol/l) when determined by equilibrium dialysis...... and 0.232 nmol/l (range 0.042-0.610 nmol/l) when calculated according to the law of mass action. This difference is insignificant. The concentrations of non-protein bound testosterone determined by the two methods correlated significantly (r=0.83; p less than 0.001). In the calculation of non...

UVA photolysis using the protein-bound sensitizers present in human lens

International Nuclear Information System (INIS)

Ortwerth, B.J.; Olesen, P.R.

1994-01-01

This research was undertaken to demonstrate that the protein-bound chromophores in aged human lens can act as sensitizers for protein damage by UVA light. The water-insoluble (WI) proteins from pooled human and bovine lenses were solubilized by sonication in water and illuminated with UV light similar in output to that transmitted by the cornea. Analysis of the irradiated proteins showed a linear decrease in sulfhydryl groups with a 30% loss after 2 h. No loss was seen when native α-crystallin was irradiated under the same conditions. A 25% loss of histidine residues was also observed with the human lens WI fraction, and sodium dodecyl sulfate polyacrylamide gels indicated considerable protein cross-linking. Similar photodamage was seen with a WI fraction from old bovine lenses. While the data show the presence of UVA sensitizers, some histidine destruction and protein cross-linking were also obtained with α-crystallin and with lysozyme which argue that part of the histidine loss in the human WISS was likely due to tryptophan acting as a sensitizer. (Author)

Crystal Structures of SlyA Protein, a Master Virulence Regulator of Salmonella, in Free and DNA-bound States

Energy Technology Data Exchange (ETDEWEB)

Dolan, Kyle T.; Duguid, Erica M.; He, Chuan (UC)

2011-11-17

SlyA is a master virulence regulator that controls the transcription of numerous genes in Salmonella enterica. We present here crystal structures of SlyA by itself and bound to a high-affinity DNA operator sequence in the slyA gene. SlyA interacts with DNA through direct recognition of a guanine base by Arg-65, as well as interactions between conserved Arg-86 and the minor groove and a large network of non-base-specific contacts with the sugar phosphate backbone. Our structures, together with an unpublished structure of SlyA bound to the small molecule effector salicylate (Protein Data Bank code 3DEU), reveal that, unlike many other MarR family proteins, SlyA dissociates from DNA without large conformational changes when bound to this effector. We propose that SlyA and other MarR global regulators rely more on indirect readout of DNA sequence to exert control over many genes, in contrast to proteins (such as OhrR) that recognize a single operator.

Protein-bound solute removal during extended multipass versus standard hemodialysis

DEFF Research Database (Denmark)

Eloot, Sunny; Van Biesen, Wim; Axelsen, Mette

2015-01-01

and middle molecules compared to standard hemodialysis (SHD). Since protein-bound solutes (PBS) exert important pathophysiological effects, we investigated whether MPHD results in improved removal of PBS as well. METHODS: A cross-over study (Clinical Trial NCT01267760) was performed in nine stable HD......), hippuric acid (HA), indole acetic acid (IAA), indoxyl sulfate (IS), and p-cresylsulfate (PCS). Dialyser extraction ratio, reduction ratio, and solute removal were calculated for these solutes. RESULTS: Already at 60 min after dialysis start, the extraction ratio in the hemodialyser was a factor 1.4-4 lower...

Competition between bound and free peptides in an ELISA-based procedure that assays peptides derived from protein digests

Directory of Open Access Journals (Sweden)

Pace Umberto

2006-05-01

Full Text Available Abstract Background We describe an ELISA-based method that can be used to identify and quantitate proteins in biological samples. In this method, peptides in solution, derived from proteolytic digests of the sample, compete with substrate-attached synthetic peptides for antibodies, also in solution, generated against the chosen peptides. The peptides used for the ELISA are chosen on the basis of their being (i products of the proteolytic (e.g. tryptic digestion of the protein to be identified and (ii unique to the target protein, as far as one can know from the published sequences. Results In this paper we describe the competition assay and we define the optimal conditions for the most effective assay. We have performed an analysis of the kinetics of interaction between the four components of the assay: the plastic substratum to which the peptide is bound, the bound peptide itself, the competing added peptide, and the antibody that is specific for the peptide and we compare the results of theoretical simulations to the actual data in some model systems. Conclusion The data suggest that the peptides bind to the plastic substratum in more than one conformation and that, once bound, the peptide displays different affinities for the antibody, depending on how it has bound to the plate

How to calculate clearance of highly protein-bound drugs during continuous venovenous hemofiltration demonstrated with flucloxacillin.

Science.gov (United States)

Meyer, Brigitte; Ahmed el Gendy, Salwa; Delle Karth, Georg; Locker, Gottfried J; Heinz, Gottfried; Jaeger, Walter; Thalhammer, Florian

2003-01-01

Flucloxacillin is an important antimicrobial drug in the treatment of infections with Staphylococcus aureus and therefore is often used in staphylococcal infections. Furthermore, flucloxacillin has a high protein binding rate as for example ceftriaxone or teicoplanin--drugs which have formerly been characterized as not being dialyzable. The pharmacokinetic parameters of 4.0 g flucloxacillin every 8 h were examined in 10 intensive care patients during continuous venovenous hemofiltration (CVVH) using a polyamide capillary hemofilter. In addition, the difficulty of calculating the hemofiltration clearance of a highly protein-bound drug is described. Flucloxacillin serum levels were significantly lowered (56.9 +/- 24.0%) even though only 15% of the drug was detected in the ultrafiltrate. Elimination half-life, total body clearance and sieving coefficient were 4.9 +/- 0.7 h, 117.2 +/- 79.1 ml/min and 0.21 +/- 0.09, respectively. These discrepancies can be explained by the high protein binding of flucloxacillin, the adsorbing property of polyamide and the equation in order to calculate hemofiltration clearance. The unbound fraction of a 4.0 g flucloxacillin dosage facilitates time above the minimum inhibitory concentration (T > MIC) of 60% only for strains up to a minimum inhibitory concentration (MIC) of 0.5 mg/l. Based on the data of this study, we conclude that intensive care patients with staphylococcal infections on CVVH should be treated with 4.0 g flucloxacillin every 8 h which was safe and well tolerated. Moreover, further studies with highly protein-bound drugs are recommended to check the classical 'hemodialysis' equation as the standard equation in calculating the CVVH clearance of highly protein-bound drugs. Copyright 2003 S. Karger AG, Basel

Crystal structure of axolotl (Ambystoma mexicanum) liver bile acid-binding protein bound to cholic and oleic acid.

Science.gov (United States)

Capaldi, Stefano; Guariento, Mara; Perduca, Massimiliano; Di Pietro, Santiago M; Santomé, José A; Monaco, Hugo L

2006-07-01

The family of the liver bile acid-binding proteins (L-BABPs), formerly called liver basic fatty acid-binding proteins (Lb-FABPs) shares fold and sequence similarity with the paralogous liver fatty acid-binding proteins (L-FABPs) but has a different stoichiometry and specificity of ligand binding. This article describes the first X-ray structure of a member of the L-BABP family, axolotl (Ambystoma mexicanum) L-BABP, bound to two different ligands: cholic and oleic acid. The protein binds one molecule of oleic acid in a position that is significantly different from that of either of the two molecules that bind to rat liver FABP. The stoichiometry of binding of cholate is of two ligands per protein molecule, as observed in chicken L-BABP. The cholate molecule that binds buried most deeply into the internal cavity overlaps well with the analogous bound to chicken L-BABP, whereas the second molecule, which interacts with the first only through hydrophobic contacts, is more external and exposed to the solvent. (c) 2006 Wiley-Liss, Inc.

Modeling the Conducting Stably-Stratified Layer of the Earth's Core

Science.gov (United States)

Petitdemange, L.; Philidet, J.; Gissinger, C.

2017-12-01

Observations of the Earth magnetic field as well as recent theoretical works tend to show that the Earth's outer liquid core is mostly comprised of a convective zone in which the Earth's magnetic field is generated - likely by dynamo action -, but also features a thin, stably stratified layer at the top of the core.We carry out direct numerical simulations by modeling this thin layer as an axisymmetric spherical Couette flow for a stably stratified fluid embedded in a dipolar magnetic field. The dynamo region is modeled by a conducting inner core rotating slightly faster than the insulating mantle due to magnetic torques acting on it, such that a weak differential rotation (low Rossby limit) can develop in the stably stratified layer.In the case of a non-stratified fluid, the combined action of the differential rotation and the magnetic field leads to the well known regime of `super-rotation', in which the fluid rotates faster than the inner core. Whereas in the classical case, this super-rotation is known to vanish in the magnetostrophic limit, we show here that the fluid stratification significantly extends the magnitude of the super-rotation, keeping this phenomenon relevant for the Earth core. Finally, we study how the shear layers generated by this new state might give birth to magnetohydrodynamic instabilities or waves impacting the secular variations or jerks of the Earth's magnetic field.

Molecular Cloning and Characterization of Growth Factor Receptor Bound-Protein in Clonorchis sinensis

Science.gov (United States)

Bai, Xuelian; Lee, Ji-Yun; Kim, Tae Im; Dai, Fuhong; Lee, Tae-Jin; Hong, Sung-Jong

2014-01-01

Background Clonorchis sinensis causes clonorchiasis, a potentially serious disease. Growth factor receptor-bound protein 2 (Grb2) is a cytosolic protein conserved among animals and plays roles in cellular functions such as meiosis, organogenesis and energy metabolism. In the present study, we report first molecular characters of growth factor receptor bound-protein (CsGrb2) from C. sinensis as counter part of Grb2 from animals and its possible functions in development and organogenesis of C. sinensis. Methodology/Principal Findings A CsGrb2 cDNA clone retrieved from the C. sinensis transcriptome encoded a polypeptide with a SH3-SH2-SH3 structure. Recombinant CsGrb2 was bacterially produced and purified to homogeneity. Native CsGrb2 with estimated molecular weight was identified from C. sinensis adult extract by western blotting using a mouse immune serum to recombinant CsGrb2. CsGrb2 transcripts was more abundant in the metacercariae than in the adults. Immunohistochemical staining showed that CsGrb2 was localized to the suckers, mesenchymal tissues, sperms in seminal receptacle and ovary in the adults, and abundantly expressed in most organs of the metacercariae. Recombinant CsGrb2 was evaluated to be little useful as a serodiagnostic reagent for C. sinesis human infections. Conclusion Grb2 protein found in C. sinensis was conserved among animals and suggested to play a role in the organogenesis, energy metabolism and mitotic spermatogenesis of C. sinensis. These findings from C. sinensis provide wider understanding on diverse function of Grb2 in lower animals such as platyhelminths. PMID:24454892

Molecular cloning and characterization of growth factor receptor bound-protein in Clonorchis sinensis.

Directory of Open Access Journals (Sweden)

Xuelian Bai

Full Text Available BACKGROUND: Clonorchis sinensis causes clonorchiasis, a potentially serious disease. Growth factor receptor-bound protein 2 (Grb2 is a cytosolic protein conserved among animals and plays roles in cellular functions such as meiosis, organogenesis and energy metabolism. In the present study, we report first molecular characters of growth factor receptor bound-protein (CsGrb2 from C. sinensis as counter part of Grb2 from animals and its possible functions in development and organogenesis of C. sinensis. METHODOLOGY/PRINCIPAL FINDINGS: A CsGrb2 cDNA clone retrieved from the C. sinensis transcriptome encoded a polypeptide with a SH3-SH2-SH3 structure. Recombinant CsGrb2 was bacterially produced and purified to homogeneity. Native CsGrb2 with estimated molecular weight was identified from C. sinensis adult extract by western blotting using a mouse immune serum to recombinant CsGrb2. CsGrb2 transcripts was more abundant in the metacercariae than in the adults. Immunohistochemical staining showed that CsGrb2 was localized to the suckers, mesenchymal tissues, sperms in seminal receptacle and ovary in the adults, and abundantly expressed in most organs of the metacercariae. Recombinant CsGrb2 was evaluated to be little useful as a serodiagnostic reagent for C. sinesis human infections. CONCLUSION: Grb2 protein found in C. sinensis was conserved among animals and suggested to play a role in the organogenesis, energy metabolism and mitotic spermatogenesis of C. sinensis. These findings from C. sinensis provide wider understanding on diverse function of Grb2 in lower animals such as platyhelminths.

Turbulent circulation above the surface heat source in stably stratified atmosphere

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Kurbatskii, A. F.; Kurbatskaya, L. I.

2016-10-01

The 3-level RANS approach for simulating a turbulent circulation over the heat island in a stably stratified environment under nearly calm conditions is formulated. The turbulent kinetic energy its spectral consumption (dissipation) and the dispersion of turbulent fluctuations of temperature are found from differential equations, thus the correct modeling of transport processes in the interface layer with the counter-gradient heat flux is assured. The three-parameter turbulence RANS approach minimizes difficulties in simulating the turbulent transport in a stably stratified environment and reduces efforts needed for the numerical implementation of the 3-level RANS approach. Numerical simulation of the turbulent structure of the penetrative convection over the heat island under conditions of stably stratified atmosphere demonstrates that the three-equation model is able to predict the thermal circulation induced by the heat island. The temperature distribution, root-mean-square fluctuations of the turbulent velocity and temperature fields and spectral turbulent kinetic energy flux are in good agreement with the experimental data. The model describes such thin physical effects, as a crossing of vertical profiles of temperature of a thermal plume with the formation of the negative buoyancy area testifying to development of the dome-shaped form at the top part of a plume in the form of "hat".

Absorption and retention of free and milk protein-bound cyano- and hydroxocobalamins

DEFF Research Database (Denmark)

Kornerup, Linda Skibsted; Juul, Christian Bredgaard; Fedosov, Sergey

2016-01-01

alone or bound to milk protein. Materials and methods We synthesized labeled OH[57Co]Cbl from commercially available CN[57Co]Cbl. Recombinant bovine transcobalamin (rbTC) was produced in yeast and skimmed milk obtained off the shelf. Male Wistar rats (250–300 g) received labeled Cbl by gastric gavage...... and CNCbl are absorbed equally well, but much more OHCbl accumulated in the liver. Benefits of oral supplementation with OHCbl compared to CNCbl should be investigated....

Evidence for the Existence of One Antenna-Associated, Lipid-Dissolved and Two Protein-Bound Pools of Diadinoxanthin Cycle Pigments in Diatoms[C][W

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Lepetit, Bernard; Volke, Daniela; Gilbert, Matthias; Wilhelm, Christian; Goss, Reimund

2010-01-01

We studied the localization of diadinoxanthin cycle pigments in the diatoms Cyclotella meneghiniana and Phaeodactylum tricornutum. Isolation of pigment protein complexes revealed that the majority of high-light-synthesized diadinoxanthin and diatoxanthin is associated with the fucoxanthin chlorophyll protein (FCP) complexes. The characterization of intact cells, thylakoid membranes, and pigment protein complexes by absorption and low-temperature fluorescence spectroscopy showed that the FCPs contain certain amounts of protein-bound diadinoxanthin cycle pigments, which are not significantly different in high-light and low-light cultures. The largest part of high-light-formed diadinoxanthin cycle pigments, however, is not bound to antenna apoproteins but located in a lipid shield around the FCPs, which is copurified with the complexes. This lipid shield is primarily composed of the thylakoid membrane lipid monogalactosyldiacylglycerol. We also show that the photosystem I (PSI) fraction contains a tightly connected FCP complex that is enriched in protein-bound diadinoxanthin cycle pigments. The peripheral FCP and the FCP associated with PSI are composed of different apoproteins. Tandem mass spectrometry analysis revealed that the peripheral FCP is composed mainly of the light-harvesting complex protein Lhcf and also significant amounts of Lhcr. The PSI fraction, on the other hand, shows an enrichment of Lhcr proteins, which are thus responsible for the diadinoxanthin cycle pigment binding. The existence of lipid-dissolved and protein-bound diadinoxanthin cycle pigments in the peripheral antenna and in PSI is discussed with respect to different specific functions of the xanthophylls. PMID:20935178

Free and protein-bound cobalamin absorption in healthy middle-aged and older subjects.

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van Asselt, D Z; van den Broek, W J; Lamers, C B; Corstens, F H; Hoefnagels, W H

1996-08-01

To study free- and protein-bound cobalamin absorption and the correlation with atrophic gastritis in healthy middle-aged and older subjects. A cross-sectional study. Fifty-two healthy subjects, aged 26 to 87 years, apparently free from conditions known to influence the cobalamin status. Middle-aged subjects were defined as those younger than 65 years of age (median age 57 years) and older subjects as those 65 years and older (median age 75 years). Protein-bound cobalamin absorption was assessed by 48-hour urinary excretion method following oral administration of scrambled egg yolk, labeled in vivo with 57 Co-cobalamin by injecting a hen with 57 Co-cyanocobalamin. The percentage of 57 Co-cobalamin bound to protein was 65%. Free cobalamin absorption was assessed by 48-hour urinary excretion method following oral administration of crystalline 57 Co-cyanocobalamin. Plasma cobalamin, folate and fasting plasma gastrin, and pepsinogen A and C concentrations were determined. The median urinary excretion of egg yolk 57 Co-cobalamin in middle-aged subjects was 12.3% (25th and 75th percentiles 10.5%-14.5%) compared with 11.7% (25th and 75th percentiles 9.8%-13.6%) in older subjects (P = .283). The median urinary excretion after administration of free 57 Co-cobalamin in middle-aged subjects was 25.7% (25th and 75th percentiles 20.6%-30.7%) compared with 27.9% (25th and 75th percentiles 21.4%-34.5%) in older subjects (P = .694). Neither egg yolk nor free 57 Co-cobalamin excretion correlated with age. A ratio of pepsinogen A to pepsinogen C less than 1.6, indicating atrophic gastritis, was found in 13 subjects. Within the atrophic gastritis group, 11 subjects had a pepsinogen A concentration greater than or equal to 17 micrograms/L, indicating mild to moderate atrophic gastritis, and two subjects had a pepsinogen A concentration less than 17 micrograms/L, indicating severe atrophic gastritis or gastric atrophy. All subjects had normal fasting plasma gastrin concentrations. Free

An albumin-fixed membrane for the removal of protein-bound toxins

International Nuclear Information System (INIS)

Ge Dongtao; Wu Dewang; Shi Wei; Ma Yuanyuan; Tian Xiangdong; Liang Pengfei; Zhang Qiqing

2006-01-01

Established methods for kidney dialysis do not work for liver failure because kidney dialysis removes only water-soluble toxins, while the liver normally removes albumin-bound toxins. In the present study, a polysulfone dialysis membrane with a -OH reactive group was prepared by hydrolyzing the chloromethylated polysulfone membrane, and the bovine serum albumin molecules were fixed into the membrane with 1,1'-carbonyldiimidazole activation. The content of albumin of the albumin-fixed membrane was 121.3 mg (g membrane) -1 . The albumin-fixed dialysis membranes were used to remove protein-bound toxins, bilirubin, from the bilirubin-albumin solution. The transfer rate of bilirubin of the albumin-fixed membrane was obviously higher compared to the normal dialysis membrane. The clearance of bilirubin with the albumin-fixed membrane was 49.8%. The albumin-fixed membrane can easily be regenerated by the bovine serum albumin and NaOH solution. Regeneration of the membrane suggested good mechanical and chemical stability, as well as good clearance of bilirubin. In addition, the effects of membrane thickness and bilirubin initial concentration on the removal of bilirubin were discussed

Characterization of calcium signals in human induced pluripotent stem cell-derived dentate gyrus neuronal progenitors and mature neurons, stably expressing an advanced calcium indicator protein.

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Vőfély, Gergő; Berecz, Tünde; Szabó, Eszter; Szebényi, Kornélia; Hathy, Edit; Orbán, Tamás I; Sarkadi, Balázs; Homolya, László; Marchetto, Maria C; Réthelyi, János M; Apáti, Ágota

2018-04-01

Pluripotent stem cell derived human neuronal progenitor cells (hPSC-NPCs) and their mature neuronal cell culture derivatives may efficiently be used for central nervous system (CNS) drug screening, including the investigation of ligand-induced calcium signalization. We have established hippocampal NPC cultures derived from human induced PSCs, which were previously generated by non-integrating Sendai virus reprogramming. Using established protocols these NPCs were differentiated into hippocampal dentate gyrus neurons. In order to study calcium signaling without the need of dye loading, we have stably expressed an advanced calcium indicator protein (GCaMP6fast) in the NPCs using the Sleeping Beauty transposon system. We observed no significant effects of the long-term GCaMP6 expression on NPC morphology, gene expression pattern or neural differentiation capacity. In order to compare the functional properties of GCaMP6-expressing neural cells and the corresponding parental cells loaded with calcium indicator dye Fluo-4, a detailed characterization of calcium signals was performed. We found that the calcium signals induced by ATP, glutamate, LPA, or proteases - were similar in these two systems. Moreover, the presence of the calcium indicator protein allowed for a sensitive, repeatable detection of changes in calcium signaling during the process of neurogenesis and neuronal maturation. Copyright © 2018 Elsevier Inc. All rights reserved.

Studying repair of a single protein-bound nick in vivo using the Flp-nick system

DEFF Research Database (Denmark)

Nielsen, Ida; Andersen, Anni Hangaard; Bjergbæk, Lotte

2012-01-01

The Flp-nick system is a simple in vivo system developed for studying the cellular responses to a protein-bound nick at a single genomic site in the budding yeast Saccharomyces cerevisiae. The Flp-nick system takes advantage of a mutant Flp recombinase that can introduce a nick at a specific Flp ...

Role of protein-bound carbonyl groups in the formation of advanced glycation endproducts.

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Liggins, J; Furth, A J

1997-08-22

Several mechanisms have been postulated for the formation of advanced glycation endproducts (AGEs) from glycated proteins; they all feature protein-bound carbonyl intermediates. Using 2,4-dinitrophenylhydrazine (DNPH), we have detected these intermediates on bovine serum albumin, lysozyme and beta-lactoglobulin after in vitro glycation by glucose or fructose. Carbonyls were formed in parallel with AGE-fluorophores, via oxidative Maillard reactions. Neither Amadori nor Heyns products contributed to the DNPH reaction. Fluorophore and carbonyl yields were much enhanced in lipid-associated proteins, but both groups could also be detected in lipid-free proteins. When pre-glycated proteins were incubated in the absence of free sugar, carbonyl groups were rapidly lost in a first-order reaction, while fluorescence continued to develop beyond the 21 days of incubation. Another unexpected finding was that not all carbonyl groups were blocked by aminoguanidine, although there was complete inhibition of reactions leading to AGE-fluorescence. It is suggested that carbonyls acting as fluorophore precursors react readily with aminoguanidine, while others are resistant to this hydrazine, possibly because they are involved in ring closure. Factors influencing the relative rates of acyclisation and hydrazone formation are discussed, together with possible implications for antiglycation therapy.

Interactions between macromolecule-bound antioxidants and Trolox during liposome autoxidation

DEFF Research Database (Denmark)

Celik, Ecem Evrim; Amigo Rubio, Jose Manuel; Andersen, Mogens Larsen

2017-01-01

The interactions between free and macromolecule-bound antioxidants were investigated in order to evaluate their combined effects on the antioxidant environment. Dietary fiber (DF), protein and lipid-bound antioxidants, obtained from whole wheat, soybean and olive oil products, respectively and Tr...... of logistic function was successfully used for modelling the oxidation curve of liposomes. Principal component analysis revealed two separate phases of liposome autoxidation.......The interactions between free and macromolecule-bound antioxidants were investigated in order to evaluate their combined effects on the antioxidant environment. Dietary fiber (DF), protein and lipid-bound antioxidants, obtained from whole wheat, soybean and olive oil products, respectively...... of the simple addition effects of Trolox and bound antioxidants with measured values on lipid oxidation revealed synergetic interactions for DF and refined olive oil-bound antioxidants, and antagonistic interactions for protein and extra virgin olive oil-bound antioxidants with Trolox. A generalized version...

Production of Active Nonglycosylated Recombinant B-Chain of Type-2 Ribosome-Inactivating Protein from Viscum articulatum and Its Biological Effects on Peripheral Blood Mononuclear Cells

Directory of Open Access Journals (Sweden)

Tzu-Li Lu

2011-01-01

Full Text Available Type-2 ribosome-inactivating proteins, composed of a toxic A-chain and lectin-like B-chain, display various biological functions, including cytotoxicity and immunomodulation. We here cloned the lectin-like B-chain encoding fragment of a newly identified type-2 RIP gene, articulatin gene, from Viscum articulatum, into a bacterial expression vector to obtain nonglycosylated recombinant protein expressed in inclusion bodies. After purification and protein refolding, soluble refolded recombinant articulatin B-chain (rATB showed lectin activity specific toward galactoside moiety and was stably maintained while stored in low ionic strength solution. Despite lacking glycosylation, rATB actively bound leukocytes with preferential binding to monocytes and in vitro stimulated PBMCs to release cytokines without obvious cytotoxicity. These results implicated such a B-chain fragment as a potential immunomodulator.

Sensitivity of the Geomagnetic Octupole to a Stably Stratified Layer in the Earth's Core

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Yan, C.; Stanley, S.

2017-12-01

The presence of a stably stratified layer at the top of the core has long been proposed for Earth, based on evidence from seismology and geomagnetic secular variation. Geodynamo modeling offers a unique window to inspect the properties and dynamics in Earth's core. For example, numerical simulations have shown that magnetic field morphology is sensitive to the presence of stably stratified layers in a planet's core. Here we use the mMoSST numerical dynamo model to investigate the effects of a thin stably stratified layer at the top of the fluid outer core in Earth on the resulting large-scale geomagnetic field morphology. We find that the existence of a stable layer has significant influence on the octupolar component of the magnetic field in our models, whereas the quadrupole doesn't show an obvious trend. This suggests that observations of the geomagnetic field can be applied to provide information of the properties of this plausible stable layer, such as how thick and how stable this layer could be. Furthermore, we have examined whether the dominant thermal signature from mantle tomography at the core-mantle boundary (CMB) (a degree & order 2 spherical harmonic) can influence our results. We found that this heat flux pattern at the CMB has no outstanding effects on the quadrupole and octupole magnetic field components. Our studies suggest that if there is a stably stratified layer at the top of the Earth's core, it must be limited in terms of stability and thickness, in order to be compatible with the observed paleomagnetic record.

Delineation of the GPRC6A Receptor Signaling Pathways Using a Mammalian Cell Line Stably Expressing the Receptor

DEFF Research Database (Denmark)

Jacobsen, Stine Engesgaard; Nørskov-Lauritsen, Lenea; Thomsen, Alex Rojas Bie

2013-01-01

receptor has been suggested to couple to multiple G protein classes albeit via indirect methods. Thus, the exact ligand preferences and signaling pathways are yet to be elucidated. In the present study, we generated a Chinese hamster ovary (CHO) cell line that stably expresses mouse GPRC6A. In an effort...... and divalent cations, and for the first time, we conclusively show that these responses are mediated through the Gq pathway. We were not able to confirm previously published data demonstrating Gi- and Gs-mediated signaling; neither could we detect agonistic activity of testosterone and osteocalcin. Generation...... of the stable CHO cell line with robust receptor responsiveness and optimization of the highly sensitive homogeneous time resolved fluorescence technology allow fast assessment of Gq activation without previous manipulations like cotransfection of mutated G proteins. This cell-based assay system for GPRC6A...

Free and Protein-Bound Maillard Reaction Products in Beer: Method Development and a Survey of Different Beer Types.

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Hellwig, Michael; Witte, Sophia; Henle, Thomas

2016-09-28

The Maillard reaction is important for beer color and flavor, but little is known about the occurrence of individual glycated amino acids in beer. Therefore, seven Maillard reaction products (MRPs), namely, fructosyllysine, maltulosyllysine, pyrraline, formyline, maltosine, MG-H1, and argpyrimidine, were synthesized and quantitated in different types of beer (Pilsner, dark, bock, wheat, and nonalcoholic beers) by HPLC-ESI-MS/MS in the multiple reaction monitoring mode through application of the standard addition method. Free MRPs were analyzed directly. A high molecular weight fraction was isolated by dialysis and hydrolyzed enzymatically prior to analysis. Maltulosyllysine was quantitated for the first time in food. The most important free MRPs in beer are fructosyllysine (6.8-27.0 mg/L) and maltulosyllysine (3.7-21.8 mg/L). Beer contains comparatively high amounts of late-stage free MRPs such as pyrraline (0.2-1.6 mg/L) and MG-H1 (0.3-2.5 mg/L). Minor amounts of formyline (4-230 μg/L), maltosine (6-56 μg/L), and argpyrimidine (0.1-4.1 μg/L) were quantitated. Maltulosyllysine was the most significant protein-bound MRP, but both maltulosyllysine and fructosyllysine represent only 15-60% of the total protein-bound lysine-derived Amadori products. Differences in the patterns of protein-bound and free individual MRPs and the ratios between them were identified, which indicate differences in their chemical, biochemical, and microbiological stabilities during the brewing process.

Large eddy simulation of turbulent and stably-stratified flows

International Nuclear Information System (INIS)

Fallon, Benoit

1994-01-01

The unsteady turbulent flow over a backward-facing step is studied by mean of Large Eddy Simulations with structure function sub grid model, both in isothermal and stably-stratified configurations. Without stratification, the flow develops highly-distorted Kelvin-Helmholtz billows, undergoing to helical pairing, with A-shaped vortices shed downstream. We show that forcing injected by recirculation fluctuations governs this oblique mode instabilities development. The statistical results show good agreements with the experimental measurements. For stably-stratified configurations, the flow remains more bi-dimensional. We show with increasing stratification, how the shear layer growth is frozen by inhibition of pairing process then of Kelvin-Helmholtz instabilities, and the development of gravity waves or stable density interfaces. Eddy structures of the flow present striking analogies with the stratified mixing layer. Additional computations show the development of secondary Kelvin-Helmholtz instabilities on the vorticity layers between two primary structures. This important mechanism based on baroclinic effects (horizontal density gradients) constitutes an additional part of the turbulent mixing process. Finally, the feasibility of Large Eddy Simulation is demonstrated for industrial flows, by studying a complex stratified cavity. Temperature fluctuations are compared to experimental measurements. We also develop three-dimensional un-stationary animations, in order to understand and visualize turbulent interactions. (author) [fr

Immunochemical characterization of rhesus proteins with antibodies raised against synthetic peptides.

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Hermand, P; Mouro, I; Huet, M; Bloy, C; Suyama, K; Goldstein, J; Cartron, J P; Bailly, P

1993-07-15

Rabbit polyclonal antibodies were raised against synthetic peptides corresponding to hydrophilic regions of the human Rhesus (Rh) IX cDNA-encoded polypeptide predicted to be extracellularly or intracellularly exposed in the topologic model of the Rh blood group protein. Four antibodies encompassing residues 33-45 (MPC1), 224-233 (MPC4), 390-404 (MPC6), and 408-416 (MPC8) were characterized and compared with a polyclonal anti-Rh protein obtained by immunization with purified Rh proteins. All antibodies had specificity for authentic Rh polypeptides and reacted on Western blot with Rh proteins immunoprecipitated with human monoclonal anti-RhD, -c, and -E. MPC1, but not the other antibodies, agglutinated all human erythrocytes except Rhnull and Rhmod cells, which either lack totally or are severely deficient in Rh proteins, respectively. Immunoblotting analysis with membrane proteins from common and rare variants showed that MPC1 and MPC8 reacted in Western blot with 32-Kd Rh polypeptides from all common red blood cells except those from Rhnull and Rhmod, indicating that peptide regions 33-45 and 408-416 may be common to several if not all Rh proteins, whatever the Rh blood group specificity. MPC4 reacted only with membrane preparations from cells carrying the E antigen, whereas MPC6 recognized preferentially the Rh proteins from E and Ee preparations, suggesting that the protein encoded by the RhIXb cDNA carries the E and/or e antigen(s). Immunoadsorption experiments using inside-out or right-side-out sealed vesicules from DccEE red blood cells as competing antigen showed that the MPC6 and MPC8 antibodies bound only to the cytoplasmic side of the erythrocyte membrane, thus providing evidence for the intracellular orientation of the C-terminal 27 residues of the Rh polypeptides. Attempts to transiently or stably express the Rh polypeptides. Attempts to transiently or stably express the Rh cDNA in eukaryotic cells were largely unsuccessful, suggesting that Rh antigen

A stably expressed llama single-domain intrabody targeting Rev displays broad-spectrum anti-HIV activity.

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Boons, Eline; Li, Guangdi; Vanstreels, Els; Vercruysse, Thomas; Pannecouque, Christophe; Vandamme, Anne-Mieke; Daelemans, Dirk

2014-12-01

The HIV Rev protein mediates the transport of partially and unspliced HIV mRNA from the nucleus to the cytoplasm. Rev multimerizes on a secondary stem-loop structure present in the viral intron-containing mRNA species and recruits the cellular karyopherin CRM1 to export viral mRNAs from the nucleus to the cytoplasm. Previously we have identified a single-domain intrabody (Nb(190)), derived from a llama heavy-chain antibody, which efficiently inhibits Rev multimerization and suppresses the production of infectious virus. We recently mapped the epitope of this nanobody and demonstrated that Rev residues K20 and Y23 are crucial for interaction while residues V16, H53 and L60 are important to a lesser extent. Here, we generated cell lines stably expressing Nb(190) and assessed the capacity of these cell lines to suppress the replication of different HIV-1 subtypes. These cells stably expressing the single-domain antibody are protected from virus-induced cytopathogenic effect even in the context of high multiplicity of infection. In addition, the replication of different subtypes of group M and one strain of group O is significantly suppressed in these cell lines. Next, we analysed the natural variations of Rev amino acids in sequence samples from HIV-1 infected patients worldwide and assessed the effect of Nb(190) on the most prevalent polymorphisms occurring at the key epitope positions (K20 and Y23) in Rev. We found that Nb(190) was able to suppress the function of these Rev variants except for the K20N mutant, which was present in only 0.7% of HIV-1 sequence populations (n = 4632). Cells stably expressing the single-domain intrabody Nb(190) are protected against virus-induced cytopathogenic effect and display a selective survival advantage upon infection. In addition, Nb(190) suppresses the replication of a wide range of different HIV-1 subtypes. Large-scale sequence analysis reveals that the Nb(190) epitope positions in Rev are well conserved across major HIV-1

Scaffold-free 3D bio-printed human liver tissue stably maintains metabolic functions useful for drug discovery.

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Kizawa, Hideki; Nagao, Eri; Shimamura, Mitsuru; Zhang, Guangyuan; Torii, Hitoshi

2017-07-01

The liver plays a central role in metabolism. Although many studies have described in vitro liver models for drug discovery, to date, no model has been described that can stably maintain liver function. Here, we used a unique, scaffold-free 3D bio-printing technology to construct a small portion of liver tissue that could stably maintain drug, glucose, and lipid metabolism, in addition to bile acid secretion. This bio-printed normal human liver tissue maintained expression of several kinds of hepatic drug transporters and metabolic enzymes that functioned for several weeks. The bio-printed liver tissue displayed glucose production via cAMP/protein kinase A signaling, which could be suppressed with insulin. Bile acid secretion was also observed from the printed liver tissue, and it accumulated in the culture medium over time. We observed both bile duct and sinusoid-like structures in the bio-printed liver tissue, which suggested that bile acid secretion occurred via a sinusoid-hepatocyte-bile duct route. These results demonstrated that our bio-printed liver tissue was unique, because it exerted diverse liver metabolic functions for several weeks. In future, we expect our bio-printed liver tissue to be applied to developing new models that can be used to improve preclinical predictions of long-term toxicity in humans, generate novel targets for metabolic liver disease, and evaluate biliary excretion in drug development.

A Physiologically Based Pharmacokinetic Model to Predict the Pharmacokinetics of Highly Protein-Bound Drugs and Impact of Errors in Plasma Protein Binding

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Ye, Min; Nagar, Swati; Korzekwa, Ken

2015-01-01

Predicting the pharmacokinetics of highly protein-bound drugs is difficult. Also, since historical plasma protein binding data was often collected using unbuffered plasma, the resulting inaccurate binding data could contribute to incorrect predictions. This study uses a generic physiologically based pharmacokinetic (PBPK) model to predict human plasma concentration-time profiles for 22 highly protein-bound drugs. Tissue distribution was estimated from in vitro drug lipophilicity data, plasma protein binding, and blood: plasma ratio. Clearance was predicted with a well-stirred liver model. Underestimated hepatic clearance for acidic and neutral compounds was corrected by an empirical scaling factor. Predicted values (pharmacokinetic parameters, plasma concentration-time profile) were compared with observed data to evaluate model accuracy. Of the 22 drugs, less than a 2-fold error was obtained for terminal elimination half-life (t1/2, 100% of drugs), peak plasma concentration (Cmax, 100%), area under the plasma concentration-time curve (AUC0–t, 95.4%), clearance (CLh, 95.4%), mean retention time (MRT, 95.4%), and steady state volume (Vss, 90.9%). The impact of fup errors on CLh and Vss prediction was evaluated. Errors in fup resulted in proportional errors in clearance prediction for low-clearance compounds, and in Vss prediction for high-volume neutral drugs. For high-volume basic drugs, errors in fup did not propagate to errors in Vss prediction. This is due to the cancellation of errors in the calculations for tissue partitioning of basic drugs. Overall, plasma profiles were well simulated with the present PBPK model. PMID:26531057

Structure of the protein core of translation initiation factor 2 in apo, GTP-bound and GDP-bound forms

International Nuclear Information System (INIS)

Simonetti, Angelita; Marzi, Stefano; Fabbretti, Attilio; Hazemann, Isabelle; Jenner, Lasse; Urzhumtsev, Alexandre; Gualerzi, Claudio O.; Klaholz, Bruno P.

2013-01-01

The crystal structures of the eubacterial translation initiation factor 2 in apo form and with bound GDP and GTP reveal conformational changes upon nucleotide binding and hydrolysis, notably of the catalytically important histidine in the switch II region. Translation initiation factor 2 (IF2) is involved in the early steps of bacterial protein synthesis. It promotes the stabilization of the initiator tRNA on the 30S initiation complex (IC) and triggers GTP hydrolysis upon ribosomal subunit joining. While the structure of an archaeal homologue (a/eIF5B) is known, there are significant sequence and functional differences in eubacterial IF2, while the trimeric eukaryotic IF2 is completely unrelated. Here, the crystal structure of the apo IF2 protein core from Thermus thermophilus has been determined by MAD phasing and the structures of GTP and GDP complexes were also obtained. The IF2–GTP complex was trapped by soaking with GTP in the cryoprotectant. The structures revealed conformational changes of the protein upon nucleotide binding, in particular in the P-loop region, which extend to the functionally relevant switch II region. The latter carries a catalytically important and conserved histidine residue which is observed in different conformations in the GTP and GDP complexes. Overall, this work provides the first crystal structure of a eubacterial IF2 and suggests that activation of GTP hydrolysis may occur by a conformational repositioning of the histidine residue

Structure of the protein core of translation initiation factor 2 in apo, GTP-bound and GDP-bound forms

Energy Technology Data Exchange (ETDEWEB)

Simonetti, Angelita [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale - INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Marzi, Stefano [Architecture et Réactivité de l’ARN, UPR 9002 CNRS, IBMC (Institute of Molecular and Cellular Biology), 15 Rue R. Descartes, 67084 Strasbourg, France, Université de Strasbourg, 67000 Strasbourg (France); Fabbretti, Attilio [University of Camerino, 62032 Camerino (Monaco) (Italy); Hazemann, Isabelle; Jenner, Lasse [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale -INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Urzhumtsev, Alexandre [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale - INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Université de Lorraine, 54506 Vandoeuvre-lès-Nancy (France); Gualerzi, Claudio O. [University of Camerino, 62032 Camerino (Monaco) (Italy); Klaholz, Bruno P., E-mail: klaholz@igbmc.fr [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale - INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France)

2013-06-01

The crystal structures of the eubacterial translation initiation factor 2 in apo form and with bound GDP and GTP reveal conformational changes upon nucleotide binding and hydrolysis, notably of the catalytically important histidine in the switch II region. Translation initiation factor 2 (IF2) is involved in the early steps of bacterial protein synthesis. It promotes the stabilization of the initiator tRNA on the 30S initiation complex (IC) and triggers GTP hydrolysis upon ribosomal subunit joining. While the structure of an archaeal homologue (a/eIF5B) is known, there are significant sequence and functional differences in eubacterial IF2, while the trimeric eukaryotic IF2 is completely unrelated. Here, the crystal structure of the apo IF2 protein core from Thermus thermophilus has been determined by MAD phasing and the structures of GTP and GDP complexes were also obtained. The IF2–GTP complex was trapped by soaking with GTP in the cryoprotectant. The structures revealed conformational changes of the protein upon nucleotide binding, in particular in the P-loop region, which extend to the functionally relevant switch II region. The latter carries a catalytically important and conserved histidine residue which is observed in different conformations in the GTP and GDP complexes. Overall, this work provides the first crystal structure of a eubacterial IF2 and suggests that activation of GTP hydrolysis may occur by a conformational repositioning of the histidine residue.

Dynamic features of apo and bound HIV-Nef protein reveal the anti-HIV dimerization inhibition mechanism.

Science.gov (United States)

Moonsamy, Suri; Bhakat, Soumendranath; Soliman, Mahmoud E S

2015-01-01

The first account on the dynamic features of Nef or negative factor, a small myristoylated protein located in the cytoplasm believes to increase HIV-1 viral titer level, is reported herein. Due to its major role in HIV-1 pathogenicity, Nef protein is considered an emerging target in anti-HIV drug design and discovery process. In this study, comparative long-range all-atom molecular dynamics simulations were employed for apo and bound protein to unveil molecular mechanism of HIV-Nef dimerization and inhibition. Results clearly revealed that B9, a newly discovered Nef inhibitor, binds at the dimeric interface of Nef protein and caused significant separation between orthogonally opposed residues, namely Asp108, Leu112 and Gln104. Large differences in magnitudes were observed in the radius of gyration (∼1.5 Å), per-residue fluctuation (∼2 Å), C-alpha deviations (∼2 Å) which confirm a comparatively more flexible nature of apo conformation due to rapid dimeric association. Compared to the bound conformer, a more globally correlated motion in case of apo structure of HIV-Nef confirms the process of dimeric association. This clearly highlights the process of inhibition as a result of ligand binding. The difference in principal component analysis (PCA) scatter plot and per-residue mobility plot across first two normal modes further justifies the same findings. The in-depth dynamic analyses of Nef protein presented in this report would serve crucial in understanding its function and inhibition mechanisms. Information on inhibitor binding mode would also assist in designing of potential inhibitors against this important HIV target.

Scanning a DNA molecule for bound proteins using hybrid magnetic and optical tweezers.

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Marijn T J van Loenhout

Full Text Available The functional state of the genome is determined by its interactions with proteins that bind, modify, and move along the DNA. To determine the positions and binding strength of proteins localized on DNA we have developed a combined magnetic and optical tweezers apparatus that allows for both sensitive and label-free detection. A DNA loop, that acts as a scanning probe, is created by looping an optically trapped DNA tether around a DNA molecule that is held with magnetic tweezers. Upon scanning the loop along the λ-DNA molecule, EcoRI proteins were detected with ~17 nm spatial resolution. An offset of 33 ± 5 nm for the detected protein positions was found between back and forwards scans, corresponding to the size of the DNA loop and in agreement with theoretical estimates. At higher applied stretching forces, the scanning loop was able to remove bound proteins from the DNA, showing that the method is in principle also capable of measuring the binding strength of proteins to DNA with a force resolution of 0.1 pN/[Formula: see text]. The use of magnetic tweezers in this assay allows the facile preparation of many single-molecule tethers, which can be scanned one after the other, while it also allows for direct control of the supercoiling state of the DNA molecule, making it uniquely suitable to address the effects of torque on protein-DNA interactions.

Effects of solution chemistry and aging time on prion protein adsorption and replication of soil-bound prions.

Directory of Open Access Journals (Sweden)

Samuel E Saunders

2011-04-01

Full Text Available Prion interactions with soil may play an important role in the transmission of chronic wasting disease (CWD and scrapie. Prions are known to bind to a wide range of soil surfaces, but the effects of adsorption solution chemistry and long-term soil binding on prion fate and transmission risk are unknown. We investigated HY TME prion protein (PrP(Sc adsorption to soil minerals in aqueous solutions of phosphate buffered saline (PBS, sodium chloride, calcium chloride, and deionized water using western blotting. The replication efficiency of bound prions following adsorption in these solutions was also evaluated by protein misfolding cyclic amplification (PMCA. Aging studies investigated PrP(Sc desorption and replication efficiency up to one year following adsorption in PBS or DI water. Results indicate that adsorption solution chemistry can affect subsequent prion replication or desorption ability, especially after incubation periods of 30 d or longer. Observed effects were minor over the short-term (7 d or less. Results of long-term aging experiments demonstrate that unbound prions or prions bound to a diverse range of soil surfaces can readily replicate after one year. Our results suggest that while prion-soil interactions can vary with solution chemistry, prions bound to soil could remain a risk for transmitting prion diseases after months in the environment.

Protein-bound NAD(P)H Lifetime is Sensitive to Multiple Fates of Glucose Carbon.

Science.gov (United States)

Sharick, Joe T; Favreau, Peter F; Gillette, Amani A; Sdao, Sophia M; Merrins, Matthew J; Skala, Melissa C

2018-04-03

While NAD(P)H fluorescence lifetime imaging (FLIM) can detect changes in flux through the TCA cycle and electron transport chain (ETC), it remains unclear whether NAD(P)H FLIM is sensitive to other potential fates of glucose. Glucose carbon can be diverted from mitochondria by the pentose phosphate pathway (via glucose 6-phosphate dehydrogenase, G6PDH), lactate production (via lactate dehydrogenase, LDH), and rejection of carbon from the TCA cycle (via pyruvate dehydrogenase kinase, PDK), all of which can be upregulated in cancer cells. Here, we demonstrate that multiphoton NAD(P)H FLIM can be used to quantify the relative concentrations of recombinant LDH and malate dehydrogenase (MDH) in solution. In multiple epithelial cell lines, NAD(P)H FLIM was also sensitive to inhibition of LDH and PDK, as well as the directionality of LDH in cells forced to use pyruvate versus lactate as fuel sources. Among the parameters measurable by FLIM, only the lifetime of protein-bound NAD(P)H (τ 2 ) was sensitive to these changes, in contrast to the optical redox ratio, mean NAD(P)H lifetime, free NAD(P)H lifetime, or the relative amount of free and protein-bound NAD(P)H. NAD(P)H τ 2 offers the ability to non-invasively quantify diversions of carbon away from the TCA cycle/ETC, which may support mechanisms of drug resistance.

Alkaline polymer electrolyte fuel cells stably working at 80 °C

Science.gov (United States)

Peng, Hanqing; Li, Qihao; Hu, Meixue; Xiao, Li; Lu, Juntao; Zhuang, Lin

2018-06-01

Alkaline polymer electrolyte fuel cells are a new class of polymer electrolyte fuel cells that fundamentally enables the use of nonprecious metal catalysts. The cell performance mostly relies on the quality of alkaline polymer electrolytes, including the ionic conductivity and the chemical/mechanical stability. For a long time, alkaline polymer electrolytes are thought to be too weak in stability to allow the fuel cell to be operated at elevated temperatures, e.g., above 60 °C. In the present work, we report a progress in the state-of-the-art alkaline polymer electrolyte fuel cell technology. By using a newly developed alkaline polymer electrolyte, quaternary ammonia poly (N-methyl-piperidine-co-p-terphenyl), which simultaneously possesses high ionic conductivity and excellent chemical/mechanical stability, the fuel cell can now be stably operated at 80 °C with high power density. The peak power density reaches ca. 1.5 W/cm2 at 80 °C with Pt/C catalysts used in both the anode and the cathode. The cell works stably in a period of study over 100 h.

Transgenic Parasites Stably Expressing Full-Length Plasmodium falciparum Circumsporozoite Protein as a Model for Vaccine Down-Selection in Mice Using Sterile Protection as an Endpoint

Science.gov (United States)

Porter, Michael D.; Nicki, Jennifer; Pool, Christopher D.; DeBot, Margot; Illam, Ratish M.; Brando, Clara; Bozick, Brooke; De La Vega, Patricia; Angra, Divya; Spaccapelo, Roberta; Crisanti, Andrea; Murphy, Jittawadee R.; Bennett, Jason W.; Schwenk, Robert J.; Ockenhouse, Christian F.

2013-01-01

Circumsporozoite protein (CSP) of Plasmodium falciparum is a protective human malaria vaccine candidate. There is an urgent need for models that can rapidly down-select novel CSP-based vaccine candidates. In the present study, the mouse-mosquito transmission cycle of a transgenic Plasmodium berghei malaria parasite stably expressing a functional full-length P. falciparum CSP was optimized to consistently produce infective sporozoites for protection studies. A minimal sporozoite challenge dose was established, and protection was defined as the absence of blood-stage parasites 14 days after intravenous challenge. The specificity of protection was confirmed by vaccinating mice with multiple CSP constructs of differing lengths and compositions. Constructs that induced high NANP repeat-specific antibody titers in enzyme-linked immunosorbent assays were protective, and the degree of protection was dependent on the antigen dose. There was a positive correlation between antibody avidity and protection. The antibodies in the protected mice recognized the native CSP on the parasites and showed sporozoite invasion inhibitory activity. Passive transfer of anti-CSP antibodies into naive mice also induced protection. Thus, we have demonstrated the utility of a mouse efficacy model to down-select human CSP-based vaccine formulations. PMID:23536694

Structure of Rotavirus Outer-Layer Protein VP7 Bound with a Neutralizing Fab

Energy Technology Data Exchange (ETDEWEB)

Aoki, Scott T.; Settembre, Ethan C.; Trask, Shane D.; Greenberg, Harry B.; Harrison, Stephen C.; Dormitzer, Philip R.; (Stanford-MED); (CH-Boston)

2009-06-17

Rotavirus outer-layer protein VP7 is a principal target of protective antibodies. Removal of free calcium ions (Ca{sup 2+}) dissociates VP7 trimers into monomers, releasing VP7 from the virion, and initiates penetration-inducing conformational changes in the other outer-layer protein, VP4. We report the crystal structure at 3.4 angstrom resolution of VP7 bound with the Fab fragment of a neutralizing monoclonal antibody. The Fab binds across the outer surface of the intersubunit contact, which contains two Ca{sup 2+} sites. Mutations that escape neutralization by other antibodies suggest that the same region bears the epitopes of most neutralizing antibodies. The monovalent Fab is sufficient to neutralize infectivity. We propose that neutralizing antibodies against VP7 act by stabilizing the trimer, thereby inhibiting the uncoating trigger for VP4 rearrangement. A disulfide-linked trimer is a potential subunit immunogen.

Membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen.

Science.gov (United States)

Zhu, Haiyan; Fang, Xiaoyun; Zhang, Dongmei; Wu, Weicheng; Shao, Miaomiao; Wang, Lan; Gu, Jianxin

2016-01-01

Heat shock proteins (HSPs) were originally identified as stress-responsive proteins and serve as molecular chaperones in different intracellular compartments. Translocation of HSPs to the cell surface and release of HSPs into the extracellular space have been observed during the apoptotic process and in response to a variety of cellular stress. Here, we report that UV irradiation and cisplatin treatment rapidly induce the expression of membrane-bound Hsp60, Hsp70, and Hsp90 upstream the phosphatidylserine exposure. Membrane-bound Hsp60, Hsp70 and Hsp90 could promote the release of IL-6 and IL-1β as well as DC maturation by the evaluation of CD80 and CD86 expression. On the other hand, Hsp60, Hsp70 and Hsp90 on cells could facilitate the uptake of dying cells by bone marrow-derived dendritic cells. Lectin-like oxidized LDL receptor-1 (LOX-1), as a common receptor for Hsp60, Hsp70, and Hsp90, is response for their recognition and mediates the uptake of dying cells. Furthermore, membrane-bound Hsp60, Hsp70 and Hsp90 could promote the cross-presentation of OVA antigen from E.G7 cells and inhibition of the uptake of dying cells by LOX-1 decreases the cross-presentation of cellular antigen. Therefore, the rapid exposure of HSPs on dying cells at the early stage allows for the recognition by and confers an activation signal to the immune system.

Inhibition of MAP kinase promotes the recruitment of corepressor SMRT by tamoxifen-bound estrogen receptor alpha and potentiates tamoxifen action in MCF-7 cells

International Nuclear Information System (INIS)

Hong, Wei; Chen, Linfeng; Li, Juan; Yao, Zhi

2010-01-01

Estrogen receptor alpha (ERα), a ligand controlled transcription factor, plays an important role in breast cancer growth and endocrine therapy. Tamoxifen (TAM) antagonizes ERα activity and has been applied in breast cancer treatment. TAM-bound ERα associates with nuclear receptor-corepressors. Mitogen-activated protein kinase (MAPK) has been elucidated to result in cross-talk between growth factor and ERα mediated signaling. We show that activated MAPK represses interaction of TAM-bound ERα with silencing mediator for retinoid and thyroid hormone receptors (SMRT) and inhibits the recruitment of SMRT by ERα to certain estrogen target genes. Blockade of MAPK signaling cascade with MEK inhibitor U0126 promotes the interaction and subsequently inhibits ERα activity via enhanced recruitment of SMRT, leading to reduced expression of ERα target genes. The growth rate of MCF-7 cells was decelerated when treated with both TAM and U0126. Moreover, the growth of MCF-7 cells stably expressing SMRT showed a robust repression in the presence of TAM and U0126. These results suggest that activated MAPK signaling cascade attenuates antagonist-induced recruitment of SMRT to ERα, suggesting corepressor mediates inhibition of ERα transactivation and breast cancer cell growth by antagonist. Taken together, our finding indicates combination of antagonist and MAPK inhibitor could be a helpful approach for breast cancer therapy.

A physiologically based pharmacokinetic model to predict the pharmacokinetics of highly protein-bound drugs and the impact of errors in plasma protein binding.

Science.gov (United States)

Ye, Min; Nagar, Swati; Korzekwa, Ken

2016-04-01

Predicting the pharmacokinetics of highly protein-bound drugs is difficult. Also, since historical plasma protein binding data were often collected using unbuffered plasma, the resulting inaccurate binding data could contribute to incorrect predictions. This study uses a generic physiologically based pharmacokinetic (PBPK) model to predict human plasma concentration-time profiles for 22 highly protein-bound drugs. Tissue distribution was estimated from in vitro drug lipophilicity data, plasma protein binding and the blood: plasma ratio. Clearance was predicted with a well-stirred liver model. Underestimated hepatic clearance for acidic and neutral compounds was corrected by an empirical scaling factor. Predicted values (pharmacokinetic parameters, plasma concentration-time profile) were compared with observed data to evaluate the model accuracy. Of the 22 drugs, less than a 2-fold error was obtained for the terminal elimination half-life (t1/2 , 100% of drugs), peak plasma concentration (Cmax , 100%), area under the plasma concentration-time curve (AUC0-t , 95.4%), clearance (CLh , 95.4%), mean residence time (MRT, 95.4%) and steady state volume (Vss , 90.9%). The impact of fup errors on CLh and Vss prediction was evaluated. Errors in fup resulted in proportional errors in clearance prediction for low-clearance compounds, and in Vss prediction for high-volume neutral drugs. For high-volume basic drugs, errors in fup did not propagate to errors in Vss prediction. This is due to the cancellation of errors in the calculations for tissue partitioning of basic drugs. Overall, plasma profiles were well simulated with the present PBPK model. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Ligand-free, protein-bound technetium-99m iron-dextran enhancement of technetium pyrophosphate uptake in tumours

International Nuclear Information System (INIS)

Pojer, P.M.; Jakovljevic, A.C.; Wise, K.N.

1985-01-01

The biodistribution of technetium-99m was studied in T-cell lymphoma and selected organs of iron-dextran treated and control mice given technetium-99m pyrophosphate. The results showed that high serum iron levels increased tumour uptake of technetium pyrophosphate. This supports the hypothesis that technetium, in common with other metal-based tumour seeking radiopharmaceuticals, is transported to tumours as a ligand-free protein-bound cation. (U.K.)

ChIP-seq Analysis in R (CSAR): An R package for the statistical detection of protein-bound genomic regions

NARCIS (Netherlands)

Muino, J.M.; Kaufmann, K.; Ham, van R.C.H.J.; Angenent, G.C.; Krajewski, P.

2011-01-01

Background In vivo detection of protein-bound genomic regions can be achieved by combining chromatin-immunoprecipitation with next-generation sequencing technology (ChIP-seq). The large amount of sequence data produced by this method needs to be analyzed in a statistically proper and computationally

Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-like Proteins Support a Highly Dynamic Domain Architecture

Energy Technology Data Exchange (ETDEWEB)

Miller, Bradley R.; Drake, Eric J.; Shi, Ce; Aldrich, Courtney C.; Gulick, Andrew M. (UMM); (HWMRI)

2016-09-05

Nonribosomal peptide synthetases (NRPSs) produce a wide variety of peptide natural products. During synthesis, the multidomain NRPSs act as an assembly line, passing the growing product from one module to the next. Each module generally consists of an integrated peptidyl carrier protein, an amino acid-loading adenylation domain, and a condensation domain that catalyzes peptide bond formation. Some adenylation domains interact with small partner proteins called MbtH-like proteins (MLPs) that enhance solubility or activity. A structure of an MLP bound to an adenylation domain has been previously reported using a truncated adenylation domain, precluding any insight that might be derived from understanding the influence of the MLP on the intact adenylation domain or on the dynamics of the entire NRPS module. Here, we present the structures of the full-length NRPS EntF bound to the MLPs from Escherichia coli and Pseudomonas aeruginosa. These new structures, along with biochemical and bioinformatics support, further elaborate the residues that define the MLP-adenylation domain interface. Additionally, the structures highlight the dynamic behavior of NRPS modules, including the module core formed by the adenylation and condensation domains as well as the orientation of the mobile thioesterase domain.

A simple elution strategy for biotinylated proteins bound to streptavidin conjugated beads using excess biotin and heat.

Science.gov (United States)

Cheah, Joleen S; Yamada, Soichiro

2017-12-02

Protein-protein interactions are the molecular basis of cell signaling. Recently, proximity based biotin identification (BioID) has emerged as an alternative approach to traditional co-immunoprecipitation. In this protocol, a mutant biotin ligase promiscuously labels proximal binding partners with biotin, and resulting biotinylated proteins are purified using streptavidin conjugated beads. This approach does not require preservation of protein complexes in vitro, making it an ideal approach to identify transient or weak protein complexes. However, due to the high affinity bond between streptavidin and biotin, elution of biotinylated proteins from streptavidin conjugated beads requires harsh denaturing conditions, which are often incompatible with downstream processing. To effectively release biotinylated proteins bound to streptavidin conjugated beads, we designed a series of experiments to determine optimal binding and elution conditions. Interestingly, the concentrations of SDS and IGEPAL-CA630 during the incubation with streptavidin conjugated beads were the key to effective elution of biotinylated proteins using excess biotin and heating. This protocol provides an alternative method to isolate biotinylated proteins from streptavidin conjugated beads that is suitable for further downstream analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

Phosphorylation Regulates the Bound Structure of an Intrinsically Disordered Protein: The p53-TAZ2 Case.

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Raúl Esteban Ithuralde

Full Text Available Disordered regions and Intrinsically Disordered Proteins (IDPs are involved in critical cellular processes and may acquire a stable three-dimensional structure only upon binding to their partners. IDPs may follow a folding-after-binding process, known as induced folding, or a folding-before-binding process, known as conformational selection. The transcription factor p53 is involved in the regulation of cellular events that arise upon stress or DNA damage. The p53 domain structure is composed of an N-terminal transactivation domain (p53TAD, a DNA Binding Domain and a tetramerization domain. The activity of TAD is tightly regulated by interactions with cofactors, inhibitors and phosphorylation. To initiate transcription, p53TAD binds to the TAZ2 domain of CBP, a co-transcription factor, and undergoes a folding and binding process, as revealed by the recent NMR structure of the complex. The activity of p53 is regulated by phosphorylation at multiple sites on the TAD domain and recent studies have shown that modifications at three residues affect the binding towards TAZ2. However, we still do not know how these phosphorylations affect the structure of the bound state and, therefore, how they regulate the p53 function. In this work, we have used computational simulations to understand how phosphorylation affects the structure of the p53TAD:TAZ2 complex and regulates the recognition mechanism. Phosphorylation has been proposed to enhance binding by direct interaction with the folded protein or by changing the unbound conformation of IDPs, for example by pre-folding the protein favoring the recognition mechanism. Here, we show an interesting turn in the p53 case: phosphorylation mainly affects the bound structure of p53TAD, highlighting the complexity of IDP protein-protein interactions. Our results are in agreement with previous experimental studies, allowing a clear picture of how p53 is regulated by phosphorylation and giving new insights into how

Water-Soluble Chlorophyll Protein (WSCP) Stably Binds Two or Four Chlorophylls.

Science.gov (United States)

Palm, Daniel M; Agostini, Alessandro; Tenzer, Stefan; Gloeckle, Barbara M; Werwie, Mara; Carbonera, Donatella; Paulsen, Harald

2017-03-28

Water-soluble chlorophyll proteins (WSCPs) of class IIa from Brassicaceae form tetrameric complexes containing one chlorophyll (Chl) per apoprotein but no carotenoids. The complexes are remarkably stable toward dissociation and protein denaturation even at 100 °C and extreme pH values, and the Chls are partially protected against photooxidation. There are several hypotheses that explain the biological role of WSCPs, one of them proposing that they function as a scavenger of Chls set free upon plant senescence or pathogen attack. The biochemical properties of WSCP described in this paper are consistent with the protein acting as an efficient and flexible Chl scavenger. At limiting Chl concentrations, the recombinant WSCP apoprotein binds substoichiometric amounts of Chl (two Chls per tetramer) to form complexes that are as stable toward thermal dissociation, denaturation, and photodamage as the fully pigmented ones. If more Chl is added, these two-Chl complexes can bind another two Chls to reach the fully pigmented state. The protection of WSCP Chls against photodamage has been attributed to the apoprotein serving as a diffusion barrier for oxygen, preventing its access to triplet excited Chls and, thus, the formation of singlet oxygen. By contrast, the sequential binding of Chls by WSCP suggests a partially open or at least flexible structure, raising the question of how WSCP photoprotects its Chls without the help of carotenoids.

Specific binding of [alpha-32P]GTP to cytosolic and membrane-bound proteins of human platelets correlates with the activation of phospholipase C

International Nuclear Information System (INIS)

Lapetina, E.G.; Reep, B.R.

1987-01-01

We have assessed the binding of [alpha- 32 P]GTP to platelet proteins from cytosolic and membrane fractions. Proteins were separated by NaDodSO 4 /PAGE and electrophoretically transferred to nitrocellulose. Incubation of the nitrocellulose blots with [alpha- 32 P]GTP indicated the presence of specific and distinct GTP-binding proteins in cytosol and membranes. Binding was prevented by 10-100 nM GTP and by 100 nM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) or GDP; binding was unaffected by 1 nM-1 microM ATP. One main GTP-binding protein (29.5 kDa) was detected in the membrane fraction, while three others (29, 27, and 21 kDa) were detected in the soluble fraction. Two cytosolic GTP-binding proteins (29 and 27 kDa) were degraded by trypsin; another cytosolic protein (21 kDa) and the membrane-bound protein (29.5 kDa) were resistant to the action of trypsin. Treatment of intact platelets with trypsin or thrombin, followed by lysis and fractionation, did not affect the binding of [alpha- 32 P]GTP to the membrane-bound protein. GTP[gamma S] still stimulated phospholipase C in permeabilized platelets already preincubated with trypsin. This suggests that trypsin-resistant GTP-binding proteins might regulate phospholipase C stimulated by GTP[gamma S

Intraerythrocyte Non-Protein-Bound Iron in Children with Bronchopulmonary Pathology

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E.M. Vasilyeva

2014-12-01

Full Text Available A total of 230 children having bronchopulmonary pathology (BPP were examined. Patients were divided into 4 groups according to their intraerythrocyte non-protein- bound iron (IE-NPBI levels. We investigated the relationship of the IE-NPBI level with parameters of respiratory function (RF tests, the severity of comorbidities, and level of other free intracellular ions, such as copper, zinc, and magnesium. The pronounced increase in IE-NPBI level was typical for patients with the connective tissue dysplasia, often accompanied by mitral valve prolapse, osteopenia, and mineral metabolism violation. The severe comorbid diagnoses were typical for patients with reduced levels of IE-NPBI (chronic cor pulmonale, tuberculosis infection. The largest number of comorbidities, aggravating the underlying disease, took place in the group of patients with a significant reduction in IE-NPBI level. A significant increase in IE-NPBI level, as well as a marked reduction of IE-NPBI level, was an unfavorable factor for the underlying disease. We found a correlation between IE-NPBI level and parameters of RF-test in patients with moderate increase in IE-NPBI level.

Aluminum stimulates uptake of non-transferrin bound iron and transferrin bound iron in human glial cells

International Nuclear Information System (INIS)

Kim, Yongbae; Olivi, Luisa; Cheong, Jae Hoon; Maertens, Alex; Bressler, Joseph P.

2007-01-01

Aluminum and other trivalent metals were shown to stimulate uptake of transferrin bound iron and nontransferrin bound iron in erytholeukemia and hepatoma cells. Because of the association between aluminum and Alzheimer's Disease, and findings of higher levels of iron in Alzheimer's disease brains, the effects of aluminum on iron homeostasis were examined in a human glial cell line. Aluminum stimulated dose- and time-dependent uptake of nontransferrin bound iron and iron bound to transferrin. A transporter was likely involved in the uptake of nontransferrin iron because uptake reached saturation, was temperature-dependent, and attenuated by inhibitors of protein synthesis. Interestingly, the effects of aluminum were not blocked by inhibitors of RNA synthesis. Aluminum also decreased the amount of iron bound to ferritin though it did not affect levels of divalent metal transporter 1. These results suggest that aluminum disrupts iron homeostasis in Brain by several mechanisms including the transferrin receptor, a nontransferrin iron transporter, and ferritin

Interconversion of two GDP-bound conformations and their selection in an Arf-family small G protein.

Science.gov (United States)

Okamura, Hideyasu; Nishikiori, Masaki; Xiang, Hongyu; Ishikawa, Masayuki; Katoh, Etsuko

2011-07-13

ADP-ribosylation factor (Arf) and other Arf-family small G proteins participate in many cellular functions via their characteristic GTP/GDP conformational cycles, during which a nucleotide(∗)Mg(2+)-binding site communicates with a remote N-terminal helix. However, the conformational interplay between the nucleotides, the helix, the protein core, and Mg(2+) has not been fully delineated. Herein, we report a study of the dynamics of an Arf-family protein, Arl8, under various conditions by means of NMR relaxation spectroscopy. The data indicated that, when GDP is bound, the protein core, which does not include the N-terminal helix, reversibly transition between an Arf-family GDP form and another conformation that resembles the Arf-family GTP form. Additionally, we found that the N-terminal helix and Mg(2+), respectively, stabilize the aforementioned former and latter conformations in a population-shift manner. Given the dynamics of the conformational changes, we can describe the Arl8 GTP/GDP cycle in terms of an energy diagram. Copyright © 2011 Elsevier Ltd. All rights reserved.

pMD-Membrane: A Method for Ligand Binding Site Identification in Membrane-Bound Proteins.

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Priyanka Prakash

2015-10-01

Full Text Available Probe-based or mixed solvent molecular dynamics simulation is a useful approach for the identification and characterization of druggable sites in drug targets. However, thus far the method has been applied only to soluble proteins. A major reason for this is the potential effect of the probe molecules on membrane structure. We have developed a technique to overcome this limitation that entails modification of force field parameters to reduce a few pairwise non-bonded interactions between selected atoms of the probe molecules and bilayer lipids. We used the resulting technique, termed pMD-membrane, to identify allosteric ligand binding sites on the G12D and G13D oncogenic mutants of the K-Ras protein bound to a negatively charged lipid bilayer. In addition, we show that differences in probe occupancy can be used to quantify changes in the accessibility of druggable sites due to conformational changes induced by membrane binding or mutation.

Optimisation of the Factor VIII yield in mammalian cell cultures by reducing the membrane bound fraction

DEFF Research Database (Denmark)

Kolind, Mille Petersen; Nørby, Peder Lisby; Berchtold, Martin Werner

2011-01-01

and forms the tenase complex together with clotting Factor IX. In vitro, during serum free production of recombinant FVIII (rFVIII), production cells also expose PS, and since vWF is not present to hinder interaction of secreted rFVIII with PS, rFVIII is partly associated with the cell membrane...... of active membrane bound rFVIII to the culture medium. Moreover, the attachment of rFVIII to cell membranes of un-transfected HEK293 cells was studied in the presence of compounds that competes for interactions between rFVIII and PS. Competitive assays between iodinated rFVIII (¹²5I-rFVIII) and annexin V...... or ortho-phospho-L-serine (OPLS) demonstrated that annexin V and OPLS were able to reduce the membrane bound fraction of rFVIII by 70% and 30%, respectively. Finally, adding OPLS to CHO cells stably expressing FVIII increased the yield by 50%. Using this new knowledge, the recovery of rFVIII could...

Binding of monoclonal antibody to protein antigen in fluid phase or bound to solid supports

Energy Technology Data Exchange (ETDEWEB)

Kennel, S J

1982-01-01

Rat monoclonal antibody (MoAb) to fragment D (FgD) of human fibrinogen was used to characterize the direct binding of antibody to protein in solution or bound to solid supports. Purified IgG, F(ab')/sub 2/ and Fab' were prepared from ascites fluid of hybridoma 104-14B which is a fusion product of spleen cells from a rat immunized with FgD and the mouse myeloma cell line, P3-X63-Ag8. Two-dimensional electrophoresis of radioiodinated antibody preparations demonstrated the presence of hybrid immunoglobulin molecules, but only structures having rat heavy and rat light chains had active antibody combinig sites. The affinity constant for IgG as well as F(ab')/sub 2/ and Fab', 6x10/sup 9/ M/sup -1/, was identical when tested using fluid phase antigen (/sup 125/I-labeled FgD). Affinity constants determined for direct binding of iodinated IgG using FgD immobilized on solid supports showed a slight dependence on the antigen concentration used in the measurement. These values ranged from 0.5x10/sup 9/ M/sup -1/ at high antigen concentrations (1.3x10/sup -7/ M) to 9x10/sup 9/ M/sup -1/ at low antigen concentration (1.3x10/sup -10/ M). Binding constants for F(ab')/sub 2/ and Fab' gave similar results indicating that binding was homogeneous and univalent. The capacity of solid state antigen to bind antibody varied with the method used to bind FgD to the solid support. FgD bound directly to polystyrene plates was least efficient at binding labeled antibody; FgD bound to plates through intermediate carriers poly(L-lysine) was only slightly more efficient, while antigen bound to Sepharose beads by cyanogen bromide activation was the most active.

Microtubule reorganization in tobacco BY-2 cells stably expressing GFP-MBD

Science.gov (United States)

Granger, C. L.; Cyr, R. J.

2000-01-01

Microtubule organization plays an important role in plant morphogenesis; however, little is known about how microtubule arrays transit from one organized state to another. The use of a genetically incorporated fluorescent marker would allow long-term observation of microtubule behavior in living cells. Here, we have characterized a Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cell line that had been stably transformed with a gfp-mbd construct previously demonstrated to label microtubules (J. Marc et al., 1998, Plant Cell 10: 1927-1939). Fluorescence levels were low, but interphase and mitotic microtubule arrays, as well as the transitions between these arrays, could be observed in individual gfp-mbd-transformed cells. By comparing several attributes of transformed and untransformed cells it was concluded that the transgenic cells are not adversely affected by low-level expression of the transgene and that these cells will serve as a useful and accurate model system for observing microtubule reorganization in vivo. Indeed, some initial observations were made that are consistent with the involvement of motor proteins in the transition between the spindle and phragmoplast arrays. Our observations also support the role of the perinuclear region in nucleating microtubules at the end of cell division with a progressive shift of these microtubules and/or nucleating activity to the cortex to form the interphase cortical array.

A photophysical study of two fluorogen-activating proteins bound to their cognate fluorogens

Energy Technology Data Exchange (ETDEWEB)

Gaiotto, Tiziano [Los Alamos National Laboratory; Nguyen, Hau B [Los Alamos National Laboratory; Jung, Jaemyeong [Los Alamos National Laboratory; Bradbury, Andrew M [Los Alamos National Laboratory; Gnanakaran, S. [Los Alamos National Laboratory; Schmidt, Jurgen G [Los Alamos National Laboratory; Waldo, Geoffrey S [Los Alamos National Laboratory; Goodwin, Peter M [Los Alamos National Laboratory

2010-12-14

We are exploring the feasibility of using recently developed flu orogen-activating proteins (FAPs) as reporters for single-molecule imaging. FAPs are single-chain antibodies choosen to specifically bind small chromophoric molecules termed f1uorogens. Upon binding to its cognate FAP the fluorescence quantum yield of the fluorogen can increase substantially giving rise to a fluorescent complex. Based on the seminal work of Szent-Gyorgyi et al. (Nature Biotechnology, Volume 26, Number 2, pp 235-240, 2008) we have chosen to study two fluorogen-activating single-chain antibodies, HL 1.0.1-TOI and H6-MG bound to their cognate fluorogens, thiazole orange and malachite green derivatives, respectively. Here we use fluorescence correlation spectroscopy study the photophysics of these fluorescent complexes.

Characterization of new cell line stably expressing CHI3L1 oncogene

Directory of Open Access Journals (Sweden)

Chekhonin V. P.

2011-06-01

Full Text Available Aim. To characterize the immortalized 293 cell line after stable transfection with human oncogene (CHI3L1. Methods. 293 cells, stably transfected with pcDNA3.1_CHI3L1, and 293 cells, stably transfected with pcDNA3.1 as a negative control, were used throughout all experiments. The clones of CHI3L1-expressing 293 cells and 293 cells, transfected with pcDNA3.1, were analyzed by immunofluorescence and confocal microscopy. Cell proliferation was measured using MTT assay; analyses of ERK1/2 and AKT activation and their cellular localization were performed with anti-phospho-ERK and anti-phospho-AKT antibodies. Specific activation of MAP and PI3 kinases was measured by densitometric analysis of Western-blot signals. Results. The obtained results show quite modest ability of CHI3L1 to stimulate cell growth and reflect rather an improved cellular plating efficiency of the 293 cells stably transfected with pcDNA3.1_CHI3L1 as compared to the 293 cells transfected with an «empty» vector. ERK1/2 and AKT are activated in the 293_CHI3L1 cells. In these cells phosphorylated ERK1/2 were localized in both cell cytoplasm and nuclei while AKT only in cytoplasm. The 293_CHI3L1 cells differed from the 293 cells, transfected with an «empty» vector, in their size and ability to adhere to the culture plates. Conclusions. The overexpression of CHI3L1 is likely to have an important role in tumorigenesis via a mechanism which involves activation of PI3K and ERK1/2 pathways. The tumors which can be induced by orthotopic implantation of the transformed human cells with overexpressed human oncogene CHI3L1 into the rat brain can be used as a target for anticancer drug development.

Inhibitory profiles of spices against free and protein-bound heterocyclic amines of roast beef patties as revealed by ultra-performance liquid chromatography-tandem mass spectrometry and principal component analysis.

Science.gov (United States)

Chen, Jing; He, Zhiyong; Qin, Fang; Chen, Jie; Cao, Dongsheng; Guo, Fengxian; Zeng, Maomao

2017-11-15

The effects of various levels of chili pepper, Sichuan pepper, and black pepper on the amounts of 17 heterocyclic amines (HAs) from seven categories of both free and protein-bound states in roast beef patties were assessed by ultra-performance liquid chromatography-tandem mass spectrometry combined with principal component analysis. Three groups of HA, including imidazopyridines (DMIP), imidazoquinoxalines (MeIQx and 4,8-MeIQx), and β-carbolines (norharman and harman), were detected and quantified in both their free and protein-bound states, whereas PhIP was detected only in its free state, and imidazoquinolines (IQ, IQ[4,5-b], and MeIQ), α-carbolines (AαC and MeAαC), and phenylpyridines (Phe-P-1) were detected only in their protein-bound states. The results demonstrate that the peppers at all three levels had significant inhibitory effects on free PhIP, DMIP, MeIQx, and 4,8-DiMeIQx and could promote free norharman. Harman was significantly suppressed by chili pepper and black pepper, but enhanced by Sichuan pepper. All 11 protein-bound HAs, with the exception of IQ, IQ[4,5-b], and MeIQx with added chili pepper, were significantly reduced by the three peppers. The total amounts of the free and protein-bound states of all 11 HAs (1692.4 ± 78.9 ng g -1 ), imidazopyridines (5.5 ± 0.2 ng g -1 ), imidazoquinolines (7.2 ± 0.2 ng g -1 ), imidazoquinoxalines (6.9 ± 0.2 ng g -1 ), α-carbolines (20.1 ± 0.4 ng g -1 ), and β-carbolines (1651.7 ± 79.5 ng g -1 ) were suppressed by each level of all of the three peppers except for 0.5% and 1.0% chili pepper. Our findings may facilitate the inhibition of HA formation in the processing of meat products.

Recombinant HT{sub m4} gene, protein and assays

Science.gov (United States)

Lim, B.; Adra, C.N.; Lelias, J.M.

1996-09-03

The invention relates to a recombinant DNA molecule which encodes a HT{sub m4} protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT{sub m4} protein and a recombinant HT{sub m4} protein. The invention also relates to a method for detecting the presence of a hereditary atopy. 2 figs.

Crystal structure of Helicobacter pylori neutrophil-activating protein with a di-nuclear ferroxidase center in a zinc or cadmium-bound form

Energy Technology Data Exchange (ETDEWEB)

Yokoyama, Hideshi, E-mail: h-yokoya@u-shizuoka-ken.ac.jp [School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Tsuruta, Osamu; Akao, Naoya; Fujii, Satoshi [School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan)

2012-06-15

Highlights: Black-Right-Pointing-Pointer Structures of a metal-bound Helicobacter pylori neutrophil-activating protein were determined. Black-Right-Pointing-Pointer Two zinc ions were tetrahedrally coordinated by ferroxidase center (FOC) residues. Black-Right-Pointing-Pointer Two cadmium ions were coordinated in a trigonal-bipyramidal and octahedral manner. Black-Right-Pointing-Pointer The second metal ion was more weakly coordinated than the first at the FOC. Black-Right-Pointing-Pointer A zinc ion was found in one negatively-charged pore suitable as an ion path. -- Abstract: Helicobacter pylori neutrophil-activating protein (HP-NAP) is a Dps-like iron storage protein forming a dodecameric shell, and promotes adhesion of neutrophils to endothelial cells. The crystal structure of HP-NAP in a Zn{sup 2+}- or Cd{sup 2+}-bound form reveals the binding of two zinc or two cadmium ions and their bridged water molecule at the ferroxidase center (FOC). The two zinc ions are coordinated in a tetrahedral manner to the conserved residues among HP-NAP and Dps proteins. The two cadmium ions are coordinated in a trigonal-bipyramidal and distorted octahedral manner. In both structures, the second ion is more weakly coordinated than the first. Another zinc ion is found inside of the negatively-charged threefold-related pore, which is suitable for metal ions to pass through.

Overlapping ETS and CRE Motifs (G/CCGGAAGTGACGTCA) Preferentially Bound by GABPα and CREB Proteins

Science.gov (United States)

Chatterjee, Raghunath; Zhao, Jianfei; He, Ximiao; Shlyakhtenko, Andrey; Mann, Ishminder; Waterfall, Joshua J.; Meltzer, Paul; Sathyanarayana, B. K.; FitzGerald, Peter C.; Vinson, Charles

2012-01-01

Previously, we identified 8-bps long DNA sequences (8-mers) that localize in human proximal promoters and grouped them into known transcription factor binding sites (TFBS). We now examine split 8-mers consisting of two 4-mers separated by 1-bp to 30-bps (X4-N1-30-X4) to identify pairs of TFBS that localize in proximal promoters at a precise distance. These include two overlapping TFBS: the ETS⇔ETS motif (C/GCCGGAAGCGGAA) and the ETS⇔CRE motif (C/GCGGAAGTGACGTCAC). The nucleotides in bold are part of both TFBS. Molecular modeling shows that the ETS⇔CRE motif can be bound simultaneously by both the ETS and the B-ZIP domains without protein-protein clashes. The electrophoretic mobility shift assay (EMSA) shows that the ETS protein GABPα and the B-ZIP protein CREB preferentially bind to the ETS⇔CRE motif only when the two TFBS overlap precisely. In contrast, the ETS domain of ETV5 and CREB interfere with each other for binding the ETS⇔CRE. The 11-mer (CGGAAGTGACG), the conserved part of the ETS⇔CRE motif, occurs 226 times in the human genome and 83% are in known regulatory regions. In vivo GABPα and CREB ChIP-seq peaks identified the ETS⇔CRE as the most enriched motif occurring in promoters of genes involved in mRNA processing, cellular catabolic processes, and stress response, suggesting that a specific class of genes is regulated by this composite motif. PMID:23050235

Structure of a rare non-standard sequence k-turn bound by L7Ae protein

Science.gov (United States)

Huang, Lin; Lilley, David M.J.

2014-01-01

Kt-23 from Thelohania solenopsae is a rare RNA kink turn (k-turn) where an adenine replaces the normal guanine at the 2n position. L7Ae is a member of a strongly conserved family of proteins that bind a range of k-turn structures in the ribosome, box C/D and H/ACA small nucleolar RNAs and U4 small nuclear RNA. We have solved the crystal structure of T. solenopsae Kt-23 RNA bound to Archeoglobus fulgidus L7Ae protein at a resolution of 2.95 Å. The protein binds in the major groove displayed on the outer face of the k-turn, in a manner similar to complexes with standard k-turn structures. The k-turn adopts a standard N3 class conformation, with a single hydrogen bond from A2b N6 to A2n N3. This contrasts with the structure of the same sequence located in the SAM-I riboswitch, where it adopts an N1 structure, showing the inherent plasticity of k-turn structure. This potentially can affect any tertiary interactions in which the RNA participates. PMID:24482444

Identification of peptides from foot‐and‐mouth disease virus structural proteins bound by class I swine leukocyte antigen (SLA) alleles, SLA‐1*0401 and SLA‐2*0401

DEFF Research Database (Denmark)

Pedersen, Lasse Eggers; Harndahl, M.; Nielsen, Morten

2013-01-01

within the structural proteins of foot‐and‐mouth disease virus (FMDV), strain A24 were analyzed as candidate T‐cell epitopes. Peptides predicted by the NetMHCpan were tested in ELISA for binding to the SLA‐1*0401 and SLA‐2*0401 major histocompatibility complex class I proteins. Four of the 10 predicted...... FMDV peptides bound to SLA‐2*0401, whereas five of the nine predicted FMDV peptides bound to SLA‐1*0401. These methods provide the characterization of T‐cell epitopes in response to pathogens in more detail. The development of such approaches to analyze vaccine performance will contribute to a more...

Pressurized liquid extraction-assisted mussel cytosol preparation for the determination of metals bound to metallothionein-like proteins

International Nuclear Information System (INIS)

Santiago-Rivas, Sandra; Moreda-Pineiro, Antonio; Bermejo-Barrera, Pilar; Moreda-Pineiro, Jorge; Alonso-Rodriguez, Elia; Muniategui-Lorenzo, Soledad; Lopez-Mahia, Purificacion; Prada-Rodriguez, Dario

2007-01-01

The possibilities of pressurized liquid extraction (PLE) have been novelty tested to assist the cytosol preparation from wet mussel soft tissue before the determination of metals bound to metallothionein-like proteins (MLPs). Results obtained after PLE were compared with those obtained after a classical blending procedure for mussel cytosolic preparation. Isoforms MLP-1 (retention time of 4.1 min) and MLP-2 (retention time of 7.4 min) were separated by anion exchange high-performance liquid chromatography (HPLC) and the concentrations of Ba, Cu, Mn, Sr and Zn bound to MLP isoforms were directly measured by inductively coupled plasma-atomic emission spectrometry (ICP-OES) as a multi-element detector. The optimized PLE-assisted mussel cytosol preparation has consisted of one extraction cycle at room temperature and 1500 psi for 2 min. Since separation between the solid mussel residue and the extract (cytosol) is performed by the PLE system, the cytosol preparation method is faster than conventional cytosol preparation methods by cutting/blending using Ultraturrax or Stomacher devices

Analysis of protein-bound metabolites of furazolidone and furaltadone in pig liver by high performance liquid chromatography and liquid chromatography mass spectrometry

NARCIS (Netherlands)

Horne, E.; Cadogan, A.; O'Keeffe, M.; Hoogenboom, L.A.P.

1996-01-01

Studies undertaken using radiolabelled furazolidone have demonstrated the covalent binding of residues of the drug to cellular protein in vivo. A portion of these bound residues and those formed by furaltadone, a related nitrofuran drug, possess intact side-chains, 3-amino-2-oxazolidinone (AOZ) and

Functions that Protect Escherichia coli from Tightly Bound DNA-Protein Complexes Created by Mutant EcoRII Methyltransferase.

Science.gov (United States)

Henderson, Morgan L; Kreuzer, Kenneth N

2015-01-01

Expression of mutant EcoRII methyltransferase protein (M.EcoRII-C186A) in Escherichia coli leads to tightly bound DNA-protein complexes (TBCs), located sporadically on the chromosome rather than in tandem arrays. The mechanisms behind the lethality induced by such sporadic TBCs are not well studied, nor is it clear whether very tight binding but non-covalent complexes are processed in the same way as covalent DNA-protein crosslinks (DPCs). Using 2D gel electrophoresis, we found that TBCs induced by M.EcoRII-C186A block replication forks in vivo. Specific bubble molecules were detected as spots on the 2D gel, only when M.EcoRII-C186A was induced, and a mutation that eliminates a specific EcoRII methylation site led to disappearance of the corresponding spot. We also performed a candidate gene screen for mutants that are hypersensitive to TBCs induced by M.EcoRII-C186A. We found several gene products necessary for protection against these TBCs that are known to also protect against DPCs induced with wild-type M.EcoRII (after 5-azacytidine incorporation): RecA, RecBC, RecG, RuvABC, UvrD, FtsK, XerCD and SsrA (tmRNA). In contrast, the RecFOR pathway and Rep helicase are needed for protection against TBCs but not DPCs induced by M.EcoRII. We propose that stalled fork processing by RecFOR and RecA promotes release of tightly bound (but non-covalent) blocking proteins, perhaps by licensing Rep helicase-driven dissociation of the blocking M.EcoRII-C186A. Our studies also argued against the involvement of several proteins that might be expected to protect against TBCs. We took the opportunity to directly compare the sensitivity of all tested mutants to two quinolone antibiotics, which target bacterial type II topoisomerases and induce a unique form of DPC. We uncovered rep, ftsK and xerCD as novel quinolone hypersensitive mutants, and also obtained evidence against the involvement of a number of functions that might be expected to protect against quinolones.

Functions that Protect Escherichia coli from Tightly Bound DNA-Protein Complexes Created by Mutant EcoRII Methyltransferase.

Directory of Open Access Journals (Sweden)

Morgan L Henderson

Full Text Available Expression of mutant EcoRII methyltransferase protein (M.EcoRII-C186A in Escherichia coli leads to tightly bound DNA-protein complexes (TBCs, located sporadically on the chromosome rather than in tandem arrays. The mechanisms behind the lethality induced by such sporadic TBCs are not well studied, nor is it clear whether very tight binding but non-covalent complexes are processed in the same way as covalent DNA-protein crosslinks (DPCs. Using 2D gel electrophoresis, we found that TBCs induced by M.EcoRII-C186A block replication forks in vivo. Specific bubble molecules were detected as spots on the 2D gel, only when M.EcoRII-C186A was induced, and a mutation that eliminates a specific EcoRII methylation site led to disappearance of the corresponding spot. We also performed a candidate gene screen for mutants that are hypersensitive to TBCs induced by M.EcoRII-C186A. We found several gene products necessary for protection against these TBCs that are known to also protect against DPCs induced with wild-type M.EcoRII (after 5-azacytidine incorporation: RecA, RecBC, RecG, RuvABC, UvrD, FtsK, XerCD and SsrA (tmRNA. In contrast, the RecFOR pathway and Rep helicase are needed for protection against TBCs but not DPCs induced by M.EcoRII. We propose that stalled fork processing by RecFOR and RecA promotes release of tightly bound (but non-covalent blocking proteins, perhaps by licensing Rep helicase-driven dissociation of the blocking M.EcoRII-C186A. Our studies also argued against the involvement of several proteins that might be expected to protect against TBCs. We took the opportunity to directly compare the sensitivity of all tested mutants to two quinolone antibiotics, which target bacterial type II topoisomerases and induce a unique form of DPC. We uncovered rep, ftsK and xerCD as novel quinolone hypersensitive mutants, and also obtained evidence against the involvement of a number of functions that might be expected to protect against quinolones.

Human Serum Protein-Bound iodine and Protein Fractions at ...

African Journals Online (AJOL)

Iodine profile of Nigerians at different ages in both sexes and in pregnant women, and under narcotic influence, such as alcoholism, cigarette smoking and marijuana addiction were studied. Their serum total protein, albumin and globulin concentrations were also determined. Results of the study showed that serum protein ...

Establishment of a human cell line stably overexpressing mouse Nip45 and characterization of Nip45 subcellular localization

Energy Technology Data Exchange (ETDEWEB)

Hashiguchi, Kohtaro; Ozaki, Masumi [Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto (Japan); Kuraoka, Isao [Biological Chemistry Group, Division of Chemistry, Graduate School of Engineering Science, Osaka University, Osaka (Japan); Saitoh, Hisato, E-mail: hisa@kumamoto-u.ac.jp [Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto (Japan); Department of New Frontier Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto (Japan); Global COE (Centers of Excellence) Program, Global Initiative Center for Pulsed Power Engineering, Kumamoto University, Kumamoto (Japan)

2013-01-04

Highlights: Black-Right-Pointing-Pointer A human cell line expressing a mouse Nip45 has facilitated Nip45 analysis. Black-Right-Pointing-Pointer Nip45 does not effectively inhibit polySUMOylation in vivo. Black-Right-Pointing-Pointer Nip45 interacts directly with SUMO and SUMO chains. Black-Right-Pointing-Pointer Nip45 accumulates at PML bodies in response to proteasome inhibition. -- Abstract: The nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 2 interacting protein, Nfatc2ip (Nip45), has been implicated as a crucial coordinator of the immune response and of cellular differentiation in humans and mice, and contains SUMO-like domains in its C-terminal region. However, the significance of its N-terminal region and its correlation to the SUMO modification pathway remain largely uncharacterized. In this study, a human cultured cell line was established, in which FLAG-tagged mouse Nip45 (FLAG-mNip45) was stably overexpressed. Under standard, non-stressful conditions, we detected FLAG-mNip45 diffusely distributed in the nucleus. Intriguingly, proteasome inhibition by MG132 caused FLAG-mNip45, together with SUMOylated proteins, to localize in nuclear domains associated with promyelocytic leukemia protein. Finally, using an in vitro binding assay, we showed interaction of the N-terminal region of mNip45 with both free SUMO-3 and SUMO-3 chains, indicating that Nip45 may, in part, exert its function via interaction with SUMO/SUMOylated proteins. Taken together, our study provides novel information on a poorly characterized mammalian protein and suggests that our newly established cell line will be useful for elucidating the physiological role of Nip45.

A Study of Lipid- and Protein- Bound Sialic Acids for the Diagnosis of Bladder Cancer and Their Relationships with the Severity of Malignancy

Directory of Open Access Journals (Sweden)

Shima Habibi

2014-05-01

Full Text Available Background: The gold standard for detection of bladder cancer is cystoscopy, which is an invasive and complicated procedure. Our study was conducted to find a tumor marker with high specificity, sensitivity, and accuracy for the diagnosis of bladder cancer. Methods: Serum samples were collected from 58 bladder cancer patients and 60 healthy control subjects. Levels of lipid-bound sialic acid (LBSA, and protein-bound sialic acid (PBSA were measured spectrophotometrically by Aminoff’s method. Results: Mean levels of both markers were found to be significantly higher in the patients than the healthy controls. Positive correlations were observed between serum levels of lipid- (r=0.283, p<0.05 and protein- bound (r=0.56, p<0.05 sialic acids and the grade of malignancy. To differentiate patients with bladder tumors from healthy controls, cut-offpoints were determined for each of the two parameters based on Receiver Operating Characteristic (ROC curve analysis (LBSA=21.25 mg/dL, PBSA=6.15 mg/dL. The data showed good sensitivities (LBSA=89%, PBSA=79%, specificities (LBSA=70%, PBSA=70% and accuracies (LBSA=83%, PBSA=81% for both markers. Conclusion: Measuring serum LBSA and PBSA by this simple, reproducible, noninvasive, and inexpensive method can accurately discriminate cancer patients from healthy individuals.

The Role of Physical Therapists in Living Environment Maintenance of the Home-bound Elderly Disabled.

Science.gov (United States)

Takemasa, Seiichi; Abe, Yoko; Nagao, Toru; Murakami, Masahito; Koeda, Hideki; Naruse, Susumu; Gotou, Makoto; Uesugi, Masayuki; Inoue, Yuri; Nanba, Yoshihumi

2013-07-01

[Purpose] This study explored the roles of physical therapists (PTs) in living environment maintenance, which is essential for living securely and stably at home, and examines how physical therapists can fulfill these roles more efficiently and effectively. [Subjects and Methods] A questionnaire on living environment maintenance was given to PTs working at randomly selected hospitals, health care facilities for the elderly requiring long-term care, home-visit nursing stations, and other such facilities and directly providing physical therapy to the home-bound elderly disabled. The subjects of the study were 77 PTs who returned valid responses. [Results] For awareness of systems for living environment maintenance, PTs were more aware of the system based on the Long-Term Care (LTC) Insurance Act than the system based on the Act on Welfare for the Home-Bound Elderly Disabled. PTs who have worked at two or more types of medical, welfare, and intermediate institutions were more aware of such systems than PTs who have worked at only one type. For PTs handling living environment maintenance for the home-bound elderly disabled, approximately 80% of respondents answered that they have handled some living environment maintenance, and PTs with longer clinical experience have handled more living environment maintenance cases. [Conclusion] The results demonstrated that PTs understand their living environment maintenance work well and handle the work. The results, however, also suggested that educational and operational improvements are urgently required for PTs handling living environment maintenance essential for the lives of the home-bound elderly disabled.

Steric Pressure among Membrane-Bound Polymers Opposes Lipid Phase Separation.

Science.gov (United States)

Imam, Zachary I; Kenyon, Laura E; Carrillo, Adelita; Espinoza, Isai; Nagib, Fatema; Stachowiak, Jeanne C

2016-04-19

Lipid rafts are thought to be key organizers of membrane-protein complexes in cells. Many proteins that interact with rafts have bulky polymeric components such as intrinsically disordered protein domains and polysaccharide chains. Therefore, understanding the interaction between membrane domains and membrane-bound polymers provides insights into the roles rafts play in cells. Multiple studies have demonstrated that high concentrations of membrane-bound polymeric domains create significant lateral steric pressure at membrane surfaces. Furthermore, our recent work has shown that lateral steric pressure at membrane surfaces opposes the assembly of membrane domains. Building on these findings, here we report that membrane-bound polymers are potent suppressors of membrane phase separation, which can destabilize lipid domains with substantially greater efficiency than globular domains such as membrane-bound proteins. Specifically, we created giant vesicles with a ternary lipid composition, which separated into coexisting liquid ordered and disordered phases. Lipids with saturated tails and poly(ethylene glycol) (PEG) chains conjugated to their head groups were included at increasing molar concentrations. When these lipids were sparse on the membrane surface they partitioned to the liquid ordered phase. However, as they became more concentrated, the fraction of GUVs that were phase-separated decreased dramatically, ultimately yielding a population of homogeneous membrane vesicles. Experiments and physical modeling using compositions of increasing PEG molecular weight and lipid miscibility phase transition temperature demonstrate that longer polymers are the most efficient suppressors of membrane phase separation when the energetic barrier to lipid mixing is low. In contrast, as the miscibility transition temperature increases, longer polymers are more readily driven out of domains by the increased steric pressure. Therefore, the concentration of shorter polymers required

Turbulent entrainment across turbulent-nonturbulent interfaces in stably stratified mixing layers

Science.gov (United States)

Watanabe, T.; Riley, J. J.; Nagata, K.

2017-10-01

The entrainment process in stably stratified mixing layers is studied in relation to the turbulent-nonturbulent interface (TNTI) using direct numerical simulations. The statistics are calculated with the interface coordinate in an Eulerian frame as well as with the Lagrangian fluid particles entrained from the nonturbulent to the turbulent regions. The characteristics of entrainment change as the buoyancy Reynolds number Reb decreases and the flow begins to layer. The baroclinic torque delays the enstrophy growth of the entrained fluids at small Reb, while this effect is less efficient for large Reb. The entrained particle movement within the TNTI layer is dominated by the small dissipative scales, and the rapid decay of the kinetic energy dissipation rate due to buoyancy causes the entrained particle movement relative to the interface location to become slower. Although the Eulerian statistics confirm that there exists turbulent fluid with strong vorticity or with large buoyancy frequency near the TNTI, the entrained fluid particles circumvent these regions by passing through the TNTI in strain-dominant regions or in regions with small buoyancy frequency. The multiparticle statistics show that once the nonturbulent fluid volumes are entrained, they are deformed into flattened shapes in the vertical direction and diffuse in the horizontal direction. When Reb is large enough for small-scale turbulence to exist, the entrained fluid is able to penetrate into the turbulent core region. Once the flow begins to layer with decreasing Reb, however, the entrained fluid volume remains near the outer edge of the turbulent region and forms a stably stratified layer without vertical overturning.

Insight into partial agonism by observing multiple equilibria for ligand-bound and Gs-mimetic nanobody-bound β1-adrenergic receptor.

Science.gov (United States)

Solt, Andras S; Bostock, Mark J; Shrestha, Binesh; Kumar, Prashant; Warne, Tony; Tate, Christopher G; Nietlispach, Daniel

2017-11-27

A complex conformational energy landscape determines G-protein-coupled receptor (GPCR) signalling via intracellular binding partners (IBPs), e.g., G s and β-arrestin. Using 13 C methyl methionine NMR for the β 1 -adrenergic receptor, we identify ligand efficacy-dependent equilibria between an inactive and pre-active state and, in complex with G s -mimetic nanobody, between more and less active ternary complexes. Formation of a basal activity complex through ligand-free nanobody-receptor interaction reveals structural differences on the cytoplasmic receptor side compared to the full agonist-bound nanobody-coupled form, suggesting that ligand-induced variations in G-protein interaction underpin partial agonism. Significant differences in receptor dynamics are observed ranging from rigid nanobody-coupled states to extensive μs-to-ms timescale dynamics when bound to a full agonist. We suggest that the mobility of the full agonist-bound form primes the GPCR to couple to IBPs. On formation of the ternary complex, ligand efficacy determines the quality of the interaction between the rigidified receptor and an IBP and consequently the signalling level.

Crystallization and preliminary X-ray diffraction analysis of phospholipid-bound Sfh1p, a member of the Saccharomyces cerevisiae Sec14p-like phosphatidylinositol transfer protein family

International Nuclear Information System (INIS)

Schaaf, Gabriel; Betts, Laurie; Garrett, Teresa A.; Raetz, Christian R. H.; Bankaitis, Vytas A.

2006-01-01

Yeast Sfh1p, a close homolog of the Sec14p phosphatidylinositol transfer protein, was crystallized in the absence of detergent. X-ray data have been collected to 2.5 Å. Sec14p is the major phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer protein in the budding yeast Saccharomyces cerevisiae and is the founding member of a large eukaryotic protein superfamily. This protein catalyzes the exchange of either PtdIns or PtdCho between membrane bilayers in vitro and this exchange reaction requires no external input of energy or of other protein cofactors. Despite the previous elucidation of the crystal structure of a detergent-bound form of Sec14p, the conformational changes that accompany the phospholipid-exchange reaction remain undefined. Moreover, a structural appreciation of how Sec14p or its homologs bind their various phospholipid substrates remains elusive. Here, the purification and crystallization of yeast Sfh1p, the protein most closely related to Sec14p, are reported. A combination of electrospray ionization mass-spectrometry and collision-induced decomposition mass-spectrometry methods indicate that recombinant Sfh1p loads predominantly with phosphatidylethanolamine. Unlike phospholipid-bound forms of Sec14p, this form of Sfh1p crystallizes readily in the absence of detergent. Sfh1p crystals diffract to 2.5 Å and belong to the orthorhombic primitive space group P2 1 2 1 2 1 , with unit-cell parameters a = 49.40, b = 71.55, c = 98.21 Å, α = β = γ = 90°. One Sfh1p molecule is present in the asymmetric unit (V M = 2.5 Å 3 Da −1 ; V s = 50%). Crystallization of a phospholipid-bound Sec14p-like protein is a critical first step in obtaining the first high-resolution picture of how proteins of the Sec14p superfamily bind their phospholipid ligands. This information will significantly extend our current understanding of how Sec14p-like proteins catalyze phospholipid exchange

Crystal Structures of Apo and Metal-Bound Forms of the UreE Protein from Helicobacter pylori: Role of Multiple Metal Binding Sites

Energy Technology Data Exchange (ETDEWEB)

Shi, Rong; Munger, Christine; Asinas, Abdalin; Benoit, Stephane L.; Miller, Erica; Matte, Allan; Maier, Robert J.; Cygler, Miroslaw (McGill); (Georgia); (Biotech Res.)

2010-10-22

The crystal structure of the urease maturation protein UreE from Helicobacter pylori has been determined in its apo form at 2.1 {angstrom} resolution, bound to Cu{sup 2+} at 2.7 {angstrom} resolution, and bound to Ni{sup 2+} at 3.1 {angstrom} resolution. Apo UreE forms dimers, while the metal-bound enzymes are arranged as tetramers that consist of a dimer of dimers associated around the metal ion through coordination by His102 residues from each subunit of the tetramer. Comparison of independent subunits from different crystal forms indicates changes in the relative arrangement of the N- and C-terminal domains in response to metal binding. The improved ability of engineered versions of UreE containing hexahistidine sequences at either the N-terminal or C-terminal end to provide Ni{sup 2+} for the final metal sink (urease) is eliminated in the H102A version. Therefore, the ability of the improved Ni{sup 2+}-binding versions to deliver more nickel is likely an effect of an increased local concentration of metal ions that can rapidly replenish transferred ions bound to His102.

How Diverse are the Protein-Bound Conformations of Small-Molecule Drugs and Cofactors?

Science.gov (United States)

Friedrich, Nils-Ole; Simsir, Méliné; Kirchmair, Johannes

2018-03-01

Knowledge of the bioactive conformations of small molecules or the ability to predict them with theoretical methods is of key importance to the design of bioactive compounds such as drugs, agrochemicals and cosmetics. Using an elaborate cheminformatics pipeline, which also evaluates the support of individual atom coordinates by the measured electron density, we compiled a complete set (“Sperrylite Dataset”) of high-quality structures of protein-bound ligand conformations from the PDB. The Sperrylite Dataset consists of a total of 10,936 high-quality structures of 4548 unique ligands. Based on this dataset, we assessed the variability of the bioactive conformations of 91 small molecules—each represented by a minimum of ten structures—and found it to be largely independent of the number of rotatable bonds. Sixty-nine molecules had at least two distinct conformations (defined by an RMSD greater than 1 Å). For a representative subset of 17 approved drugs and cofactors we observed a clear trend for the formation of few clusters of highly similar conformers. Even for proteins that share a very low sequence identity, ligands were regularly found to adopt similar conformations. For cofactors, a clear trend for extended conformations was measured, although in few cases also coiled conformers were observed. The Sperrylite Dataset is available for download from http://www.zbh.uni-hamburg.de/sperrylite_dataset.

Nutritional supplements modulate fluorescent protein-bound advanced glycation endproducts and digestive enzymes related to type 2 diabetes mellitus.

Science.gov (United States)

Koch, Emily R; Deo, Permal

2016-09-01

Chronic hyperglycemia enhances the formation of advanced glycation endproducts (AGEs) and reactive oxygen species (ROS), contributing to diabetic complications. Thus, controlling blood glucose levels, inhibiting the formation of AGEs and reducing ROS are key therapeutic targets in early stage type 2 diabetes. The inhibitory effects of seven commercial liquid nutritional supplements against carbohydrate hydrolysing enzymes, α-amylase and α-glucosidase, was determined by dinitrosalicylic (DNS) reagent and p-nitrophenyl-α-D-glucopyranoside solution, respectively. Antiglycation activity was determined using the formation of fluorescent protein-bound AGEs. Total phenolic and flavonoid content and antioxidant properties (1,1-diphenyl-2-picrylhydrazyl antioxidant activity (DPPH) and ferric reducing antioxidant power (FRAP)) were determined for correlation among these components and inhibitory activities. Samoan noni juice showed the greatest inhibitory effects against α-amylase, whereas chlorophyll extracts showed the greatest inhibitory effect against α-glucosidase. Inhibition of α-glucosidase correlated with TFC (r(2) = 0.766; p 1) and FRAP (r(2) = 0.750; p 1) whereas no correlation was observed for α-amylase inhibition. All supplements inhibited fluorescent protein-bound AGEs, with the greatest effect exerted by Olive Leaf Extract, Blood Sugar Support (IC50 = 0.5 mg/ml). The IC50 values negatively correlated with TPC (r(2) = -0.707; p 1) and DPPH scavenging activities (r(2) = 0.515; p nutritional supplements in managing and treating type 2 diabetes mellitus.

Quantitative Evaluation of Myostatin Gene in Stably Transfected Caprine Fibroblast Cells by Anti-Myostatin shRNA.

Science.gov (United States)

Jain, Sudhir Kumar; Jain, Hemlata; Kumar, Dharmendra; Bedekar, Megha Kadam; Pandey, Akhilesh Kumar; Sarkhel, Bikash Chandra

2015-09-01

Skeletal muscle is the major component of lean tissue that is used for consumption, and myostatin is a negative regulator of skeletal muscle growth. Downregulation of this gene therefore offers a strategy for developing superior animals with enhanced muscle growth. Knockdown of myostatin was achieved by RNA interference technology. The anti-myostatin shRNA were designed and stably transfected in caprine fibroblast cells. The reduced expression of target gene was achieved and measured in clonal fibroblast cells by real-time PCR. Two single-cell clones induced significant decrease of myostatin gene expression by 73.96 and 72.66 %, respectively (P < 0.05). To ensure the appropriate growth of transfected cell, seven media were tested. The best suited media was used for transfected fibroblast cell proliferation. The findings suggest that shRNA provides a novel potential tool for gene knockdown and these stably transfected cells can be used as the donor cells for animal cloning.

PROTEIN TARGETING TO STARCH is required for localising GRANULE-BOUND STARCH SYNTHASE to starch granules and for normal amylose synthesis in Arabidopsis.

Directory of Open Access Journals (Sweden)

David Seung

2015-02-01

Full Text Available The domestication of starch crops underpinned the development of human civilisation, yet we still do not fully understand how plants make starch. Starch is composed of glucose polymers that are branched (amylopectin or linear (amylose. The amount of amylose strongly influences the physico-chemical behaviour of starchy foods during cooking and of starch mixtures in non-food manufacturing processes. The GRANULE-BOUND STARCH SYNTHASE (GBSS is the glucosyltransferase specifically responsible for elongating amylose polymers and was the only protein known to be required for its biosynthesis. Here, we demonstrate that PROTEIN TARGETING TO STARCH (PTST is also specifically required for amylose synthesis in Arabidopsis. PTST is a plastidial protein possessing an N-terminal coiled coil domain and a C-terminal carbohydrate binding module (CBM. We discovered that Arabidopsis ptst mutants synthesise amylose-free starch and are phenotypically similar to mutants lacking GBSS. Analysis of granule-bound proteins showed a dramatic reduction of GBSS protein in ptst mutant starch granules. Pull-down assays with recombinant proteins in vitro, as well as immunoprecipitation assays in planta, revealed that GBSS physically interacts with PTST via a coiled coil. Furthermore, we show that the CBM domain of PTST, which mediates its interaction with starch granules, is also required for correct GBSS localisation. Fluorescently tagged Arabidopsis GBSS, expressed either in tobacco or Arabidopsis leaves, required the presence of Arabidopsis PTST to localise to starch granules. Mutation of the CBM of PTST caused GBSS to remain in the plastid stroma. PTST fulfils a previously unknown function in targeting GBSS to starch. This sheds new light on the importance of targeting biosynthetic enzymes to sub-cellular sites where their action is required. Importantly, PTST represents a promising new gene target for the biotechnological modification of starch composition, as it is

A germline chromothripsis event stably segregating in 11 individuals through three generations

DEFF Research Database (Denmark)

Bertelsen, Birgitte; Nazaryan-Petersen, Lusine; Sun, Wei

2016-01-01

PURPOSE: Parentally transmitted germ-line chromothripsis (G-CTH) has been identified in only a few cases. Most of these rearrangements were stably transmitted, in an unbalanced form, from a healthy mother to her child with congenital abnormalities probably caused by de novo copy-number changes...... of the DNA damage response, may be related to G-CTH formation. CONCLUSION: G-CTH rearrangements are not always associated with abnormal phenotypes and may be misinterpreted as balanced two-way translocations, suggesting that G-CTH is an underdiagnosed phenomenon.Genet Med 18 5, 494-500....

G protein- and agonist-bound serotonin 5-HT2A receptor model activated by steered molecular dynamics simulations

DEFF Research Database (Denmark)

Ísberg, Vignir; Balle, Thomas; Sander, Tommy

2011-01-01

molecular dynamics (MD) simulations. The driving force for the transformation was the addition of several known intermolecular and receptor interhelical hydrogen bonds enforcing the necessary helical and rotameric movements. Subsquent MD simulations without constraints confirmed the stability......A 5-HT(2A) receptor model was constructed by homology modeling based on the ß(2)-adrenergic receptor and the G protein-bound opsin crystal structures. The 5-HT(2A) receptor model was transferred into an active conformation by an agonist ligand and a G(aq) peptide in four subsequent steered...

Dissociation and purification of the endogenous membrane-bound Vo complex from Pichia pastoris.

Science.gov (United States)

Li, Sumei; Hong, Tao; Wang, Kun; Lu, Yinghong; Zhou, Min

2017-10-01

Most proteins occur and function in complexes rather than as isolated entities in membranes. In most cases macromolecules with multiple subunits are purified from endogenous sources. In this study, an endogenous membrane-protein complex was obtained from Pichia pastoris, which can be grown at high densities to significantly improve the membrane protein yield. We successfully isolated the membrane-bound Vo complex of V-ATPase from P. pastoris using a fusion FLAG tag attached to the C-terminus of subunit a to generate the vph-tag strain, which was used for dissociation and purification. After FLAG affinity and size exclusion chromatography purification, the production quantity and purity of the membrane-bound Vo complex was 20 μg l -1 and >98%, respectively. The subunits of the endogenous membrane-bound Vo complex observed in P. pastoris were similar to those obtained from S. cerevisiae, as demonstrated by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Therefore, successful dissociation and purification of the membrane-bound Vo complex at a high purity and sufficient quantity was achieved via a rapid and simple procedure that can be used to obtain the endogenous membrane-protein complexes from P. pastoris. Copyright © 2017 Elsevier Inc. All rights reserved.

Grb-IR: A SH2-Domain-Containing Protein that Binds to the Insulin Receptor and Inhibits Its Function

Science.gov (United States)

Liu, Feng; Roth, Richard A.

1995-10-01

To identify potential signaling molecules involved in mediating insulin-induced biological responses, a yeast two-hybrid screen was performed with the cytoplasmic domain of the human insulin receptor (IR) as bait to trap high-affinity interacting proteins encoded by human liver or HeLa cDNA libraries. A SH2-domain-containing protein was identified that binds with high affinity in vitro to the autophosphorylated IR. The mRNA for this protein was found by Northern blot analyses to be highest in skeletal muscle and was also detected in fat by PCR. To study the role of this protein in insulin signaling, a full-length cDNA encoding this protein (called Grb-IR) was isolated and stably expressed in Chinese hamster ovary cells overexpressing the human IR. Insulin treatment of these cells resulted in the in situ formation of a complex of the IR and the 60-kDa Grb-IR. Although almost 75% of the Grb-IR protein was bound to the IR, it was only weakly tyrosine-phosphorylated. The formation of this complex appeared to inhibit the insulin-induced increase in tyrosine phosphorylation of two endogenous substrates, a 60-kDa GTPase-activating-protein-associated protein and, to a lesser extent, IR substrate 1. The subsequent association of this latter protein with phosphatidylinositol 3-kinase also appeared to be inhibited. These findings raise the possibility that Grb-IR is a SH2-domain-containing protein that directly complexes with the IR and serves to inhibit signaling or redirect the IR signaling pathway.

The human multidrug resistance-associated protein MRP is a plasma membrane drug-efflux pump

NARCIS (Netherlands)

Zaman, G. J.; Flens, M. J.; van Leusden, M. R.; de Haas, M.; Mülder, H. S.; Lankelma, J.; Pinedo, H. M.; Scheper, R. J.; Baas, F.; Broxterman, H. J.

1994-01-01

The multidrug-resistance associated protein MRP is a 180- to 195-kDa membrane protein associated with resistance of human tumor cells to cytotoxic drugs. We have investigated how MRP confers drug resistance in SW-1573 human lung carcinoma cells by generating a subline stably transfected with an

Production of recombinant cholesterol oxidase containing covalently bound FAD in Escherichia coli

Directory of Open Access Journals (Sweden)

Molla Gianluca

2010-04-01

Full Text Available Abstract Background Cholesterol oxidase is an alcohol dehydrogenase/oxidase flavoprotein that catalyzes the dehydrogenation of C(3-OH of cholesterol. It has two major biotechnological applications, i.e. in the determination of serum (and food cholesterol levels and as biocatalyst providing valuable intermediates for industrial steroid drug production. Cholesterol oxidases of type I are those containing the FAD cofactor tightly but not covalently bound to the protein moiety, whereas type II members contain covalently bound FAD. This is the first report on the over-expression in Escherichia coli of type II cholesterol oxidase from Brevibacterium sterolicum (BCO. Results Design of the plasmid construct encoding the mature BCO, optimization of medium composition and identification of the best cultivation/induction conditions for growing and expressing the active protein in recombinant E. coli cells, concurred to achieve a valuable improvement: BCO volumetric productivity was increased from ~500 up to ~25000 U/L and its crude extract specific activity from 0.5 up to 7.0 U/mg protein. Interestingly, under optimal expression conditions, nearly 55% of the soluble recombinant BCO is produced as covalently FAD bound form, whereas the protein containing non-covalently bound FAD is preferentially accumulated in insoluble inclusion bodies. Conclusions Comparison of our results with those published on non-covalent (type I COs expressed in recombinant form (either in E. coli or Streptomyces spp., shows that the fully active type II BCO can be produced in E. coli at valuable expression levels. The improved over-production of the FAD-bound cholesterol oxidase will support its development as a novel biotool to be exploited in biotechnological applications.

Turbulent fluxes in stably stratified boundary layers

International Nuclear Information System (INIS)

L'vov, Victor S; Procaccia, Itamar; Rudenko, Oleksii

2008-01-01

We present here an extended version of an invited talk we gave at the international conference 'Turbulent Mixing and Beyond'. The dynamical and statistical description of stably stratified turbulent boundary layers with the important example of the stable atmospheric boundary layer in mind is addressed. Traditional approaches to this problem, based on the profiles of mean quantities, velocity second-order correlations and dimensional estimates of the turbulent thermal flux, run into a well-known difficulty, predicting the suppression of turbulence at a small critical value of the Richardson number, in contradiction to observations. Phenomenological attempts to overcome this problem suffer from various theoretical inconsistencies. Here, we present an approach taking into full account all the second-order statistics, which allows us to respect the conservation of total mechanical energy. The analysis culminates in an analytic solution of the profiles of all mean quantities and all second-order correlations, removing the unphysical predictions of previous theories. We propose that the approach taken here is sufficient to describe the lower parts of the atmospheric boundary layer, as long as the Richardson number does not exceed an order of unity. For much higher Richardson numbers, the physics may change qualitatively, requiring careful consideration of the potential Kelvin-Helmoholtz waves and their interaction with the vortical turbulence.

Turbulent circulation above the surface heat source in a stably stratified environment

Science.gov (United States)

Kurbatskii, A. F.; Kurbatskaya, L. I.

2016-09-01

The results of the numerical modeling of turbulent structure of the penetrating convection above the urban heat island with a small aspect ratio in a stably stratified medium at rest are presented. The gradient diffusion representations for turbulent momentum and heat fluxes are used, which depend on three parameters — the turbulence kinetic energy, the velocity of its spectral expenditure, and the dispersion of temperature fluctuations. These parameters are found from the closed differential equations of balance in the RANS approach of turbulence description. The distributions of averaged velocity and temperature fields as well as turbulent characteristics agree well with measurement data.

Microstructure of Turbulence in the Stably Stratified Boundary Layer

Science.gov (United States)

Sorbjan, Zbigniew; Balsley, Ben B.

2008-11-01

The microstructure of a stably stratified boundary layer, with a significant low-level nocturnal jet, is investigated based on observations from the CASES-99 campaign in Kansas, U.S.A. The reported, high-resolution vertical profiles of the temperature, wind speed, wind direction, pressure, and the turbulent dissipation rate, were collected under nocturnal conditions on October 14, 1999, using the CIRES Tethered Lifting System. Two methods for evaluating instantaneous (1-sec) background profiles are applied to the raw data. The background potential temperature is calculated using the “bubble sort” algorithm to produce a monotonically increasing potential temperature with increasing height. Other scalar quantities are smoothed using a running vertical average. The behaviour of background flow, buoyant overturns, turbulent fluctuations, and their respective histograms are presented. Ratios of the considered length scales and the Ozmidov scale are nearly constant with height, a fact that can be applied in practice for estimating instantaneous profiles of the dissipation rate.

[Establishment and application of a Vero cell line stably expressing raccoon dog SLAM, the cellular receptor of canine distemper virus].

Science.gov (United States)

Zhao, Jianjun; Yan, Ruxun; Zhang, Hailing; Zhang, Lei; Hu, Bo; Bai, Xue; Shao, Xiqun; Chai, Xiuli; Yan, Xijun; Wu, Wei

2012-12-04

The signaling lymphocyte activation molecule (SLAM, also known as CD150), is used as a cellular receptor by canine distemper virus (CDV). Wild-type strains of CDVs can be isolated and propagated efficiently in non-lymphoid cells expressing this protein. Our aim is to establish a Vero cells expressing raccoon dog SLAM (rSLAM) to efficiently isolate CDV from pathological samples. A eukaryotic expression plasmid, pIRES2-EGFP-rSLAMhis, containing rSLAM gene fused with six histidine-coding sequence, EGFP gene, and neomycin resistance gene was constructed. After transfection with the plasmid, a stable cell line, Vero-rSLAM, was screened from Vero cells with the identification of EGFP reporter and G418 resistance. Three CD positive specimens from infected foxes and raccoon dogs were inoculated to Vero-rSLAM cells for CDV isolation. Foxes and raccoon dogs were inoculated subcutaneously LN (10)fl strain with 4 x 10(2.39)TCID50 dose to evaluate pathogenicity of CDV isolations. The rSLAMh fused gene was shown to transcript and express stably in Vero-rSLAM cells by RT-PCR and Immunohistochemistry assay. Three CDV strains were isolated successfully in Vero-rSLAM cells 36 -48 hours after inoculation with spleen or lung specimens from foxes and raccoon dogs with distemper. By contrast, no CDV was recovered from those CD positive specimens when Vero cells were used for virus isolation. Infected foxes and raccoon dogs with LN(10)f1 strain all showed typical CD symptoms and high mortality (2/3 for foxes and 3/3 for raccoon dogs) in 22 days post challenge. Our results indicate that Vero-rSLAM cells stably expressing raccoon dog SLAM are highly sensitive to CDV in clinical specimens and the CDV isolation can maintain high virulence to its host animals.

Determination of total, and bound Se in sera by INAA

International Nuclear Information System (INIS)

Spate, V.L.; Mason, M.M.; Reams, C.L.; Baskett, C.K.; Morris, J.S.; Mills, D.S.

1994-01-01

The comparison between the total selenium in serum ('total Se') with that which is apparently bound to high molecular weight (> 12.000 D) species, presumably proteins ('bound Se') was reported. Nine hundred seventy seven (977) serum samples arising out of a population-based epidemiological study were prepared in duplicate for the determination of 'total Se' by pipeting directly into irradiation vials; and separate duplicate aliquots were dialyzed against DI water for the determination of 'bound Se'. All samples were analyzed by neutron activation analysis via 77m Se (17.4s) A small dialyzable Se component (∼ 6%) ('free Se'), defined as the difference between the 'total Se' minus the 'bound Se', was identified. (author) 2 refs.; 3 figs.; 5 tabs

Interferon-inducible MyD88 protein inhibits hepatitis B virus replication

International Nuclear Information System (INIS)

Xiong Wei; Wang Xun; Liu Xiaoying; Xiang Li; Zheng Lingjie; Yuan Zhenghong

2004-01-01

Myeloid differential primary response protein (MyD88) is a critical component in the signaling cascade through Toll-like receptors (TLRs) and is induced by α interferon (IFN-α). To examine the role of MyD88 in the antiviral activity of IFN-α against hepatitis B virus (HBV), we established MyD88 stably expressing cell lines and studied HBV replication in these lines after transient transfection. The levels of HBV proteins and viral replicative intermediates were effectively reduced in MyD88-expressing cells. A significant reduction of total and cytoplasmic viral RNAs in MyD88 stably expressing cells was also observed. Using a nuclear factor-κB (NF-κB) dependent reporter assay, it was shown that activation of NF-κB was moderately increased in the presence of expression of MyD88, and further significantly increased by co-expression of HBV. These results suggest a novel mechanism for the inhibition of HBV replication by IFN-α via expression of MyD88 protein involving activation of NF-κB signaling pathway and downregulation of viral transcription

Conformational Sampling in Template-Free Protein Loop Structure Modeling: An Overview

OpenAIRE

Li, Yaohang

2013-01-01

Accurately modeling protein loops is an important step to predict three-dimensional structures as well as to understand functions of many proteins. Because of their high flexibility, modeling the three-dimensional structures of loops is difficult and is usually treated as a “mini protein folding problem” under geometric constraints. In the past decade, there has been remarkable progress in template-free loop structure modeling due to advances of computational methods as well as stably increas...

High current relativistic beam propagates stably in gas surrounded by nonconducting walls

International Nuclear Information System (INIS)

Clark, J.C.

1977-01-01

LLL has been studying the propagation of high current electron beams for a number of years to understand their behavior for use in a variety of experimental uses. Our latest experiments have shown that a mildly relativistic electron beam of 10 to 15 kA and a pulse width of 30 to 40 ns can propagate stably and with no net current transfer in insulating tubes filled with neutral gases. These experiments have been performed in the Magnetic Fusion Energy program where Electronics Engineering has been operating an electron beam accelerator, designing some of the diagnostics, such as laser interferometers, and performing the experiments. This article briefly describes our experimental observations

A Lithium-Air Battery Stably Working at High Temperature with High Rate Performance.

Science.gov (United States)

Pan, Jian; Li, Houpu; Sun, Hao; Zhang, Ye; Wang, Lie; Liao, Meng; Sun, Xuemei; Peng, Huisheng

2018-02-01

Driven by the increasing requirements for energy supply in both modern life and the automobile industry, the lithium-air battery serves as a promising candidate due to its high energy density. However, organic solvents in electrolytes are likely to rapidly vaporize and form flammable gases under increasing temperatures. In this case, serious safety problems may occur and cause great harm to people. Therefore, a kind of lithium-air that can work stably under high temperature is desirable. Herein, through the use of an ionic liquid and aligned carbon nanotubes, and a fiber shaped design, a new type of lithium-air battery that can effectively work at high temperatures up to 140 °C is developed. Ionic liquids can offer wide electrochemical windows and low vapor pressures, as well as provide high thermal stability for lithium-air batteries. The aligned carbon nanotubes have good electric and heat conductivity. Meanwhile, the fiber format can offer both flexibility and weavability, and realize rapid heat conduction and uniform heat distribution of the battery. In addition, the high temperature has also largely improved the specific powers by increasing the ionic conductivity and catalytic activity of the cathode. Consequently, the lithium-air battery can work stably at 140 °C with a high specific current of 10 A g -1 for 380 cycles, indicating high stability and good rate performance at high temperatures. This work may provide an effective paradigm for the development of high-performance energy storage devices. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Physicochemical characterization of mineral (iron/zinc) bound caseinate and their mineral uptake in Caco-2 cells.

Science.gov (United States)

Shilpashree, B G; Arora, Sumit; Kapila, Suman; Sharma, Vivek

2018-08-15

Milk proteins (especially caseins) are widely accepted as good vehicle for the delivery of various bioactive compounds including minerals. Succinylation is one of the most acceptable chemical modification techniques to enhance the mineral binding ability of caseins. Addition of minerals to succinylated proteins may alter their physicochemical and biochemical properties. Physicochemical characteristics of succinylated sodium caseinate (S.NaCN)-mineral (iron/zinc) complexes were elucidated. Chromatographic behaviour and fluorescence intensity confirmed the structural modification of S.NaCN upon binding with minerals. The bound mineral from protein complexes showed significantly higher (P < 0.05) in vitro bioavailability (mineral uptake) than mineral salts in Caco-2 cells. Also, iron bound S.NaCN showed higher cellular ferritin formation than iron in its free form. These mineral bound protein complexes with improved bioavailability could safely replace inorganic fortificants in various functional food formulations. Copyright © 2018 Elsevier Ltd. All rights reserved.

Reduced protein bound uraemic toxins in vegetarian kidney failure patients treated by haemodiafiltration.

Science.gov (United States)

Kandouz, Sakina; Mohamed, Ali Shendi; Zheng, Yishan; Sandeman, Susan; Davenport, Andrew

2016-10-01

Introduction Indoxyl sulfate (IS) and p cresyl sulfate (PCS) are protein bound toxins which accumulate with chronic kidney disease. Haemodiafiltration (HDF) increases middle molecule clearances and has been suggested to increase IS and PCS clearance. We therefore wished to establish whether higher convective clearances with HDF would reduce IS and PCS concentrations. Methods We measured total plasma IS and PCS in a cohort of 138 CKD5d patients treated by On-line HDF (Ol-HDF), by high pressure liquid chromatography. Findings Mean patient age was 64.6 ± 16.5 years, 60.1% male, 57.3% diabetic, median dialysis vintage 25.9 months (12.4-62.0). The mean ICS concentration was 79.8 ± 56.4 umol/L and PCS 140.3 ± 101.8 umol/L. On multivariate analysis, IS was associated with serum albumin (β 4.31,P vegetarian diet(β-28.3, P = 0.048) and PCS negatively with log C reactive protein (β-75.8, P vegetarian diet (β-109, P = 0.001). Vegetarian patients had lower IS and PCS levels (median 41.5 (24.2-71.9) vs. 78.1 (49.5-107.5) and PCS (41.6 (14.2-178.3) vs. 127.3 (77.4-205.6) µmol/L, respectively, P Vegetarian patients had lower preOl-HDF serum urea, and phosphate (13.8 ±3.8 vs. 18.4 ± 5.2 mmol/L, and 1.33 ± 0.21 vs. 1.58 ± 0.45 mmol/L), and estimated urea nitrogen intake (1.25 ± 0.28 vs. 1.62 ± 0.5 g/kg/day), respectively, all P vegetarian diet had reduced IS and PCS concentrations. Although this could be due to differences in dietary protein intake, a vegetarian diet may also potentially reduce IS and PCS production by the intestinal microbiome. © 2016 International Society for Hemodialysis.

Curcumin inhibits hepatitis B virus infection by down-regulating cccDNA-bound histone acetylation.

Science.gov (United States)

Wei, Zhi-Qiang; Zhang, Yong-Hong; Ke, Chang-Zheng; Chen, Hong-Xia; Ren, Pan; He, Yu-Lin; Hu, Pei; Ma, De-Qiang; Luo, Jie; Meng, Zhong-Ji

2017-09-14

To investigate the potential effect of curcumin on hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and the underlying mechanism. A HepG2.2.15 cell line stably transfected with HBV was treated with curcumin, and HBV surface antigen (HBsAg) and e antigen (HBeAg) expression levels were assessed by ELISA. Intracellular HBV DNA replication intermediates and cccDNA were detected by Southern blot and real-time PCR, respectively. The acetylation levels of histones H3 and H4 were measured by Western blot. H3/H4-bound cccDNA was detected by chromatin immunoprecipitation (ChIP) assays. The deacetylase inhibitors trichostatin A and sodium butyrate were used to study the mechanism of action for curcumin. Additionally, short interfering RNAs (siRNAs) targeting HBV were tested along with curcumin. Curcumin treatment led to time- and dose-dependent reductions in HBsAg and HBeAg expression and significant reductions in intracellular HBV DNA replication intermediates and HBV cccDNA. After treatment with 20 μmol/L curcumin for 2 d, HBsAg and cccDNA levels in HepG2.2.15 cells were reduced by up to 57.7% ( P curcumin, accompanied by reductions in H3- and H4-bound cccDNA. Furthermore, the deacetylase inhibitors trichostatin A and sodium butyrate could block the effects of curcumin. Additionally, transfection of siRNAs targeting HBV enhanced the inhibitory effects of curcumin. Curcumin inhibits HBV gene replication via down-regulation of cccDNA-bound histone acetylation and has the potential to be developed as a cccDNA-targeting antiviral agent for hepatitis B.

Circuit lower bounds in bounded arithmetics

Czech Academy of Sciences Publication Activity Database

Pich, Ján

2015-01-01

Roč. 166, č. 1 (2015), s. 29-45 ISSN 0168-0072 R&D Projects: GA AV ČR IAA100190902 Keywords : bounded arithmetic * circuit lower bounds Subject RIV: BA - General Mathematics Impact factor: 0.582, year: 2015 http://www.sciencedirect.com/science/article/pii/S0168007214000888

Determination of the total concentration of highly protein-bound drugs in plasma by on-line dialysis and column liquid chromatography: application to non-steroidal anti-inflammatory drugs.

NARCIS (Netherlands)

Herraez-Hernandez, R.; van de Merbel, N.C.; Brinkman, U.A.T.

1995-01-01

The potential of on-line dialysis as a sample preparation procedure for compounds highly bound to plasma proteins is evaluated, using non-steroidal anti-inflammatory drugs as model compounds and column liquid chromatography as the separation technique. Different strategies to reduce the degree of

Determination of the total concentration of highly protein-bound drugs in plasma by on-line dialysis and column liquid chromatography : application to non-steroidal anti-inflammatory drugs

NARCIS (Netherlands)

Herráez-Hernández, R; van de Merbel, N C; Brinkman, U A

1995-01-01

The potential of on-line dialysis as a sample preparation procedure for compounds highly bound to plasma proteins is evaluated, using non-steroidal anti-inflammatory drugs as model compounds and column liquid chromatography as the separation technique. Different strategies to reduce the degree of

Determination of Free-Form and Peptide Bound Pyrraline in the Commercial Drinks Enriched with Different Protein Hydrolysates

Directory of Open Access Journals (Sweden)

Zhili Liang

2016-07-01

Full Text Available Pyrraline, a causative factor for the recent epidemics of diabetes and cardiovascular disease, is also employed as an indicator to evaluate heat damage and formation of advanced glycation end-products (AGEs in foods. Peptide-enriched drinks (PEDs are broadly consumed worldwide due to rapid rate of absorption and perceived health effects. It can be hypothesized that PED is an important source of pyrraline, especially peptide bound pyrraline (Pep-Pyr. In this study we determined free-form pyrraline (Free-Pyr and Pep-Pyr in drinks enriched with whey protein hydrolysate (WPH, soy protein hydrolysate (SPH and collagen protein hydrolysate (CPH. A detection method was developed using ultrahigh-performance liquid chromatography with UV-visible detector coupled with tandem mass spectrometry after solid-phase extraction (SPE. The SPE led to excellent recovery rates ranging between 93.2% and 98.5% and a high reproducibility with relative standard deviations (RSD of <5%. The limits of detection and quantification obtained were 30.4 and 70.3 ng/mL, respectively. Pep-Pyr was identified as the most abundant form (above 96 percent of total pyrraline, whereas Free-Pyr was present in a small proportion (less than four percent of total pyrraline. The results indicate that PED is an important extrinsic source of pyrraline, especially Pep-Pyr. As compared with CPH- and SPH-enriched drinks, WPH-enriched drinks contained high content of Pep-Pyr. The Pep-Pyr content is associated with the distribution of peptide lengths and the amino acid compositions of protein in PEDs.

The Three-Dimensional Solution Structure of the Src Homology Domain-2 of the Growth Factor Receptor-Bound Protein-2

International Nuclear Information System (INIS)

Senior, Mary M.; Frederick, Anne F.; Black, Stuart; Murgolo, Nicholas J.; Perkins, Louise M.; Wilson, Oswald; Snow, Mark E.; Wang Yusen

1998-01-01

A set of high-resolution three-dimensional solution structures of the Src homology region-2 (SH2) domain of the growth factor receptor-bound protein-2 was determined using heteronuclear NMR spectroscopy. The NMR data used in this study were collected on a stable monomeric protein solution that was free of protein aggregates and proteolysis. The solution structure was determined based upon a total of 1439 constraints, which included 1326 nuclear Overhauser effect distance constraints, 70 hydrogen bond constraints, and 43 dihedral angle constraints. Distance geometry-simulated annealing calculations followed by energy minimization yielded a family of 18 structures that converged to a root-mean-square deviation of 1.09 A for all backbone atoms and 0.40 A for the backbone atoms of the central β-sheet. The core structure of the SH2 domain contains an antiparallel β-sheet flanked by two parallel α-helices displaying an overall architecture that is similar to other known SH2 domain structures. This family of NMR structures is compared to the X-ray structure and to another family of NMR solution structures determined under different solution conditions

Markers of protein oxidation

DEFF Research Database (Denmark)

Headlam, Henrietta A; Davies, Michael Jonathan

2004-01-01

Exposure of proteins to radicals in the presence of O2 gives both side-chain oxidation and backbone fragmentation. These processes can be interrelated, with initial side-chain oxidation giving rise to backbone damage via transfer reactions. We have shown previously that alkoxyl radicals formed...... of this process depends on the extent of oxidation at C-3 compared with other sites. HO*, generated by gamma radiolysis, gave the highest total carbonyl yield, with protein-bound carbonyls predominating over released. In contrast, metal ion/H2O2 systems, gave more released than bound carbonyls, with this ratio...... modulated by EDTA. This is ascribed to metal ion-protein interactions affecting the sites of initial oxidation. Hypochlorous acid gave low concentrations of released carbonyls, but high yields of protein-bound material. The peroxyl radical generator 2,2'-azobis(2-amidinopropane) hydrochloride...

Preparation of Sulfobetaine-Grafted PVDF Hollow Fiber Membranes with a Stably Anti-Protein-Fouling Performance

Directory of Open Access Journals (Sweden)

Qian Li

2014-04-01

Full Text Available Based on a two-step polymerization method, two sulfobetaine-based zwitterionic monomers, including 3-(methacryloylamino propyl-dimethyl-(3-sulfopropyl ammonium hydroxide (MPDSAH and 2-(methacryloyloxyethyl ethyl-dimethyl-(3-sulfopropyl ammonium (MEDSA, were successfully grafted from poly(vinylidene fluoride (PVDF hollow fiber membrane surfaces in the presence of N,N′-methylene bisacrylamide (MBAA as a cross-linking agent. The mechanical properties of the PVDF membrane were improved by the zwitterionic surface layers. The surface hydrophilicity of PVDF membranes was significantly enhanced and the polyMPDSAH-g-PVDF membrane showed a higher hydrophilicity due to the higher grafting amount. Compared to the polyMEDSA-g-PVDF membrane, the polyMPDSAH-g-PVDF membrane showed excellent significantly better anti-protein-fouling performance with a flux recovery ratio (RFR higher than 90% during the cyclic filtration of a bovine serum albumin (BSA solution. The polyMPDSAH-g-PVDF membrane showed an obvious electrolyte-responsive behavior and its protein-fouling-resistance performance was improved further during the filtration of the protein solution with 100 mmol/L of NaCl. After cleaned with a membrane cleaning solution for 16 days, the grafted MPDSAH layer on the PVDF membrane could be maintain without any chang; however, the polyMEDSA-g-PVDF membrane lost the grafted MEDSA layer after this treatment. Therefore, the amide group of sulfobetaine, which contributed significantly to the higher hydrophilicity and stability, was shown to be imperative in modifying the PVDF membrane for a stable anti-protein-fouling performance via the two-step polymerization method.

Small-angle X-ray scattering analysis reveals the ATP-bound monomeric state of the ATPase domain from the homodimeric MutL endonuclease, a GHKL phosphotransferase superfamily protein.

Science.gov (United States)

Iino, Hitoshi; Hikima, Takaaki; Nishida, Yuya; Yamamoto, Masaki; Kuramitsu, Seiki; Fukui, Kenji

2015-05-01

DNA mismatch repair is an excision system that removes mismatched bases chiefly generated by replication errors. In this system, MutL endonucleases direct the excision reaction to the error-containing strand of the duplex by specifically incising the newly synthesized strand. Both bacterial homodimeric and eukaryotic heterodimeric MutL proteins belong to the GHKL ATPase/kinase superfamily that comprises the N-terminal ATPase and C-terminal dimerization regions. Generally, the GHKL proteins show large ATPase cycle-dependent conformational changes, including dimerization-coupled ATP binding of the N-terminal domain. Interestingly, the ATPase domain of human PMS2, a subunit of the MutL heterodimer, binds ATP without dimerization. The monomeric ATP-bound state of the domain has been thought to be characteristic of heterodimeric GHKL proteins. In this study, we characterized the ATP-bound state of the ATPase domain from the Aquifex aeolicus MutL endonuclease, which is a homodimeric GHKL protein unlike the eukaryotic MutL. Gel filtration, dynamic light scattering, and small-angle X-ray scattering analyses clearly showed that the domain binds ATP in a monomeric form despite its homodimeric nature. This indicates that the uncoupling of dimerization and ATP binding is a common feature among bacterial and eukaryotic MutL endonucleases, which we suggest is closely related to the molecular mechanisms underlying mismatch repair.

Development of immobilized membrane-based affinity columns for use in the online characterization of membrane bound proteins and for targeted affinity isolations

International Nuclear Information System (INIS)

Moaddel, Ruin; Wainer, Irving W.

2006-01-01

Membranes obtained from cell lines that express or do not express a target membrane bound protein have been immobilized on a silica-based liquid chromatographic support or on the surface of an activated glass capillary. The resulting chromatographic columns have been placed in liquid chromatographic systems and used to characterize the target proteins and to identify small molecules that bind to the target. Membranes containing ligand gated ion channels, G-protein coupled receptors and drug transporters have been prepared and characterized. If a marker ligand has been identified for the target protein, frontal or zonal displacement chromatographic techniques can be used to determine binding affinities (K d values) and non-linear chromatography can be used to assess the association (k on ) and dissociation (k off ) rate constants and the thermodynamics of the binding process. Membrane-based affinity columns have been created using membranes from a cell line that does not express the target protein (control) and the same cell line that expresses the target protein (experimental) after genomic transfection. The resulting columns can be placed in a parallel chromatography system and the differential retention between the control and experimental columns can be used to identify small molecules and protein that bind to the target protein. These applications will be illustrated using columns created using cellular membranes containing nicotinic acetylcholine receptors and the drug transporter P-glycoprotein

Development of immobilized membrane-based affinity columns for use in the online characterization of membrane bound proteins and for targeted affinity isolations

Energy Technology Data Exchange (ETDEWEB)

Moaddel, Ruin [Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825 (United States); Wainer, Irving W. [Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825 (United States)]. E-mail: Wainerir@grc.nia.nih.gov

2006-03-30

Membranes obtained from cell lines that express or do not express a target membrane bound protein have been immobilized on a silica-based liquid chromatographic support or on the surface of an activated glass capillary. The resulting chromatographic columns have been placed in liquid chromatographic systems and used to characterize the target proteins and to identify small molecules that bind to the target. Membranes containing ligand gated ion channels, G-protein coupled receptors and drug transporters have been prepared and characterized. If a marker ligand has been identified for the target protein, frontal or zonal displacement chromatographic techniques can be used to determine binding affinities (K {sub d} values) and non-linear chromatography can be used to assess the association (k {sub on}) and dissociation (k {sub off}) rate constants and the thermodynamics of the binding process. Membrane-based affinity columns have been created using membranes from a cell line that does not express the target protein (control) and the same cell line that expresses the target protein (experimental) after genomic transfection. The resulting columns can be placed in a parallel chromatography system and the differential retention between the control and experimental columns can be used to identify small molecules and protein that bind to the target protein. These applications will be illustrated using columns created using cellular membranes containing nicotinic acetylcholine receptors and the drug transporter P-glycoprotein.

Perceptron Mistake Bounds

OpenAIRE

Mohri, Mehryar; Rostamizadeh, Afshin

2013-01-01

We present a brief survey of existing mistake bounds and introduce novel bounds for the Perceptron or the kernel Perceptron algorithm. Our novel bounds generalize beyond standard margin-loss type bounds, allow for any convex and Lipschitz loss function, and admit a very simple proof.

Removal of Bound Triton X-100 from Purified Bovine Heart Cytochrome bc1

OpenAIRE

Varhač, Rastislav; Robinson, Neal C.; Musatov, Andrej

2009-01-01

Cytochrome bc1 isolated from Triton X-100 solubilized mitochondrial membranes contains up to 120 nmol of Triton X-100 bound per nmol of the enzyme. Purified cytochrome bc1 is fully active; however, protein bound Triton X-100 significantly interferes with structural studies of the enzyme. Removal of Triton X-100 bound to bovine cytochrome bc1 was accomplished by incubation with Bio-Beads SM-2 in presence of sodium cholate. Sodium cholate is critical since it does not interfere with the adsorpt...

Optimised purification and characterisation of lipid transfer protein 1 (LTP1) and its lipid-bound isoform LTP1b from barley malt.

Science.gov (United States)

Nieuwoudt, Melanie; Lombard, Nicolaas; Rautenbach, Marina

2014-08-15

In beer brewing, brewers worldwide strive to obtain product consistency in terms of flavour, colour and foam. Important proteins contributing to beer foam are lipid transfer proteins (LTPs), in particular LTP1 and its lipid-bound isoform LTP1b, which are known to transport lipids in vivo and prevent lipids from destabilising the beer foam. LTP1 and LTP1b were successfully purified using only five purification steps with a high purified protein yield (160 mg LTP1 and LTP1b from 200 g barley). Circular dichroism of LTP1 and LTP1b confirmed that both proteins are highly tolerant to high temperatures (>90 °C) and are pH stable, particularly at a neutral to a more basic pH. Only LTP1 exhibited antiyeast and thermo-stable lytic activity, while LTP1b was inactive, indicating that the fatty acid moiety compromised the antimicrobial activity of LTP1. This lack in antiyeast activity and the positive foam properties of LTP1b would benefit beer fermentation and quality. Copyright © 2014 Elsevier Ltd. All rights reserved.

Cells determine cell density using a small protein bound to a unique tissue-specific phospholipid

Directory of Open Access Journals (Sweden)

Christopher J. Petzold

2013-10-01

bone cofactor was identified as a lipid containing a ceramide phosphate, a single chained glycerol lipid and a linker. Tendon uses a different cofactor made up of two fatty acid chains linked directly to the phosphate yielding a molecule about half the size. Moreover, adding the tendon factor/cofactor to osteosarcoma cells causes them to stop growing, which is opposite to its role with tendon cells. Thus, the cofactor is cell type specific both in composition and in the triggered response. Further support of its proposed role came from frozen sections from 5 week old mice where an antibody to the factor stained strongly at the growing ends of the tendon as predicted. In conclusion, the molecule needed for cell density signaling is a small protein bound to a unique, tissue-specific phospholipid yielding a membrane associated but diffusible molecule. Signal transduction is postulated to occur by an increased ordering of the plasma membrane as the concentration of this protein/lipid increases with cell density.

Apo and ligand-bound structures of ModA from the archaeon Methanosarcina acetivorans

International Nuclear Information System (INIS)

Chan, Sum; Giuroiu, Iulia; Chernishof, Irina; Sawaya, Michael R.; Chiang, Janet; Gunsalus, Robert P.; Arbing, Mark A.; Perry, L. Jeanne

2010-01-01

Crystal structures of ModA from M. acetivorans in the apo and ligand-bound conformations confirm domain rotation upon ligand binding. The trace-element oxyanion molybdate, which is required for the growth of many bacterial and archaeal species, is transported into the cell by an ATP-binding cassette (ABC) transporter superfamily uptake system called ModABC. ModABC consists of the ModA periplasmic solute-binding protein, the integral membrane-transport protein ModB and the ATP-binding and hydrolysis cassette protein ModC. In this study, X-ray crystal structures of ModA from the archaeon Methanosarcina acetivorans (MaModA) have been determined in the apoprotein conformation at 1.95 and 1.69 Å resolution and in the molybdate-bound conformation at 2.25 and 2.45 Å resolution. The overall domain structure of MaModA is similar to other ModA proteins in that it has a bilobal structure in which two mixed α/β domains are linked by a hinge region. The apo MaModA is the first unliganded archaeal ModA structure to be determined: it exhibits a deep cleft between the two domains and confirms that upon binding ligand one domain is rotated towards the other by a hinge-bending motion, which is consistent with the ‘Venus flytrap’ model seen for bacterial-type periplasmic binding proteins. In contrast to the bacterial ModA structures, which have tetrahedral coordination of their metal substrates, molybdate-bound MaModA employs octahedral coordination of its substrate like other archaeal ModA proteins

Glycogen-bound polyphosphate kinase from the archaebacterium Sulfolobus acidocaldarius.

OpenAIRE

Skórko, R; Osipiuk, J; Stetter, K O

1989-01-01

Glycogen-bound polyphosphate kinase has been isolated from a crude extract of Sulfolobus acidocaldarius by isopycnic centrifugation in CsCl. Divalent cations (Mn2+ greater than Mg2+) stimulated the reaction. The enzyme does not require the presence of histones for its activity; it is inhibited strongly by phosphate and slightly by fluoride. The protein from the glycogen complex migrated in a sodium dodecyl sulfate-polyacrylamide gel as a 57-kilodalton protein band; after isoelectric focusing ...

Evolution of protein bound Maillard reaction end-products and free Amadori compounds in low lactose milk in presence of fructosamine oxidase I.

Science.gov (United States)

Troise, Antonio Dario; Buonanno, Martina; Fiore, Alberto; Monti, Simona Maria; Fogliano, Vincenzo

2016-12-01

Thermal treatments and storage influence milk quality, particularly in low lactose milk as the higher concentration of reducing sugars can lead to the increased formation of the Maillard reaction products (MRPs). The control of the Amadori products (APs) formation is the key step to mitigate the Maillard reaction (MR) in milk. The use of fructosamine oxidases, (Faox) provided promising results. In this paper, the effects of Faox I were evaluated by monitoring the concentration of free and bound MRPs in low lactose milk during shelf life. Results showed that the enzyme reduced the formation of protein-bound MRPs down to 79% after six days at 37°C. Faox I lowered the glycation of almost all the free amino acids resulting effective on basic and polar amino acids. Data here reported corroborate previous findings on the potentiality of Faox enzymes in controlling the early stage of the MR in foods. Copyright © 2016 Elsevier Ltd. All rights reserved.

Antimetastatic effect of PSK, a protein-bound polysaccharide, against the B16-BL6 mouse melanoma.

Science.gov (United States)

Matsunaga, K; Ohhara, M; Oguchi, Y; Iijima, H; Kobayashi, H

1996-01-01

We examined the effect of PSK, a protein-bound polysaccharide, upon in vivo metastasis and in vitro invasion of the B16-BL6 mouse melanoma cells. (1) PSK suppressed in vivo artificial and spontaneous lung metastases of B16-BL6 in C57BL/6 mice. (2) PSK in a dose-dependent fashion suppressed in vitro invasion and chemotaxis of the tumor cells using filters coated with a reconstituted basement membrane. (3) PSK had little effect on DNA synthesis in tumor cells in vitro, but suppressed tumor cell adhesion to, degradation of, and haptotaxis to components of the basement membrane. (4) PSK suppressed the binding of tumor cells to components of the basement membrane. These findings suggest that PSK may suppress metastasis through inhibition of tumor cell invasion and that this effect is the result of interactions between PSK and components of the basement membrane.

Differential effects of vasopressin and phenylephrine on protein kinase C-mediated protein phosphorylations in isolated hepatocytes

International Nuclear Information System (INIS)

Cooper, R.H.; Johanson, R.A.; Wiliamson, J.R.

1986-01-01

Receptor-mediated breakdown of inositol lipids produces two intracellular signals, diacylglycerol, which activates protein kinase C, and inositol trisphosphate, which causes release of intracellular vesicular Ca 2+ . This study examined the effects of Ca 2+ -ionophores, vasopressin, phenylephrine, and phorbol ester (PMA) on hepatocyte protein phosphorylations. [ 32 P] Phosphoproteins from hepatocytes prelabeled with 32 P were resolved by 2-dimensional SDS-PAGE and corresponding autoradiographs were quantitated by densitometric analysis. The phosphorylation of five proteins, a plasma membrane bound 16 kDa protein with pI 6.4, a cytosolic 16 kDa protein with pI 5.8, and proteins with Mr's of 36 kDa, 52 kDa, and 68 kDa, could be attributed to phosphorylation by protein kinase C since the phosphorylation was stimulated by PMA. When the vasopressin concentration was varied, low vasopressin stimulated the phosphorylation of only the membrane bound 16 kDa protein of the above set of proteins, while higher vasopressin concentrations were required to stimulate the phosphorylation of all five proteins. Phenylephrine, even at supramaximal concentrations, stimulated the phosphorylation of only the membrane bound 16 kDa protein. These results suggest that phenylephrine is a less potent activator of protein kinase C than vasopressin by virtue of limited or localized diacylglycerol production

In Situ Proteolysis for Crystallization of Membrane Bound Cytochrome P450 17A1 and 17A2 Proteins from Zebrafish.

Science.gov (United States)

Lei, Li; Egli, Martin

2016-04-01

Fish and human cytochrome P450 (P450) 17A1 catalyze both steroid 17α-hydroxylation and 17α,20-lyase reactions. Fish P450 17A2 catalyzes only 17α-hydroxylation. Both enzymes are microsomal-type P450s, integral membrane proteins that bind to the membrane through their N-terminal hydrophobic segment, the signal anchor sequence. The presence of this N-terminal region renders expression of full-length proteins challenging or impossible. For some proteins, variable truncation of the signal anchor sequence precludes expression or results in poor expression levels. To crystallize P450 17A1 and 17A2 in order to gain insight into their different activities, we used an alternative N-terminal sequence to boost expression together with in situ proteolysis. Key features of our approach to identify crystallizable P450 fragments were the use of an N-terminal leader sequence, a screen composed of 12 proteases to establish optimal cleavage, variations of protease concentration in combination with an SDS-PAGE assay, and analysis of the resulting fragments using Edman sequencing. Described in this unit are protocols for vector preparation, expression, purification, and in situ proteolytic crystallization of two membrane-bound P450 proteins. Copyright © 2016 John Wiley & Sons, Inc.

Bound water at protein-protein interfaces: partners, roles and hydrophobic bubbles as a conserved motif.

Directory of Open Access Journals (Sweden)

Mostafa H Ahmed

Full Text Available There is a great interest in understanding and exploiting protein-protein associations as new routes for treating human disease. However, these associations are difficult to structurally characterize or model although the number of X-ray structures for protein-protein complexes is expanding. One feature of these complexes that has received little attention is the role of water molecules in the interfacial region.A data set of 4741 water molecules abstracted from 179 high-resolution (≤ 2.30 Å X-ray crystal structures of protein-protein complexes was analyzed with a suite of modeling tools based on the HINT forcefield and hydrogen-bonding geometry. A metric termed Relevance was used to classify the general roles of the water molecules.The water molecules were found to be involved in: a (bridging interactions with both proteins (21%, b favorable interactions with only one protein (53%, and c no interactions with either protein (26%. This trend is shown to be independent of the crystallographic resolution. Interactions with residue backbones are consistent for all classes and account for 21.5% of all interactions. Interactions with polar residues are significantly more common for the first group and interactions with non-polar residues dominate the last group. Waters interacting with both proteins stabilize on average the proteins' interaction (-0.46 kcal mol(-1, but the overall average contribution of a single water to the protein-protein interaction energy is unfavorable (+0.03 kcal mol(-1. Analysis of the waters without favorable interactions with either protein suggests that this is a conserved phenomenon: 42% of these waters have SASA ≤ 10 Å(2 and are thus largely buried, and 69% of these are within predominantly hydrophobic environments or "hydrophobic bubbles". Such water molecules may have an important biological purpose in mediating protein-protein interactions.

Virial Expansion Bounds

Science.gov (United States)

Tate, Stephen James

2013-10-01

In the 1960s, the technique of using cluster expansion bounds in order to achieve bounds on the virial expansion was developed by Lebowitz and Penrose (J. Math. Phys. 5:841, 1964) and Ruelle (Statistical Mechanics: Rigorous Results. Benjamin, Elmsford, 1969). This technique is generalised to more recent cluster expansion bounds by Poghosyan and Ueltschi (J. Math. Phys. 50:053509, 2009), which are related to the work of Procacci (J. Stat. Phys. 129:171, 2007) and the tree-graph identity, detailed by Brydges (Phénomènes Critiques, Systèmes Aléatoires, Théories de Jauge. Les Houches 1984, pp. 129-183, 1986). The bounds achieved by Lebowitz and Penrose can also be sharpened by doing the actual optimisation and achieving expressions in terms of the Lambert W-function. The different bound from the cluster expansion shows some improvements for bounds on the convergence of the virial expansion in the case of positive potentials, which are allowed to have a hard core.

Upper bounds on superpartner masses from upper bounds on the Higgs boson mass.

Science.gov (United States)

Cabrera, M E; Casas, J A; Delgado, A

2012-01-13

The LHC is putting bounds on the Higgs boson mass. In this Letter we use those bounds to constrain the minimal supersymmetric standard model (MSSM) parameter space using the fact that, in supersymmetry, the Higgs mass is a function of the masses of sparticles, and therefore an upper bound on the Higgs mass translates into an upper bound for the masses for superpartners. We show that, although current bounds do not constrain the MSSM parameter space from above, once the Higgs mass bound improves big regions of this parameter space will be excluded, putting upper bounds on supersymmetry (SUSY) masses. On the other hand, for the case of split-SUSY we show that, for moderate or large tanβ, the present bounds on the Higgs mass imply that the common mass for scalars cannot be greater than 10(11)  GeV. We show how these bounds will evolve as LHC continues to improve the limits on the Higgs mass.

Contribution of granule bound starch synthase in kernel modification ...

African Journals Online (AJOL)

The role of gbssI and gbssII genes, encoding granule bound starch synthase enzyme I and II, respectively, in quality protein maize (QPM) were studied at different days after pollination (DAP). Total RNA was used for first strand cDNA synthesis using the ImpromIISriptTM reverse transcriptase. No detectable levels of gbssI ...

Chemopreventive effects of free and bound phenolics associated to steep waters (nejayote) obtained after nixtamalization of different maize types.

Science.gov (United States)

Rojas-García, Carlos; García-Lara, Silverio; Serna-Saldivar, Sergio O; Gutiérrez-Uribe, Janet A

2012-03-01

Free and bound phenolics extracts from nejayote solids were obtained after optimally lime-cooking blue, normal white, red, normal yellow, high-carotenoid and quality protein maize types. The extraction yield ranged from 4.47 to 10.05%. Bound phenolics extracts had higher content of total phenolics, antioxidant activity and ferulic acid compared to the free phenolics extracts. In general, free phenolics extracts were less cytotoxic than the bound phenolics counterparts. Bound phenolics extracts had higher induction of quinone reductase (QR) and particularly the normal yellow nejayote exerted the highest chemopreventive index tested in Hepa1c1c7 cells. When tested for monofunctional phase 2 induction capacity in BPrc1 cells, the bound phenolics extracts of blue, normal white and quality protein nejayotes were better inducers than the normal yellow counterpart. Particularly, the free phenolics extract of the white maize nejayote induced BPrc1 cells QR and exerted a higher chemopreventive index compared to the bound phenolics extract. Therefore, the nejayote of the normal white maize was the best source of monofunctional phase 2 enzyme inducers.

Glycogen-bound polyphosphate kinase from the archaebacterium Sulfolobus acidocaldarius.

Science.gov (United States)

Skórko, R; Osipiuk, J; Stetter, K O

1989-09-01

Glycogen-bound polyphosphate kinase has been isolated from a crude extract of Sulfolobus acidocaldarius by isopycnic centrifugation in CsCl. Divalent cations (Mn2+ greater than Mg2+) stimulated the reaction. The enzyme does not require the presence of histones for its activity; it is inhibited strongly by phosphate and slightly by fluoride. The protein from the glycogen complex migrated in a sodium dodecyl sulfate-polyacrylamide gel as a 57-kilodalton protein band; after isoelectric focusing it separated into several spots in the pH range of 5.6 to 6.7.

[The Role of Membrane-Bound Heat Shock Proteins Hsp90 in Migration of Tumor Cells in vitro and Involvement of Cell Surface Heparan Sulfate Proteoglycans in Protein Binding to Plasma Membrane].

Science.gov (United States)

Snigireva, A V; Vrublevskaya, V V; Skarga, Y Y; Morenkov, O S

2016-01-01

Heat shock protein Hsp90, detected in the extracellular space and on the membrane of cells, plays an important role in cell motility, migration, invasion and metastasis of tumor cells. At present, the functional role and molecular mechanisms of Hsp90 binding to plasma membrane are not elucidated. Using isoform-specific antibodies against Hsp90, Hsp9α and Hsp90β, we showed that membrane-bound Hsp90α and Hsp90β play a significant role in migration of human fibrosarcoma (HT1080) and glioblastoma (A-172) cells in vitro. Disorders of sulfonation of cell heparan sulfates, cleavage of cell heparan. sulfates by heparinase I/III as well as treatment of cells with heparin lead to an abrupt reduction in the expression level of Hsp90 isoforms. Furthermore, heparin significantly inhibits tumor cell migration. The results obtained demonstrate that two isoforms of membrane-bound Hsp90 are involved in migration of tumor cells in vitro and that cell surface heparan sulfate proteoglycans play a pivotal role in the "anchoring" of Hsp90α and Hsp90β to the plasma membrane.

The DMM Bound

DEFF Research Database (Denmark)

Emiris, Ioannis Z.; Mourrain, Bernard; Tsigaridas, Elias

2010-01-01

) resultant by means of mixed volume, as well as recent advances on aggregate root bounds for univariate polynomials, and are applicable to arbitrary positive dimensional systems. We improve upon Canny's gap theorem [7] by a factor of O(dn-1), where d bounds the degree of the polynomials, and n is the number...... bound on the number of steps that subdivision-based algorithms perform in order to isolate all real roots of a polynomial system. This leads to the first complexity bound of Milne's algorithm [22] in 2D....

A potent transrepression domain in the retinoblastoma protein induces a cell cycle arrest when bound to E2F sites.

Science.gov (United States)

Sellers, W R; Rodgers, J W; Kaelin, W G

1995-01-01

An intact T/E1A-binding domain (the pocket) is necessary, but not sufficient, for the retinoblastoma protein (RB) to bind to DNA-protein complexes containing E2F and for RB to induce a G1/S block. Indirect evidence suggests that the binding of RB to E2F may, in addition to inhibiting E2F transactivation function, generate a complex capable of functioning as a transrepressor. Here we show that a chimera in which the E2F1 transactivation domain was replaced with the RB pocket could, in a DNA-binding and pocket-dependent manner, mimic the ability of RB to repress transcription and induce a cell cycle arrest. In contrast, a transdominant negative E2F1 mutant that is capable of blocking E2F-dependent transactivation did not. Fusion of the RB pocket to a heterologous DNA-binding domain unrelated to E2F likewise generated a transrepressor protein when scored against a suitable reporter. These results suggest that growth suppression by RB is due, at least in part, to transrepression mediated by the pocket domain bound to certain promoters via E2F. Images Fig. 4 Fig. 5 PMID:8524800

h5-HT(1B) receptor-mediated constitutive Galphai3-protein activation in stably transfected Chinese hamster ovary cells: an antibody capture assay reveals protean efficacy of 5-HT.

Science.gov (United States)

Newman-Tancredi, Adrian; Cussac, Didier; Marini, Laetitia; Touzard, Manuelle; Millan, Mark J

2003-03-01

1. Serotonin 5-HT(1B) receptors couple to G-proteins of the Gi/o family. However, their activation of specific G-protein subtypes is poorly characterised. Using an innovative antibody capture/guanosine-5'-0-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) binding strategy, we characterised Galpha(i3) subunit activation by h5-HT(1B) receptors stably expressed in Chinese hamster ovary (CHO) cells. 2. The agonists, 5-HT, alniditan and BMS181,101, stimulated Galpha(i3), whereas methiothepin and SB224,289 behaved as inverse agonists. The selective 5-HT(1B) receptor ligand, S18127, modestly stimulated Galpha(i3) and reversed the actions of both 5-HT and methiothepin. S18127 (1 micro M) also produced parallel, dextral shifts of the 5-HT and methiothepin isotherms. 3. Isotopic dilution experiments ([(35)S]GTPgammaS versus GTPgammaS) revealed high-affinity [(35)S]GTPgammaS binding to Galpha(i3) subunits in the absence of receptor ligands indicating constitutive activity. High-affinity [(35)S]GTPgammaS binding was increased 2.8-fold by 5-HT with an increase in the affinity of GTPgammaS for Galpha(i3) subunits. In contrast, methiothepin halved the number of high-affinity binding sites and decreased their affinity. 4. h5-HT(1B) receptor-mediated Galpha(i3) subunit activation was dependent on the concentration of NaCl. At 300 mM, 5-HT stimulated [(35)S]GTPgammaS binding, basal Galpha(i3) activation was low and methiothepin was inactive. In contrast, at 10 mM NaCl, basal activity was enhanced and the inverse agonist activity of methiothepin was accentuated. Under these conditions, 5-HT decreased Galpha(i3) activation. 5. In conclusion, at h5-HT(1B) receptors expressed in CHO cells: (i) inverse agonist induced inhibition of Galpha(i3), and its reversal by S18127, reveals constitutive activation of this Galpha subunit; (ii) constitutive Galpha(i3) activation can be quantified by isotopic dilution [(35)S]GTPgammaS binding and (iii) decreasing NaCl concentrations enhances Galpha(i3

Isolation and expression of the genes coding for the membrane bound transglycosylase B (MltB and the transferrin binding protein B (TbpB of the salmon pathogen Piscirickettsia salmonis

Directory of Open Access Journals (Sweden)

VIVIAN WILHELM

2004-01-01

Full Text Available We have isolated and sequenced the genes encoding the membrane bound transglycosylase B (MltB and the transferring binding protein B (TbpB of the salmon pathogen Piscirickettsia salmonis. The results of the sequence revealed two open reading frames that encode proteins with calculated molecular weights of 38,830 and 85,140. The deduced aminoacid sequences of both proteins show a significant homology to the respective protein from phylogenetically related microorganisms. Partial sequences coding the amino and carboxyl regions of MltB and a sequence of 761 base pairs encoding the amino region of TbpB have been expressed in E. coli. The strong humoral response elicited by these proteins in mouse confirmed the immunogenic properties of the recombinant proteins. A similar response was elicited by both proteins when injected intraperitoneally in Atlantic salmon. The present data indicates that these proteins are good candidates to be used in formulations to study the protective immunity of salmon to infection by P. salmonis.

Effects of soft interactions and bound mobility on diffusion in crowded environments: a model of sticky and slippery obstacles

Science.gov (United States)

Stefferson, Michael W.; Norris, Samantha L.; Vernerey, Franck J.; Betterton, Meredith D.; E Hough, Loren

2017-08-01

Crowded environments modify the diffusion of macromolecules, generally slowing their movement and inducing transient anomalous subdiffusion. The presence of obstacles also modifies the kinetics and equilibrium behavior of tracers. While previous theoretical studies of particle diffusion have typically assumed either impenetrable obstacles or binding interactions that immobilize the particle, in many cellular contexts bound particles remain mobile. Examples include membrane proteins or lipids with some entry and diffusion within lipid domains and proteins that can enter into membraneless organelles or compartments such as the nucleolus. Using a lattice model, we studied the diffusive movement of tracer particles which bind to soft obstacles, allowing tracers and obstacles to occupy the same lattice site. For sticky obstacles, bound tracer particles are immobile, while for slippery obstacles, bound tracers can hop without penalty to adjacent obstacles. In both models, binding significantly alters tracer motion. The type and degree of motion while bound is a key determinant of the tracer mobility: slippery obstacles can allow nearly unhindered diffusion, even at high obstacle filling fraction. To mimic compartmentalization in a cell, we examined how obstacle size and a range of bound diffusion coefficients affect tracer dynamics. The behavior of the model is similar in two and three spatial dimensions. Our work has implications for protein movement and interactions within cells.

Enzymatic Digestion of Chronic Wasting Disease Prions Bound to Soil

Science.gov (United States)

SAUNDERS, SAMUEL E.; BARTZ, JASON C.; VERCAUTEREN, KURT C.; BARTELT-HUNT, SHANNON L.

2010-01-01

Chronic wasting disease (CWD) and sheep scrapie can be transmitted via indirect environmental routes, and it is known that soil can serve as a reservoir of prion infectivity. Given the strong interaction between the prion protein (PrP) and soil, we hypothesized that binding to soil enhances prion resistance to enzymatic digestion, thereby facilitating prion longevity in the environment and providing protection from host degradation. We characterized the performance of a commercially available subtilisin enzyme, the Prionzyme, to degrade soil-bound and unbound CWD and HY TME PrP as a function of pH, temperature, and treatment time. The subtilisin enzyme effectively degraded PrP adsorbed to a wide range of soils and soil minerals below the limits of detection. Signal loss occurred rapidly at high pH (12.5) and within 7 d under conditions representative of the natural environment (pH 7.4, 22°C). We observed no apparent difference in enzyme effectiveness between bound and unbound CWD PrP. Our results show that although adsorbed prions do retain relative resistance to enzymatic digestion compared with other brain homogenate proteins, they can be effectively degraded when bound to soil. Our results also suggest a topical application of a subtilisin enzyme solution may be an effective decontamination method to limit disease transmission via environmental ‘hot spots’ of prion infectivity. PMID:20450190

The Common Inhalational Anesthetic Sevoflurane Induces Apoptosis and Increases β-Amyloid Protein Levels

Science.gov (United States)

Dong, Yuanlin; Zhang, Guohua; Zhang, Bin; Moir, Robert D.; Xia, Weiming; Marcantonio, Edward R.; Culley, Deborah J.; Crosby, Gregory; Tanzi, Rudolph E.; Xie, Zhongcong

2009-01-01

Objective: To assess the effects of sevoflurane, the most commonly used inhalation anesthetic, on apoptosis and β-amyloid protein (Aβ) levels in vitro and in vivo. Subjects: Naive mice, H4 human neuroglioma cells, and H4 human neuroglioma cells stably transfected to express full-length amyloid precursor protein. Interventions: Human H4 neuroglioma cells stably transfected to express full-length amyloid precursor protein were exposed to 4.1% sevoflurane for 6 hours. Mice received 2.5% sevoflurane for 2 hours. Caspase-3 activation, apoptosis, and Aβ levels were assessed. Results: Sevoflurane induced apoptosis and elevated levels of β-site amyloid precursor protein-cleaving enzyme and Aβ in vitro and in vivo. The caspase inhibitor Z-VAD decreased the effects of sevoflurane on apoptosis and Aβ. Sevoflurane-induced caspase-3 activation was attenuated by the γ-secretase inhibitor L-685,458 and was potentiated by Aβ. These results suggest that sevoflurane induces caspase activation which, in turn, enhances β-site amyloid precursor protein–cleaving enzyme and Aβ levels. Increased Aβ levels then induce further rounds of apoptosis. Conclusions: These results suggest that inhalational anesthetic sevoflurane may promote Alzheimer disease neuropathogenesis. If confirmed in human subjects, it may be prudent to caution against the use of sevoflurane as an anesthetic, especially in those suspected of possessing excessive levels of cerebral Aβ. PMID:19433662

Organically bound tritium

International Nuclear Information System (INIS)

Diabate, S.; Strack, S.

1993-01-01

Tritium released into the environment may be incorporated into organic matter. Organically bound tritium in that case will show retention times in organisms that are considerably longer than those of tritiated water which has significant consequences on dose estimates. This article reviews the most important processes of organically bound tritium production and transport through food networks. Metabolic reactions in plant and animal organisms with tritiated water as a reaction partner are of great importance in this respect. The most important production process, in quantitative terms, is photosynthesis in green plants. The translocation of organically bound tritium from the leaves to edible parts of crop plants should be considered in models of organically bound tritium behavior. Organically bound tritium enters the human body on several pathways, either from the primary producers (vegetable food) or at a higher tropic level (animal food). Animal experiments have shown that the dose due to ingestion of organically bound tritium can be up to twice as high as a comparable intake of tritiated water in gaseous or liquid form. In the environment, organically bound tritium in plants and animals is often found to have higher specific tritium concentrations than tissue water. This is not due to some tritium enrichment effects but to the fact that no equilibrium conditions are reached under natural conditions. 66 refs

Peripheral Protein Unfolding Drives Membrane Bending.

Science.gov (United States)

Siaw, Hew Ming Helen; Raghunath, Gokul; Dyer, R Brian

2018-06-20

Dynamic modulation of lipid membrane curvature can be achieved by a number of peripheral protein binding mechanisms such as hy-drophobic insertion of amphipathic helices and membrane scaffolding. Recently, an alternative mechanism was proposed in which crowding of peripherally bound proteins induces membrane curvature through steric pressure generated by lateral collisions. This effect was enhanced using intrinsically disordered proteins that possess high hydrodynamic radii, prompting us to explore whether membrane bending can be triggered by the folding-unfolding transition of surface-bound proteins. We utilized histidine-tagged human serum albumin bound to Ni-NTA-DGS containing liposomes as our model system to test this hypothesis. We found that reduction of the disulfide bonds in the protein resulted in unfolding of HSA, which subsequently led to membrane tubule formation. The frequency of tubule formation was found to be significantly higher when the proteins were unfolded while being localized to a phase-separated domain as opposed to randomly distributed in fluid phase liposomes, indicating that the steric pressure generated from protein unfolding is directly responsible for membrane deformation. Our results are critical for the design of peripheral membrane protein-immobilization strategies and open new avenues for exploring mechanisms of membrane bending driven by conformational changes of peripheral membrane proteins.

Physical Uncertainty Bounds (PUB)

Energy Technology Data Exchange (ETDEWEB)

Vaughan, Diane Elizabeth [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Preston, Dean L. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

2015-03-19

This paper introduces and motivates the need for a new methodology for determining upper bounds on the uncertainties in simulations of engineered systems due to limited fidelity in the composite continuum-level physics models needed to simulate the systems. We show that traditional uncertainty quantification methods provide, at best, a lower bound on this uncertainty. We propose to obtain bounds on the simulation uncertainties by first determining bounds on the physical quantities or processes relevant to system performance. By bounding these physics processes, as opposed to carrying out statistical analyses of the parameter sets of specific physics models or simply switching out the available physics models, one can obtain upper bounds on the uncertainties in simulated quantities of interest.

Expression of hepatitis C virus envelope protein 2 induces apoptosis in cultured mammalian cells

Institute of Scientific and Technical Information of China (English)

Li-Xin Zhu; Jing Liu; You-Hua Xie; Yu-Ying Kong; Ye Ye; Chun-Lin Wang; Guang-Di Li; Yuan Wang

2004-01-01

AIM: To explore the role of hepatitis C virus (HCV) envelope protein 2 (E2) in the induction of apoptosis.METHODS: A carboxyterminal truncated E2 (E2-661) was transiently expressed in several cultured mammalian cell lines or stably expressed in Chinese hamster ovary (CHO)cell line. Cell proliferation was assessed by 3H thymidine uptake. Apoptosis was examined by Hoechst 33258staining, flow cytometry and DNA fragmentation analysis.RESULTS: Reduced proliferation was readily observed in the E2-661 expressing cells. These cells manifested the typical features of apoptosis, including cell shrinkage,chromatin condensation and hypodiploid genomic DNA content. Similar apoptotic cell death was observed in an E2-661 stably expressing cell line.CONCLUSION: HCV E2 can induce apoptosis in cultured mammalian cells.

Effects of amantadine on the dynamics of membrane-bound influenza A M2 transmembrane peptide studied by NMR relaxation

Energy Technology Data Exchange (ETDEWEB)

Cady, Sarah D.; Hong Mei [Iowa State University, Department of Chemistry (United States)], E-mail: mhong@iastate.edu

2009-09-15

The molecular motions of membrane proteins in liquid-crystalline lipid bilayers lie at the interface between motions in isotropic liquids and in solids. Specifically, membrane proteins can undergo whole-body uniaxial diffusion on the microsecond time scale. In this work, we investigate the {sup 1}H rotating-frame spin-lattice relaxation (T{sub 1{rho}}) caused by the uniaxial diffusion of the influenza A M2 transmembrane peptide (M2TMP), which forms a tetrameric proton channel in lipid bilayers. This uniaxial diffusion was proved before by {sup 2}H, {sup 15}N and {sup 13}C NMR lineshapes of M2TMP in DLPC bilayers. When bound to an inhibitor, amantadine, the protein exhibits significantly narrower linewidths at physiological temperature. We now investigate the origin of this line narrowing through temperature-dependent {sup 1}H T{sub 1{rho}} relaxation times in the absence and presence of amantadine. Analysis of the temperature dependence indicates that amantadine decreases the correlation time of motion from 2.8 {+-} 0.9 {mu}s for the apo peptide to 0.89 {+-} 0.41 {mu}s for the bound peptide at 313 K. Thus the line narrowing of the bound peptide is due to better avoidance of the NMR time scale and suppression of intermediate time scale broadening. The faster diffusion of the bound peptide is due to the higher attempt rate of motion, suggesting that amantadine creates better-packed and more cohesive helical bundles. Analysis of the temperature dependence of ln (T{sub 1{rho}}{sup -1}) indicates that the activation energy of motion increased from 14.0 {+-} 4.0 kJ/mol for the apo peptide to 23.3 {+-} 6.2 kJ/mol for the bound peptide. This higher activation energy indicates that excess amantadine outside the protein channel in the lipid bilayer increases the membrane viscosity. Thus, the protein-bound amantadine speeds up the diffusion of the helical bundles while the excess amantadine in the bilayer increases the membrane viscosity.

Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins

DEFF Research Database (Denmark)

Weber, Daniela; Davies, Michael J.; Grune, Tilman

2015-01-01

Protein oxidation is involved in regulatory physiological events as well as in damage to tissues and is thought to play a key role in the pathophysiology of diseases and in the aging process. Protein-bound carbonyls represent a marker of global protein oxidation, as they are generated by multiple...

Discrete persistent-chain model for protein binding on DNA.

Science.gov (United States)

Lam, Pui-Man; Zhen, Yi

2011-04-01

We describe and solve a discrete persistent-chain model of protein binding on DNA, involving an extra σ(i) at a site i of the DNA. This variable takes the value 1 or 0, depending on whether or not the site is occupied by a protein. In addition, if the site is occupied by a protein, there is an extra energy cost ɛ. For a small force, we obtain analytic expressions for the force-extension curve and the fraction of bound protein on the DNA. For higher forces, the model can be solved numerically to obtain force-extension curves and the average fraction of bound proteins as a function of applied force. Our model can be used to analyze experimental force-extension curves of protein binding on DNA, and hence deduce the number of bound proteins in the case of nonspecific binding. ©2011 American Physical Society

The catalytic function of cytochrome P450 is entwined with its membrane-bound nature [version 1; referees: 4 approved

Directory of Open Access Journals (Sweden)

Carlo Barnaba

2017-05-01

Full Text Available Cytochrome P450, a family of monooxygenase enzymes, is organized as a catalytic metabolon, which requires enzymatic partners as well as environmental factors that tune its complex dynamic. P450 and its reducing counterparts—cytochrome P450-reductase and cytochrome b5—are membrane-bound proteins located in the cytosolic side of the endoplasmic reticulum. They are believed to dynamically associate to form functional complexes. Increasing experimental evidence signifies the role(s played by both protein-protein and protein-lipid interactions in P450 catalytic function and efficiency. However, the biophysical challenges posed by their membrane-bound nature have severely limited high-resolution understanding of the molecular interfaces of these interactions. In this article, we provide an overview of the current knowledge on cytochrome P450, highlighting the environmental factors that are entwined with its metabolic function. Recent advances in structural biophysics are also discussed, setting up the bases for a new paradigm in the study of this important class of membrane-bound enzymes.

h5-HT1B receptor-mediated constitutive Gαi3-protein activation in stably transfected Chinese hamster ovary cells: an antibody capture assay reveals protean efficacy of 5-HT

Science.gov (United States)

Newman-Tancredi, Adrian; Cussac, Didier; Marini, Laetitia; Touzard, Manuelle; Millan, Mark J

2003-01-01

Serotonin 5-HT1B receptors couple to G-proteins of the Gi/o family. However, their activation of specific G-protein subtypes is poorly characterised. Using an innovative antibody capture/guanosine-5′-0-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding strategy, we characterised Gαi3 subunit activation by h5-HT1B receptors stably expressed in Chinese hamster ovary (CHO) cells. The agonists, 5-HT, alniditan and BMS181,101, stimulated Gαi3, whereas methiothepin and SB224,289 behaved as inverse agonists. The selective 5-HT1B receptor ligand, S18127, modestly stimulated Gαi3 and reversed the actions of both 5-HT and methiothepin. S18127 (1 μM) also produced parallel, dextral shifts of the 5-HT and methiothepin isotherms. Isotopic dilution experiments ([35S]GTPγS versus GTPγS) revealed high-affinity [35S]GTPγS binding to Gαi3 subunits in the absence of receptor ligands indicating constitutive activity. High-affinity [35S]GTPγS binding was increased 2.8-fold by 5-HT with an increase in the affinity of GTPγS for Gαi3 subunits. In contrast, methiothepin halved the number of high-affinity binding sites and decreased their affinity. h5-HT1B receptor-mediated Gαi3 subunit activation was dependent on the concentration of NaCl. At 300 mM, 5-HT stimulated [35S]GTPγS binding, basal Gαi3 activation was low and methiothepin was inactive. In contrast, at 10 mM NaCl, basal activity was enhanced and the inverse agonist activity of methiothepin was accentuated. Under these conditions, 5-HT decreased Gαi3 activation. In conclusion, at h5-HT1B receptors expressed in CHO cells: (i) inverse agonist induced inhibition of Gαi3, and its reversal by S18127, reveals constitutive activation of this Gα subunit; (ii) constitutive Gαi3 activation can be quantified by isotopic dilution [35S]GTPγS binding and (iii) decreasing NaCl concentrations enhances Gαi3 activation and leads to protean agonist properties of 5-HT: that is a switch to inhibition of Gαi3. PMID:12684263

Acylation of cellular proteins with endogenously synthesized fatty acids

International Nuclear Information System (INIS)

Towler, D.; Glaser, L.

1986-01-01

A number of cellular proteins contain covalently bound fatty acids. Previous studies have identified myristic acid and palmitic acid covalently linked to protein, the former usually attached to proteins by an amide linkage and the latter by ester or thio ester linkages. While in a few instances specific proteins have been isolated from cells and their fatty acid composition has been determined, the most frequent approach to the identification of protein-linked fatty acids is to biosynthetically label proteins with fatty acids added to intact cells. This procedure introduces possible bias in that only a selected fraction of proteins may be labeled, and it is not known whether the radioactive fatty acid linked to the protein is identical with that which is attached to the protein when the fatty acid is derived from endogenous sources. We have examined the distribution of protein-bound fatty acid following labeling with [ 3 H]acetate, a general precursor of all fatty acids, using BC 3 H1 cells (a mouse muscle cell line) and A431 cells (a human epidermoid carcinoma). Myristate, palmitate, and stearate account for essentially all of the fatty acids linked to protein following labeling with [ 3 H]acetate, but at least 30% of the protein-bound palmitate in these cells was present in amide linkage. In BC3H1 cells, exogenous palmitate becomes covalently bound to protein such that less than 10% of the fatty acid is present in amide linkage. These data are compatible with multiple protein acylating activities specific for acceptor protein fatty acid chain length and linkage

The recombinant globular head domain of the measles virus hemagglutinin protein as a subunit vaccine against measles.

Science.gov (United States)

Lobanova, Liubov M; Eng, Nelson F; Satkunarajah, Malathy; Mutwiri, George K; Rini, James M; Zakhartchouk, Alexander N

2012-04-26

Despite the availability of live attenuated measles virus (MV) vaccines, a large number of measles-associated deaths occur among infants in developing countries. The development of a measles subunit vaccine may circumvent the limitations associated with the current live attenuated vaccines and eventually contribute to global measles eradication. Therefore, the goal of this study was to test the feasibility of producing the recombinant globular head domain of the MV hemagglutinin (H) protein by stably transfected human cells and to examine the ability of this recombinant protein to elicit MV-specific immune responses. The recombinant protein was purified from the culture supernatant of stably transfected HEK293T cells secreting a tagged version of the protein. Two subcutaneous immunizations with the purified recombinant protein alone resulted in the production of MV-specific serum IgG and neutralizing antibodies in mice. Formulation of the protein with adjuvants (polyphosphazene or alum) further enhanced the humoral immune response and in addition resulted in the induction of cell-mediated immunity as measured by the production of MV H-specific interferon gamma (IFN-γ) and interleukin 5 (IL-5) by in vitro re-stimulated splenocytes. Furthermore, the inclusion of polyphosphazene into the vaccine formulation induced a mixed Th1/Th2-type immune response. In addition, the purified recombinant protein retained its immunogenicity even after storage at 37°C for 2 weeks. Copyright © 2012 Elsevier Ltd. All rights reserved.

78 FR 18326 - Agency Information Collection Activities; Comment Request; Upward Bound and Upward Bound Math...

Science.gov (United States)

2013-03-26

...; Comment Request; Upward Bound and Upward Bound Math Science Annual Performance Report AGENCY: The Office... considered public records. Title of Collection: Upward Bound and Upward Bound Math Science Annual Performance...) and Upward Bound Math and Science (UBMS) Programs. The Department is requesting a new APR because of...

Allosteric Regulation of Proteins

Indian Academy of Sciences (India)

interactions with other proteins, or binding of small molecules. Covalent .... vealed through structural elucidation of the protein in free and oxygen-bound forms .... stance, molecular dynamic simulation of glutamine binding pro- tein shows that ...

Universal bounds on current fluctuations.

Science.gov (United States)

Pietzonka, Patrick; Barato, Andre C; Seifert, Udo

2016-05-01

For current fluctuations in nonequilibrium steady states of Markovian processes, we derive four different universal bounds valid beyond the Gaussian regime. Different variants of these bounds apply to either the entropy change or any individual current, e.g., the rate of substrate consumption in a chemical reaction or the electron current in an electronic device. The bounds vary with respect to their degree of universality and tightness. A universal parabolic bound on the generating function of an arbitrary current depends solely on the average entropy production. A second, stronger bound requires knowledge both of the thermodynamic forces that drive the system and of the topology of the network of states. These two bounds are conjectures based on extensive numerics. An exponential bound that depends only on the average entropy production and the average number of transitions per time is rigorously proved. This bound has no obvious relation to the parabolic bound but it is typically tighter further away from equilibrium. An asymptotic bound that depends on the specific transition rates and becomes tight for large fluctuations is also derived. This bound allows for the prediction of the asymptotic growth of the generating function. Even though our results are restricted to networks with a finite number of states, we show that the parabolic bound is also valid for three paradigmatic examples of driven diffusive systems for which the generating function can be calculated using the additivity principle. Our bounds provide a general class of constraints for nonequilibrium systems.

Protein-protein docking using region-based 3D Zernike descriptors

Directory of Open Access Journals (Sweden)

Sael Lee

2009-12-01

Full Text Available Abstract Background Protein-protein interactions are a pivotal component of many biological processes and mediate a variety of functions. Knowing the tertiary structure of a protein complex is therefore essential for understanding the interaction mechanism. However, experimental techniques to solve the structure of the complex are often found to be difficult. To this end, computational protein-protein docking approaches can provide a useful alternative to address this issue. Prediction of docking conformations relies on methods that effectively capture shape features of the participating proteins while giving due consideration to conformational changes that may occur. Results We present a novel protein docking algorithm based on the use of 3D Zernike descriptors as regional features of molecular shape. The key motivation of using these descriptors is their invariance to transformation, in addition to a compact representation of local surface shape characteristics. Docking decoys are generated using geometric hashing, which are then ranked by a scoring function that incorporates a buried surface area and a novel geometric complementarity term based on normals associated with the 3D Zernike shape description. Our docking algorithm was tested on both bound and unbound cases in the ZDOCK benchmark 2.0 dataset. In 74% of the bound docking predictions, our method was able to find a near-native solution (interface C-αRMSD ≤ 2.5 Å within the top 1000 ranks. For unbound docking, among the 60 complexes for which our algorithm returned at least one hit, 60% of the cases were ranked within the top 2000. Comparison with existing shape-based docking algorithms shows that our method has a better performance than the others in unbound docking while remaining competitive for bound docking cases. Conclusion We show for the first time that the 3D Zernike descriptors are adept in capturing shape complementarity at the protein-protein interface and useful for

Quivers of Bound Path Algebras and Bound Path Coalgebras

Directory of Open Access Journals (Sweden)

Dr. Intan Muchtadi

2010-09-01

Full Text Available bras and coalgebras can be represented as quiver (directed graph, and from quiver we can construct algebras and coalgebras called path algebras and path coalgebras. In this paper we show that the quiver of a bound path coalgebra (resp. algebra is the dual quiver of its bound path algebra (resp. coalgebra.

Protein translation, proteolysis and autophagy in human skeletal muscle atrophy after spinal cord injury.

Science.gov (United States)

Lundell, L S; Savikj, M; Kostovski, E; Iversen, P O; Zierath, J R; Krook, A; Chibalin, A V; Widegren, U

2018-02-08

Spinal cord injury-induced loss of skeletal muscle mass does not progress linearly. In humans, peak muscle loss occurs during the first 6 weeks postinjury, and gradually continues thereafter. The aim of this study was to delineate the regulatory events underlying skeletal muscle atrophy during the first year following spinal cord injury. Key translational, autophagic and proteolytic proteins were analysed by immunoblotting of human vastus lateralis muscle obtained 1, 3 and 12 months following spinal cord injury. Age-matched able-bodied control subjects were also studied. Several downstream targets of Akt signalling decreased after spinal cord injury in skeletal muscle, without changes in resting Akt Ser 473 and Akt Thr 308 phosphorylation or total Akt protein. Abundance of mTOR protein and mTOR Ser 2448 phosphorylation, as well as FOXO1 Ser 256 phosphorylation and FOXO3 protein, decreased in response to spinal cord injury, coincident with attenuated protein abundance of E3 ubiquitin ligases, MuRF1 and MAFbx. S6 protein and Ser 235/236 phosphorylation, as well as 4E-BP1 Thr 37/46 phosphorylation, increased transiently after spinal cord injury, indicating higher levels of protein translation early after injury. Protein abundance of LC3-I and LC3-II decreased 3 months postinjury as compared with 1 month postinjury, but not compared to able-bodied control subjects, indicating lower levels of autophagy. Proteins regulating proteasomal degradation were stably increased in response to spinal cord injury. Together, these data provide indirect evidence suggesting that protein translation and autophagy transiently increase, while whole proteolysis remains stably higher in skeletal muscle within the first year after spinal cord injury. © 2018 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Improved Energy Bound Accuracy Enhances the Efficiency of Continuous Protein Design

OpenAIRE

Roberts, Kyle E.; Donald, Bruce R.

2015-01-01

Flexibility and dynamics are important for protein function and a protein’s ability to accommodate amino acid substitutions. However, when computational protein design algorithms search over protein structures, the allowed flexibility is often reduced to a relatively small set of discrete side-chain and backbone conformations. While simplifications in scoring functions and protein flexibility are currently necessary to computationally search the vast protein sequence and conformational space,...

Fibronectin-synthesizing activity of free and membrane-bound polyribosomes from human embryonic fibroblasts and chick embryos

International Nuclear Information System (INIS)

Belkin, V.M.; Volodarskaya, S.M.

1986-01-01

The fibronectin-synthesizing activity of membrane-bound and free polyribosomes in a cell-free system was studied using immunochemical methods. It was found that fibronectin biosynthesis on membrane-bound polyribosomes from human embryonic fibroblasts accounts for 4.9% and those from 10-day-old chick embryos for 1.1% of the total amount of newly synthesized proteins, whereas on free polyribosomes it is 1.0 and 0.3%, respectively. Fibronectin monomers with a molecular weight of 220,000 were found only in the material of the cell-free system containing heavy fractions of membrane-bound polyribosomes newly synthesized in the presence of spermidine. Thus, it was shown that fibronectin is synthesized primarily on membrane-bound polyribosomes

Interrogating the architecture of protein assemblies and protein interaction networks by cross-linking mass spectrometry

NARCIS (Netherlands)

Liu, Fan; Heck, Albert J R

2015-01-01

Proteins are involved in almost all processes of the living cell. They are organized through extensive networks of interaction, by tightly bound macromolecular assemblies or more transiently via signaling nodes. Therefore, revealing the architecture of protein complexes and protein interaction

Comparison of the rate of uptake and biologic effects of retinol added to human keratinocytes either directly to the culture medium or bound to serum retinol-binding protein

International Nuclear Information System (INIS)

Hodam, J.R.; St Hilaire, P.; Creek, K.E.

1991-01-01

Retinol circulates in the plasma bound to retinol-binding protein (RBP), but the mechanism by which retinol is transferred from RBP to target cells is not known. To study retinol delivery, human keratinocytes (HKc) were incubated with [3H]retinol added directly to the culture medium or bound to RBP and the uptake of [3H]retinol was determined at various times. During the first hour of incubation, the rate of [3H]retinol accumulation by HKc was about 40 times greater when the vitamin was added directly to the media rather than bound to RBP. Although maximal uptake of [3H]retinol added directly to the culture medium occurred at 3 h, the uptake of [3H]retinol from RBP was linear with time for at least 72 h. By 57 h, cell-associated [3H]retinol was the same whether it was added directly to the culture medium or bound to RBP. Excess unlabeled retinol or pretreatment of HKc with retinol had no effect on the uptake of [3H]retinol added directly to the culture medium or bound to RBP. Apo- but not holo-RBP was capable of competing with HKc for the uptake of [3H]retinol from RBP. No specific or saturable binding of 125I-labeled RBP to HKc cultured in the absence or the presence of retinol was found. The dose response of retinol inhibition of cholesterol sulfate synthesis and phorbol ester-induced ornithine decarboxylase activity or retinol modulation of keratin expression was the same whether the retinol was delivered to HKc bound to RBP or added directly to the medium. Our data support a mechanism for retinol delivery from RBP to HKc that does not involve cell-surface RBP receptors but instead suggest that the vitamin is first slowly released from RBP and then becomes cell-associated from the aqueous phase. This mechanism is consistent with the finding that HKc respond identically to retinol whether or not it is delivered to them bound to RBP

Tritium ingestion as organically bound tritium (OBT) - incorporation in different organs of pregnant and non-pregnant rats

International Nuclear Information System (INIS)

Bhatia, A.L.; Pollaris, K.; Vandecasteele, C.M.; Kowalska, M.

1998-01-01

For a better understanding of the hazard of tritium, its bound form in the food constituents (organically bound tritium (OBT)) has not been investigated though study on tritiated water are many. Hence an evaluation of the uptake of tritium incorporated in basic constituents of food viz, proteins, carbohydrates and lipids is warranted. Present study cells with the incorporated three organically bound tritium components separated from tritiated milk powder (casein, butter and lactose). This is further compared in the organs of pregnant (after parturition) and non-pregnant rats

Photon virtual bound state

International Nuclear Information System (INIS)

Inoue, J.; Ohtaka, K.

2004-01-01

We study virtual bound states in photonics, which are a vectorial extension of electron virtual bound states. The condition for these states is derived. It is found that the Mie resonant state which satisfies the condition that the size parameter is less than the angular momentum should be interpreted as a photon virtual bound state. In order to confirm the validity of the concept, we compare the photonic density of states, the width of which represents the lifetime of the photon virtual bound states, with numerical results

Photoexcitation dynamics of nitric oxide bound ferric myoglobin probed by femtosecond IR spectroscopy

Directory of Open Access Journals (Sweden)

Park Jaehun

2013-03-01

Full Text Available Time-resolved vibrational spectra show that photolysis quantum yield of NO bound ferric myoglobin is smaller than 0.86, the deligated NO geminately rebinds with subnanosecond time scale, and the rebinding kinetics depends on protein conformation.

New low-flux mixed matrix membranes that offer superior removal of protein-bound toxins from human plasma

Science.gov (United States)

Pavlenko, Denys; van Geffen, Esmée; van Steenbergen, Mies J.; Glorieux, Griet; Vanholder, Raymond; Gerritsen, Karin G. F.; Stamatialis, Dimitrios

2016-10-01

Hemodialysis is a widely available and well-established treatment for patients with End Stage Renal Disease (ESRD). However, although life-sustaining, patient mortality rates are very high. Several recent studies corroborated the link between dialysis patients’ outcomes and elevated levels of protein-bound uremic toxins (PBUT) that are poorly removed by conventional hemodialysis. Therefore, new treatments are needed to improve their removal. Recently, our group showed that the combination of dialysis and adsorption on one membrane, the mixed matrix membrane (MMM), can effectively remove those toxins from human plasma. However, these first MMMs were rather large in diameter and their mass transport characteristics needed improvement before application in the clinical setting. Therefore, in this study we developed a new generation of MMMs that have a smaller diameter and optimized characteristics offering superior ability in removing the PBUT indoxyl sulfate (IS) and p-cresyl sulfate (pCS) in comparison to first generation MMMs (30 and 125% respectively), as well as, a commercial dialysis membrane (more than 100% better removal).

Apo and ligand-bound structures of ModA from the archaeon Methanosarcina acetivorans.

Science.gov (United States)

Chan, Sum; Giuroiu, Iulia; Chernishof, Irina; Sawaya, Michael R; Chiang, Janet; Gunsalus, Robert P; Arbing, Mark A; Perry, L Jeanne

2010-03-01

The trace-element oxyanion molybdate, which is required for the growth of many bacterial and archaeal species, is transported into the cell by an ATP-binding cassette (ABC) transporter superfamily uptake system called ModABC. ModABC consists of the ModA periplasmic solute-binding protein, the integral membrane-transport protein ModB and the ATP-binding and hydrolysis cassette protein ModC. In this study, X-ray crystal structures of ModA from the archaeon Methanosarcina acetivorans (MaModA) have been determined in the apoprotein conformation at 1.95 and 1.69 A resolution and in the molybdate-bound conformation at 2.25 and 2.45 A resolution. The overall domain structure of MaModA is similar to other ModA proteins in that it has a bilobal structure in which two mixed alpha/beta domains are linked by a hinge region. The apo MaModA is the first unliganded archaeal ModA structure to be determined: it exhibits a deep cleft between the two domains and confirms that upon binding ligand one domain is rotated towards the other by a hinge-bending motion, which is consistent with the 'Venus flytrap' model seen for bacterial-type periplasmic binding proteins. In contrast to the bacterial ModA structures, which have tetrahedral coordination of their metal substrates, molybdate-bound MaModA employs octahedral coordination of its substrate like other archaeal ModA proteins.

Competitive binding thyroid assay with improved bound-free separation step

International Nuclear Information System (INIS)

1979-01-01

A competitive binding assay is described for serum thyroid hormone using 125 I-labelled thyroid hormone and exogenous thyroid hormone binding protein. The unbound thyroid hormone is separated from thyroid hormone bound to thyroid hormone binding protein using an intermediate base anion exchange resin. This resin comprises tertiary and quaternary amine groups on a polyalkyleneamine lattice and is compressed with microcrystalline cellulose in a tablet form. The assay technique of the present invention employing an intermediate base anion resin was found to give superior results compared with alternative assay techniques used in serum thyroid hormone estimation. (UK)

Efficient expression of SRK intracellular domain by a modeling-based protein engineering.

Science.gov (United States)

Murase, Kohji; Hirano, Yoshinori; Takayama, Seiji; Hakoshima, Toshio

2017-03-01

S-locus protein kinase (SRK) is a receptor kinase that plays a critical role in self-recognition in the Brassicaceae self-incompatibility (SI) response. SRK is activated by binding of its ligand S-locus protein 11 (SP11) and subsequently induced phosphorylation of the intracellular kinase domain. However, a detailed activation mechanism of SRK is still largely unknown because of the difficulty in stably expressing SRK recombinant proteins. Here, we performed modeling-based protein engineering of the SRK kinase domain for stable expression in Escherichia coli. The engineered SRK intracellular domain was expressed about 54-fold higher production than wild type SRK, without loss of the kinase activity, suggesting it could be useful for further biochemical and structural studies. Copyright © 2016 Elsevier Inc. All rights reserved.

Hydrogen Exchange Mass Spectrometry of Functional Membrane-bound Chemotaxis Receptor Complexes

Science.gov (United States)

Koshy, Seena S.; Eyles, Stephen J.; Weis, Robert M.; Thompson, Lynmarie K.

2014-01-01

The transmembrane signaling mechanism of bacterial chemotaxis receptors is thought to involve changes in receptor conformation and dynamics. The receptors function in ternary complexes with two other proteins, CheA and CheW, that form extended membrane-bound arrays. Previous studies have shown that attractant binding induces a small (~2 Å) piston displacement of one helix of the periplasmic and transmembrane domains towards the cytoplasm, but it is not clear how this signal propagates through the cytoplasmic domain to control the kinase activity of the CheA bound at the membrane-distal tip, nearly 200 Å away. The cytoplasmic domain has been shown to be highly dynamic, which raises the question of how a small piston motion could propagate through a dynamic domain to control CheA kinase activity. To address this, we have developed a method for measuring dynamics of the receptor cytoplasmic fragment (CF) in functional complexes with CheA and CheW. Hydrogen exchange mass spectrometry (HDX-MS) measurements of global exchange of CF demonstrate that CF exhibits significantly slower exchange in functional complexes than in solution. Since the exchange rates in functional complexes are comparable to that of other proteins of similar structure, the CF appears to be a well-structured protein within these complexes, which is compatible with its role in propagating a signal that appears to be a tiny conformational change in the periplasmic and transmembrane domains of the receptor. We also demonstrate the feasibility of this protocol for local exchange measurements, by incorporating a pepsin digest step to produce peptides with 87% sequence coverage and only 20% back exchange. This method extends HDX-MS to membrane-bound functional complexes without detergents that may perturb the stability or structure of the system. PMID:24274333

Photo-oxidation of proteins and its role in cataractogenesis

DEFF Research Database (Denmark)

Davies, Michael Jonathan; Truscott, R J

2001-01-01

by the protein, or bound chromophore groups, thereby generating excited states (singlet or triplets) or radicals via photo-ionisation. The second major process involves indirect oxidation of the protein via the formation and subsequent reactions of singlet oxygen generated by the transfer of energy to ground...... state (triplet) molecular oxygen by either protein-bound, or other, chromophores. The basic principles behind these mechanisms of photo-oxidation of amino acids, peptides and proteins and the potential selectivity of damage are discussed. Emphasis is placed primarily on the intermediates...

Study on REE bound water-soluble polysaccharides in plant

International Nuclear Information System (INIS)

Wang Yuqi; Guo Fanqing; Xu Lei; Chen Hongmin; Sun Jingxin; Cao Guoyin

1999-01-01

The binding of REE with water-soluble polysaccharides (PSs) in leaves of fern Dicranopteris Dichotoma (DD) has been studied by molecular activation analysis. The cold-water-soluble and hot-water-soluble PSs in leaves of DD were obtained by using biochemical separation techniques. The PSs of non-deproteinization and deproteinization, were separated on Sephadex G-200 gel permeation chromatography. The absorption curves of elution for the PSs were obtained by colorimetry, and the proteins were detected using Coomassic brilliant G-250. Eight REEs (La, Ce, Nd, Sm, Eu, Tb, Yb and Lu) in these PSs were determined by instrumental neutron activation analysis. The results obtained show that the REEs are bound firmly with the water-soluble PSs in the plant. A measurement demonstrates that the PSs bound with REEs are mainly of smaller molecular weight (10,000 to 20,000 Dalton)

Stably engineered nanobubbles and ultrasound - An effective platform for enhanced macromolecular delivery to representative cells of the retina.

Directory of Open Access Journals (Sweden)

Sachin S Thakur

Full Text Available Herein we showcase the potential of ultrasound-responsive nanobubbles in enhancing macromolecular permeation through layers of the retina, ultimately leading to significant and direct intracellular delivery; this being effectively demonstrated across three relevant and distinct retinal cell lines. Stably engineered nanobubbles of a highly homogenous and echogenic nature were fully characterised using dynamic light scattering, B-scan ultrasound and transmission electron microscopy (TEM. The nanobubbles appeared as spherical liposome-like structures under TEM, accompanied by an opaque luminal core and darkened corona around their periphery, with both features indicative of efficient gas entrapment and adsorption, respectively. A nanobubble +/- ultrasound sweeping study was conducted next, which determined the maximum tolerated dose for each cell line. Detection of underlying cellular stress was verified using the biomarker heat shock protein 70, measured before and after treatment with optimised ultrasound. Next, with safety to nanobubbles and optimised ultrasound demonstrated, each human or mouse-derived cell population was incubated with biotinylated rabbit-IgG in the presence and absence of ultrasound +/- nanobubbles. Intracellular delivery of antibody in each cell type was then quantified using Cy3-streptavidin. Nanobubbles and optimised ultrasound were found to be negligibly toxic across all cell lines tested. Macromolecular internalisation was achieved to significant, yet varying degrees in all three cell lines. The results of this study pave the way towards better understanding mechanisms underlying cellular responsiveness to ultrasound-triggered drug delivery in future ex vivo and in vivo models of the posterior eye.

TeBG- and CBG-bound steroid hormones in rabbits are available for influx into uterus in vivo

International Nuclear Information System (INIS)

Chaudhuri, G.; Steingold, K.A.; Pardridge, W.M.; Judd, H.L.

1988-01-01

The metabolic clearance rate (MCR) of gonadal or adrenal steroid hormones in rabbits often does not bear the expected inverse relationship with hormone binding to testosterone-binding globulin (TeBG) or corticosteroid-binding globulin (CBG). This suggests TeBG or CBG may not impede steroid hormone delivery to tissues. The effects of rabbit plasma proteins on the influxes of 3 H-labeled steroids from the circulation into the rabbit uterus were measured in vivo using a tissue sampling single-injection technique. In the absence of plasma proteins, estradiol (E 2 ) and testosterone (T) were freely diffusible through the uterine microvasculature (i.e., extraction >80%). The extractions of dihydrostestosterone (DHT) and corticosterone (B) ranged from 60 to 72%, while that of cortisol (F) was reduced at 40%. Rabbit serum exerted no inhibition of the influxes of the steroids tested. The influxes of T and B greatly exceeded the rates that would be expected if only the free and albumin-bound fractions estimated in vitro were diffusible in vivo. However, the extraction of [ 3 H]corticosteroid-binding globulin or bovine [ 3 H]albumin were low, consistent with little, if any, extravascular uptake of the plasma proteins. The results indicate both albumin-bound and globulin-bound steroid hormone are available for transport into the uterus in the rabbit in vivo without significant exodus of the plasma protein, per se

Exploiting conformational ensembles in modeling protein-protein interactions on the proteome scale

Science.gov (United States)

Kuzu, Guray; Gursoy, Attila; Nussinov, Ruth; Keskin, Ozlem

2013-01-01

Cellular functions are performed through protein-protein interactions; therefore, identification of these interactions is crucial for understanding biological processes. Recent studies suggest that knowledge-based approaches are more useful than ‘blind’ docking for modeling at large scales. However, a caveat of knowledge-based approaches is that they treat molecules as rigid structures. The Protein Data Bank (PDB) offers a wealth of conformations. Here, we exploited ensemble of the conformations in predictions by a knowledge-based method, PRISM. We tested ‘difficult’ cases in a docking-benchmark dataset, where the unbound and bound protein forms are structurally different. Considering alternative conformations for each protein, the percentage of successfully predicted interactions increased from ~26% to 66%, and 57% of the interactions were successfully predicted in an ‘unbiased’ scenario, in which data related to the bound forms were not utilized. If the appropriate conformation, or relevant template interface, is unavailable in the PDB, PRISM could not predict the interaction successfully. The pace of the growth of the PDB promises a rapid increase of ensemble conformations emphasizing the merit of such knowledge-based ensemble strategies for higher success rates in protein-protein interaction predictions on an interactome-scale. We constructed the structural network of ERK interacting proteins as a case study. PMID:23590674

Seminal Plasma Proteins as Androgen Receptor Corregulators Promote Prostate Cancer Growth

Science.gov (United States)

2016-12-01

protease activity of PSA [14]. Semenogelins are expressed in other male genital organs, such as the vas def - erens, epididymis, and prostate, as well...expression and secretion in prostate cancer lines stably expressing SgI. Cell extracts (A) or acetone-precipitated proteins in con - ditioned media... con - firmed this by demonstrating the failure to detect SgI signals in the conditioned medium after culturing control LNCaP with endogenous SgI and

Curvature bound from gravitational catalysis

Science.gov (United States)

Gies, Holger; Martini, Riccardo

2018-04-01

We determine bounds on the curvature of local patches of spacetime from the requirement of intact long-range chiral symmetry. The bounds arise from a scale-dependent analysis of gravitational catalysis and its influence on the effective potential for the chiral order parameter, as induced by fermionic fluctuations on a curved spacetime with local hyperbolic properties. The bound is expressed in terms of the local curvature scalar measured in units of a gauge-invariant coarse-graining scale. We argue that any effective field theory of quantum gravity obeying this curvature bound is safe from chiral symmetry breaking through gravitational catalysis and thus compatible with the simultaneous existence of chiral fermions in the low-energy spectrum. With increasing number of dimensions, the curvature bound in terms of the hyperbolic scale parameter becomes stronger. Applying the curvature bound to the asymptotic safety scenario for quantum gravity in four spacetime dimensions translates into bounds on the matter content of particle physics models.

Redox conditions and protein oxidation in plant mitochondria

DEFF Research Database (Denmark)

Møller, Ian Max; Kasimova, Marina R.; Krab, Klaas

2005-01-01

Redox conditions and protein oxidation in plant mitochondria NAD(P)H has a central position in respiratory metabolism. It is produced by a large number of enzymes, e.g. the Krebs cycle dehydrogenases, in the mitochondrial matrix and is oxidised by, amongst others, the respiratory chain. Most...... of this NAD(P)H appears to be bound to proteins, in fact free NAD(P)H – an important parameter in metabolic regulation - has never been observed in mitochondria. We have estimated free and bound NAD(P)H in isolated plant mitochondria under different metabolic conditions. The fluorescence spectra of free...... and bound NADH was determined and used to deconvolute fluorescence spectra of actively respiring mitochondria. Most of the mitochondrial NADH is bound in states 2 and 4. The amount of free NADH is lower but relatively constant even increasing a little in state 3 where it is about equal to bound NADH...

Structural deformation upon protein-protein interaction: a structural alphabet approach.

Science.gov (United States)

Martin, Juliette; Regad, Leslie; Lecornet, Hélène; Camproux, Anne-Claude

2008-02-28

In a number of protein-protein complexes, the 3D structures of bound and unbound partners significantly differ, supporting the induced fit hypothesis for protein-protein binding. In this study, we explore the induced fit modifications on a set of 124 proteins available in both bound and unbound forms, in terms of local structure. The local structure is described thanks to a structural alphabet of 27 structural letters that allows a detailed description of the backbone. Using a control set to distinguish induced fit from experimental error and natural protein flexibility, we show that the fraction of structural letters modified upon binding is significantly greater than in the control set (36% versus 28%). This proportion is even greater in the interface regions (41%). Interface regions preferentially involve coils. Our analysis further reveals that some structural letters in coil are not favored in the interface. We show that certain structural letters in coil are particularly subject to modifications at the interface, and that the severity of structural change also varies. These information are used to derive a structural letter substitution matrix that summarizes the local structural changes observed in our data set. We also illustrate the usefulness of our approach to identify common binding motifs in unrelated proteins. Our study provides qualitative information about induced fit. These results could be of help for flexible docking.

Structural deformation upon protein-protein interaction: A structural alphabet approach

Directory of Open Access Journals (Sweden)

Lecornet Hélène

2008-02-01

Full Text Available Abstract Background In a number of protein-protein complexes, the 3D structures of bound and unbound partners significantly differ, supporting the induced fit hypothesis for protein-protein binding. Results In this study, we explore the induced fit modifications on a set of 124 proteins available in both bound and unbound forms, in terms of local structure. The local structure is described thanks to a structural alphabet of 27 structural letters that allows a detailed description of the backbone. Using a control set to distinguish induced fit from experimental error and natural protein flexibility, we show that the fraction of structural letters modified upon binding is significantly greater than in the control set (36% versus 28%. This proportion is even greater in the interface regions (41%. Interface regions preferentially involve coils. Our analysis further reveals that some structural letters in coil are not favored in the interface. We show that certain structural letters in coil are particularly subject to modifications at the interface, and that the severity of structural change also varies. These information are used to derive a structural letter substitution matrix that summarizes the local structural changes observed in our data set. We also illustrate the usefulness of our approach to identify common binding motifs in unrelated proteins. Conclusion Our study provides qualitative information about induced fit. These results could be of help for flexible docking.

Optimization of Xenon Biosensors for Detection of Protein Interactions

International Nuclear Information System (INIS)

Lowery, Thomas J.; Garcia, Sandra; Chavez, Lana; Ruiz, E.Janette; Wu, Tom; Brotin, Thierry; Dutasta, Jean-Pierre; King, David S.; Schultz, Peter G.; Pines, Alex; Wemmer, David E.

2005-08-01

Hyperpolarized 129Xe NMR can detect the presence of specific low-concentration biomolecular analytes by means of the xenon biosensor, which consists of a water-soluble, targeted cryptophane-A cage that encapsulates xenon. In this work we use the prototypical biotinylated xenon biosensor to determine the relationship between the molecular composition of the xenon biosensor and the characteristics of protein-bound resonances. The effects of diastereomer overlap, dipole-dipole coupling, chemical shift anisotropy, xenon exchange, and biosensor conformational exchange on protein-bound biosensor signal were assessed. It was found that optimal protein-bound biosensor signal can be obtained by minimizing the number of biosensor diastereomers and using a flexible linker of appropriate length. Both the linewidth and sensitivity of chemical shift to protein binding of the xenon biosensor were found to be inversely proportional to linker length

Deeply bound pionic atom

International Nuclear Information System (INIS)

Toki, Hiroshi; Yamazaki, Toshimitsu

1989-01-01

The standard method of pionic atom formation does not produce deeply bound pionic atoms. A study is made on the properties of deeply bound pionic atom states by using the standard pion-nucleus optical potential. Another study is made to estimate the cross sections of the formation of ls pionic atom states by various methods. The pion-nucleus optical potential is determined by weakly bound pionic atom states and pion nucleus scattering. Although this potential may not be valid for deeply bound pionic atoms, it should provide some hint on binding energies and level widths of deeply bound states. The width of the ls state comes out to be 0.3 MeV and is well separated from the rest. The charge dependence of the ls state is investigated. The binding energies and the widths increase linearly with Z azbove a Z of 30. The report then discusses various methods to populate deeply bound pionic atoms. In particular, 'pion exchange' reactions are proposed. (n, pπ) reaction is discussed first. The cross section is calculated by assuming the in- and out-going nucleons on-shell and the produced pion in (n1) pionic atom states. Then, (n, dπ - ) cross sections are estimated. (p, 2 Heπ - ) reaction would have cross sections similar to the cross section of (n, dπ - ) reaction. In conclusion, it seems best to do (n, p) experiment on heavy nuclei for deeply bound pionic atom. (Nogami, K.)

21-Methylpyrenyl-cholesterol stably and specifically associates with lipoprotein peripheral hemi-membrane: A new labelling tool

Energy Technology Data Exchange (ETDEWEB)

Gaibelet, Gérald [INSERM U563, CHU Purpan, Toulouse (France); CEA, SB2SM and UMR8221 CNRS, IBiTec-Saclay, Gif-sur-Yvette (France); Tercé, François [Université Toulouse III, UMR 1048, Toulouse (France); INSERM U1048, Toulouse (France); Bertrand-Michel, Justine [Université Toulouse III, UMR 1048, Toulouse (France); INSERM U1048, Lipidomic Platform Metatoul, Toulouse (France); Allart, Sophie [Plateau Technique d’Imagerie Cellulaire, INSERM U1043, Toulouse (France); Azalbert, Vincent [Université Toulouse III, UMR 1048, Toulouse (France); INSERM U1048, Toulouse (France); Lecompte, Marie-France [INSERM U563, Faculté de Médecine de Rangueil, Toulouse (France); Collet, Xavier [Université Toulouse III, UMR 1048, Toulouse (France); INSERM U1048, Toulouse (France); Orlowski, Stéphane, E-mail: stephane.orlowski@cea.fr [INSERM U563, CHU Purpan, Toulouse (France); CEA, SB2SM and UMR8221 CNRS, IBiTec-Saclay, Gif-sur-Yvette (France)

2013-11-01

Highlights: •21-Methylpyrenyl-cholesterol specifically and stably associates to lipoproteins. •It is not esterified by LCAT, and thus reliably labels their peripheral hemi-membrane. •HDL vs. LDL are well distinguishable by various fluorescent labelling characteristics. •LDL peripheral hemi-membrane harbors cholesterol-rich ordered lipid (micro)domains. •Cultured cells can be stained by such labelled lipoproteins-mediated delivery. -- Abstract: Lipoproteins are important biological components. However, they have few convenient fluorescent labelling probes currently reported, and their physiological reliability can be questioned. We compared the association of two fluorescent cholesterol derivatives, 22-nitrobenzoxadiazole-cholesterol (NBD-Chol) and 21-methylpyrenyl-cholesterol (Pyr-met-Chol), to serum lipoproteins and to purified HDL and LDL. Both lipoproteins could be stably labelled by Pyr-met-Chol, but virtually not by NBD-Chol. At variance with NBD-Chol, LCAT did not esterify Pyr-met-Chol. The labelling characteristics of lipoproteins by Pyr-met-Chol were well distinguishable between HDL and LDL, regarding dializability, associated probe amount and labelling kinetics. We took benefit of the pyrene labelling to approach the structural organization of LDL peripheral hemi-membrane, since Pyr-met-Chol-labelled LDL, but not HDL, presented a fluorescence emission of pyrene excimers, indicating that the probe was present in an ordered lipid micro-environment. Since the peripheral membrane of LDL contains more sphingomyelin (SM) than HDL, this excimer formation was consistent with the existence of cholesterol- and SM-enriched lipid microdomains in LDL, as already suggested in model membranes of similar composition and reminiscent to the well-described “lipid rafts” in bilayer membranes. Finally, we showed that Pyr-met-Chol could stain cultured PC-3 cells via lipoprotein-mediated delivery, with a staining pattern well different to that observed with NBD

21-Methylpyrenyl-cholesterol stably and specifically associates with lipoprotein peripheral hemi-membrane: A new labelling tool

International Nuclear Information System (INIS)

Gaibelet, Gérald; Tercé, François; Bertrand-Michel, Justine; Allart, Sophie; Azalbert, Vincent; Lecompte, Marie-France; Collet, Xavier; Orlowski, Stéphane

2013-01-01

Highlights: •21-Methylpyrenyl-cholesterol specifically and stably associates to lipoproteins. •It is not esterified by LCAT, and thus reliably labels their peripheral hemi-membrane. •HDL vs. LDL are well distinguishable by various fluorescent labelling characteristics. •LDL peripheral hemi-membrane harbors cholesterol-rich ordered lipid (micro)domains. •Cultured cells can be stained by such labelled lipoproteins-mediated delivery. -- Abstract: Lipoproteins are important biological components. However, they have few convenient fluorescent labelling probes currently reported, and their physiological reliability can be questioned. We compared the association of two fluorescent cholesterol derivatives, 22-nitrobenzoxadiazole-cholesterol (NBD-Chol) and 21-methylpyrenyl-cholesterol (Pyr-met-Chol), to serum lipoproteins and to purified HDL and LDL. Both lipoproteins could be stably labelled by Pyr-met-Chol, but virtually not by NBD-Chol. At variance with NBD-Chol, LCAT did not esterify Pyr-met-Chol. The labelling characteristics of lipoproteins by Pyr-met-Chol were well distinguishable between HDL and LDL, regarding dializability, associated probe amount and labelling kinetics. We took benefit of the pyrene labelling to approach the structural organization of LDL peripheral hemi-membrane, since Pyr-met-Chol-labelled LDL, but not HDL, presented a fluorescence emission of pyrene excimers, indicating that the probe was present in an ordered lipid micro-environment. Since the peripheral membrane of LDL contains more sphingomyelin (SM) than HDL, this excimer formation was consistent with the existence of cholesterol- and SM-enriched lipid microdomains in LDL, as already suggested in model membranes of similar composition and reminiscent to the well-described “lipid rafts” in bilayer membranes. Finally, we showed that Pyr-met-Chol could stain cultured PC-3 cells via lipoprotein-mediated delivery, with a staining pattern well different to that observed with NBD

Bounding species distribution models

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Thomas J. STOHLGREN, Catherine S. JARNEVICH, Wayne E. ESAIAS,Jeffrey T. MORISETTE

2011-10-01

Full Text Available Species distribution models are increasing in popularity for mapping suitable habitat for species of management concern. Many investigators now recognize that extrapolations of these models with geographic information systems (GIS might be sensitive to the environmental bounds of the data used in their development, yet there is no recommended best practice for “clamping” model extrapolations. We relied on two commonly used modeling approaches: classification and regression tree (CART and maximum entropy (Maxent models, and we tested a simple alteration of the model extrapolations, bounding extrapolations to the maximum and minimum values of primary environmental predictors, to provide a more realistic map of suitable habitat of hybridized Africanized honey bees in the southwestern United States. Findings suggest that multiple models of bounding, and the most conservative bounding of species distribution models, like those presented here, should probably replace the unbounded or loosely bounded techniques currently used [Current Zoology 57 (5: 642–647, 2011].

Bounding Species Distribution Models

Science.gov (United States)

Stohlgren, Thomas J.; Jarnevich, Cahterine S.; Morisette, Jeffrey T.; Esaias, Wayne E.

2011-01-01

Species distribution models are increasing in popularity for mapping suitable habitat for species of management concern. Many investigators now recognize that extrapolations of these models with geographic information systems (GIS) might be sensitive to the environmental bounds of the data used in their development, yet there is no recommended best practice for "clamping" model extrapolations. We relied on two commonly used modeling approaches: classification and regression tree (CART) and maximum entropy (Maxent) models, and we tested a simple alteration of the model extrapolations, bounding extrapolations to the maximum and minimum values of primary environmental predictors, to provide a more realistic map of suitable habitat of hybridized Africanized honey bees in the southwestern United States. Findings suggest that multiple models of bounding, and the most conservative bounding of species distribution models, like those presented here, should probably replace the unbounded or loosely bounded techniques currently used [Current Zoology 57 (5): 642-647, 2011].

Fluorogen-activating proteins: beyond classical fluorescent proteins

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Shengnan Xu

2018-05-01

Full Text Available Fluorescence imaging is a powerful technique for the real-time noninvasive monitoring of protein dynamics. Recently, fluorogen activating proteins (FAPs/fluorogen probes for protein imaging were developed. Unlike the traditional fluorescent proteins (FPs, FAPs do not fluoresce unless bound to their specific small-molecule fluorogens. When using FAPs/fluorogen probes, a washing step is not required for the removal of free probes from the cells, thus allowing rapid and specific detection of proteins in living cells with high signal-to-noise ratio. Furthermore, with different fluorogens, living cell multi-color proteins labeling system was developed. In this review, we describe about the discovery of FAPs, the design strategy of FAP fluorogens, the application of the FAP technology and the advances of FAP technology in protein labeling systems. KEY WORDS: Fluorogen activating proteins, Fluorogens, Genetically encoded sensors, Fluorescence imaging, Molecular imaging

Protein-protein docking using region-based 3D Zernike descriptors.

Science.gov (United States)

Venkatraman, Vishwesh; Yang, Yifeng D; Sael, Lee; Kihara, Daisuke

2009-12-09

Protein-protein interactions are a pivotal component of many biological processes and mediate a variety of functions. Knowing the tertiary structure of a protein complex is therefore essential for understanding the interaction mechanism. However, experimental techniques to solve the structure of the complex are often found to be difficult. To this end, computational protein-protein docking approaches can provide a useful alternative to address this issue. Prediction of docking conformations relies on methods that effectively capture shape features of the participating proteins while giving due consideration to conformational changes that may occur. We present a novel protein docking algorithm based on the use of 3D Zernike descriptors as regional features of molecular shape. The key motivation of using these descriptors is their invariance to transformation, in addition to a compact representation of local surface shape characteristics. Docking decoys are generated using geometric hashing, which are then ranked by a scoring function that incorporates a buried surface area and a novel geometric complementarity term based on normals associated with the 3D Zernike shape description. Our docking algorithm was tested on both bound and unbound cases in the ZDOCK benchmark 2.0 dataset. In 74% of the bound docking predictions, our method was able to find a near-native solution (interface C-alphaRMSD 3D Zernike descriptors are adept in capturing shape complementarity at the protein-protein interface and useful for protein docking prediction. Rigorous benchmark studies show that our docking approach has a superior performance compared to existing methods.

Factor VII and protein C are phosphatidic acid-binding proteins.

Science.gov (United States)

Tavoosi, Narjes; Smith, Stephanie A; Davis-Harrison, Rebecca L; Morrissey, James H

2013-08-20

Seven proteins in the human blood clotting cascade bind, via their GLA (γ-carboxyglutamate-rich) domains, to membranes containing exposed phosphatidylserine (PS), although with membrane binding affinities that vary by 3 orders of magnitude. Here we employed nanodiscs of defined phospholipid composition to quantify the phospholipid binding specificities of these seven clotting proteins. All bound preferentially to nanobilayers in which PS headgroups contained l-serine versus d-serine. Surprisingly, however, nanobilayers containing phosphatidic acid (PA) bound substantially more of two of these proteins, factor VIIa and activated protein C, than did equivalent bilayers containing PS. Consistent with this finding, liposomes containing PA supported higher proteolytic activity by factor VIIa and activated protein C toward their natural substrates (factors X and Va, respectively) than did PS-containing liposomes. Moreover, treating activated human platelets with phospholipase D enhanced the rates of factor X activation by factor VIIa in the presence of soluble tissue factor. We hypothesize that factor VII and protein C bind preferentially to the monoester phosphate of PA because of its accessibility and higher negative charge compared with the diester phosphates of most other phospholipids. We further found that phosphatidylinositol 4-phosphate, which contains a monoester phosphate attached to its myo-inositol headgroup, also supported enhanced enzymatic activity of factor VIIa and activated protein C. We conclude that factor VII and protein C bind preferentially to monoester phosphates, which may have implications for the function of these proteases in vivo.

Construction of a CD147 Lentiviral Expression Vector and Establishment of Its Stably Transfected A549 Cell Line

Directory of Open Access Journals (Sweden)

Shaoxing YANG

2012-12-01

Full Text Available Background and objective CD147, a type of transmembrane glycoprotein embedded on the surface of tumor cells, can promote tumor invasion and metastasis. This aim of this study is to construct a CD147 lentiviral expression vector, establish its stably transfected A549 cell line, and observe the effect of CD147 on MMP-9 proliferation as well as on the invasive ability of human lung adenocarcinoma cells. Methods Full-length CD147 gene was amplified by real-time polymerase chain reaction (RT-PCR, inserted into a pEGFP vector to construct pEGFP-CD147 and pEGFP vectors, and then transfected into 293FT cells to precede the lentivirus equipment package. Subsequently, we collected the lentivirus venom to infect the A549 cells and establish a stable, overexpressed cell line named A549-CD147. The mRNA expression of MMP-9 was examined by RT-PCR. The proliferation and invasive ability of the human lung cancer cells before and after transfection were examined by the CCK-8 and Transwell methods. Results A CD147 lentiviral expression vector (pEGFP-CD147 was successfully constructed by restrictive enzyme digestion and plasmid sequencing. RT-PCR and Western blot analyses revealed increased mRNA and protein expression of CD147 gene in cells transfected with pEGFP-CD147 compared with the control groups. Therefore, the A549-CD147 cell line was successfully established through the experiment. The mRNA expression of MMP-9 also significantly increased after the upregulation of CD147 expression. Meanwhile, CCK-8 and Transwell assays indicated that the proliferation and invasive ability significantly increased in the A549-CD147 cells. Conclusion A lentiviral CD147 expression vector and its A549 cell line (A549-CD14 were successfully constructed. CD147 overexpression upregulated the protein expression of MMP-9, and strengthened the proliferation and invasive ability of human lung adenocarcinoma cells.

An Enzymatic Treatment of Soil-Bound Prions Effectively Inhibits Replication ▿

Science.gov (United States)

Saunders, Samuel E.; Bartz, Jason C.; Vercauteren, Kurt C.; Bartelt-Hunt, Shannon L.

2011-01-01

Chronic wasting disease (CWD) and scrapie can be transmitted through indirect environmental routes, possibly via soil, and a practical decontamination strategy for prion-contaminated soil is currently unavailable. In the laboratory, an enzymatic treatment under environmentally relevant conditions (22°C, pH 7.4) can degrade soil-bound PrPSc below the limits of Western blot detection. We developed and used a quantitative serial protein misfolding cyclic amplification (PMCA) protocol to characterize the amplification efficiency of treated soil samples relative to controls of known infectious titer. Our results suggest large (104- to >106-fold) decreases in soil-bound prion infectivity following enzyme treatment, demonstrating that a mild enzymatic treatment could effectively reduce the risk of prion disease transmission via soil or other environmental surfaces. PMID:21571886

Labeling schemes for bounded degree graphs

DEFF Research Database (Denmark)

Adjiashvili, David; Rotbart, Noy Galil

2014-01-01

We investigate adjacency labeling schemes for graphs of bounded degree Δ = O(1). In particular, we present an optimal (up to an additive constant) log n + O(1) adjacency labeling scheme for bounded degree trees. The latter scheme is derived from a labeling scheme for bounded degree outerplanar...... graphs. Our results complement a similar bound recently obtained for bounded depth trees [Fraigniaud and Korman, SODA 2010], and may provide new insights for closing the long standing gap for adjacency in trees [Alstrup and Rauhe, FOCS 2002]. We also provide improved labeling schemes for bounded degree...

Doing the math

Science.gov (United States)

Knoblach, Barbara; Rachubinski, Richard A

2013-01-01

The formation of membrane contact sites between cellular organelles is required for proper organelle communication and maintenance in the compartmentalized eukaryotic cell. We recently identified a tether that links peroxisomes to the cortical ER in the yeast, Saccharomyces cerevisiae. The tether is made up of the peroxisome biogenic protein Pex3p and the peroxisome inheritance factor Inp1p, and is formed by Inp1p-mediated linkage of ER-bound Pex3p and peroxisomal Pex3p. Here we discuss how this tether is fine-tuned to ensure that peroxisomes are stably maintained over generations of yeast cells. PMID:24567780

The quantification of free Amadori compounds and amino acids allows to model the bound Maillard reaction products formation in soybean products

NARCIS (Netherlands)

Troise, Antonio Dario; Wiltafsky, Markus; Fogliano, Vincenzo; Vitaglione, Paola

2018-01-01

The quantification of protein bound Maillard reaction products (MRPs) is still a challenge in food chemistry. Protein hydrolysis is the bottleneck step: it is time consuming and the protein degradation is not always complete. In this study, the quantitation of free amino acids and Amadori products

Membrane-bound 2,3-diphosphoglycerate phosphatase of human erythrocytes.

Science.gov (United States)

Schröter, W; Neuvians, M

1970-12-01

Gradual osmotic hemolysis of human erythrocytes reduces the cell content of whole protein, hemoglobin, 2,3-diphosphoglycerate and triosephosphate isomerase extensively, but not that of membrane protein and 2,3-diphosphoglycerate phosphatase. After the refilling of the ghosts with 2,3-diphosphoglycerate and reconstitution of the membrane, the 2,3-diphosphoglycerate phosphatase activity equals that of intact red cells. The membrane-bound 2,3-diphosphoglycerate phosphatase can be activated by sodium hyposulfite. The enzyme system of ghosts seems to differ from that of intact red cells with regard to the optima of pH and temperature. It remains to be elucidated if the membrane binding of the 2,3-diphosphoglycerate phosphatase is related to the transfer of inorganic phosphate across the red cell membrane.

Structure of the Z Ring-associated Protein, ZapD, Bound to the C-terminal Domain of the Tubulin-like Protein, FtsZ, Suggests Mechanism of Z Ring Stabilization through FtsZ Cross-linking.

Science.gov (United States)

Schumacher, Maria A; Huang, Kuo-Hsiang; Zeng, Wenjie; Janakiraman, Anuradha

2017-03-03

Cell division in most bacteria is mediated by the tubulin-like FtsZ protein, which polymerizes in a GTP-dependent manner to form the cytokinetic Z ring. A diverse repertoire of FtsZ-binding proteins affects FtsZ localization and polymerization to ensure correct Z ring formation. Many of these proteins bind the C-terminal domain (CTD) of FtsZ, which serves as a hub for FtsZ regulation. FtsZ ring-associated proteins, ZapA-D (Zaps), are important FtsZ regulatory proteins that stabilize FtsZ assembly and enhance Z ring formation by increasing lateral assembly of FtsZ protofilaments, which then form the Z ring. There are no structures of a Zap protein bound to FtsZ; therefore, how these proteins affect FtsZ polymerization has been unclear. Recent data showed ZapD binds specifically to the FtsZ CTD. Thus, to obtain insight into the ZapD-CTD interaction and how it may mediate FtsZ protofilament assembly, we determined the Escherichia coli ZapD-FtsZ CTD structure to 2.67 Å resolution. The structure shows that the CTD docks within a hydrophobic cleft in the ZapD helical domain and adopts an unusual structure composed of two turns of helix separated by a proline kink. FtsZ CTD residue Phe-377 inserts into the ZapD pocket, anchoring the CTD in place and permitting hydrophobic contacts between FtsZ residues Ile-374, Pro-375, and Leu-378 with ZapD residues Leu-74, Trp-77, Leu-91, and Leu-174. The structural findings were supported by mutagenesis coupled with biochemical and in vivo studies. The combined data suggest that ZapD acts as a molecular cross-linking reagent between FtsZ protofilaments to enhance FtsZ assembly. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine

Energy Technology Data Exchange (ETDEWEB)

Pletnev, Vladimir Z., E-mail: vzpletnev@gmail.com; Pletneva, Nadya V.; Lukyanov, Konstantin A.; Souslova, Ekaterina A.; Fradkov, Arkady F.; Chudakov, Dmitry M.; Chepurnykh, Tatyana; Yampolsky, Ilia V. [Russian Academy of Sciences, Moscow (Russian Federation); Wlodawer, Alexander [National Cancer Institute, Frederick, MD 21702 (United States); Dauter, Zbigniew [National Cancer Institute, Argonne, IL 60439 (United States); Pletnev, Sergei, E-mail: vzpletnev@gmail.com [National Cancer Institute, Argonne, IL 60439 (United States); SAIC-Frederick, Argonne, IL 60439 (United States); Russian Academy of Sciences, Moscow (Russian Federation)

2013-09-01

The crystal structure of the novel red emitting fluorescent protein from lancelet Branchiostoma lanceolatum (Chordata) revealed an unusual five residues cyclic unit comprising Gly58-Tyr59-Gly60 chromophore, the following Phe61 and Tyr62 covalently bound to chromophore Tyr59. A key property of proteins of the green fluorescent protein (GFP) family is their ability to form a chromophore group by post-translational modifications of internal amino acids, e.g. Ser65-Tyr66-Gly67 in GFP from the jellyfish Aequorea victoria (Cnidaria). Numerous structural studies have demonstrated that the green GFP-like chromophore represents the ‘core’ structure, which can be extended in red-shifted proteins owing to modifications of the protein backbone at the first chromophore-forming position. Here, the three-dimensional structures of green laGFP (λ{sub ex}/λ{sub em} = 502/511 nm) and red laRFP (λ{sub ex}/λ{sub em} ≃ 521/592 nm), which are fluorescent proteins (FPs) from the lancelet Branchiostoma lanceolatum (Chordata), were determined together with the structure of a red variant laRFP-ΔS83 (deletion of Ser83) with improved folding. Lancelet FPs are evolutionarily distant and share only ∼20% sequence identity with cnidarian FPs, which have been extensively characterized and widely used as genetically encoded probes. The structure of red-emitting laRFP revealed three exceptional features that have not been observed in wild-type fluorescent proteins from Cnidaria reported to date: (i) an unusual chromophore-forming sequence Gly58-Tyr59-Gly60, (ii) the presence of Gln211 at the position of the conserved catalytic Glu (Glu222 in Aequorea GFP), which proved to be crucial for chromophore formation, and (iii) the absence of modifications typical of known red chromophores and the presence of an extremely unusual covalent bond between the Tyr59 C{sup β} atom and the hydroxyl of the proximal Tyr62. The impact of this covalent bond on the red emission and the large Stokes shift (�

High Efficacy but Low Potency of δ-Opioid Receptor-G Protein Coupling in Brij-58-Treated, Low-Density Plasma Membrane Fragments.

Science.gov (United States)

Roubalova, Lenka; Vosahlikova, Miroslava; Brejchova, Jana; Sykora, Jan; Rudajev, Vladimir; Svoboda, Petr

2015-01-01

HEK293 cells stably expressing PTX-insensitive δ-opioid receptor-Gi1α (C351I) fusion protein were homogenized, treated with low concentrations of non-ionic detergent Brij-58 at 0°C and fractionated by flotation in sucrose density gradient. In optimum range of detergent concentrations (0.025-0.05% w/v), Brij-58-treated, low-density membranes exhibited 2-3-fold higher efficacy of DADLE-stimulated, high-affinity [32P]GTPase and [35S]GTPγS binding than membranes of the same density prepared in the absence of detergent. The potency of agonist DADLE response was significantly decreased. At high detergent concentrations (>0.1%), the functional coupling between δ-opioid receptors and G proteins was completely diminished. The same detergent effects were measured in plasma membranes isolated from PTX-treated cells. Therefore, the effect of Brij-58 on δ-opioid receptor-G protein coupling was not restricted to the covalently bound Gi1α within δ-opioid receptor-Gi1α fusion protein, but it was also valid for PTX-sensitive G proteins of Gi/Go family endogenously expressed in HEK293 cells. Characterization of the direct effect of Brij-58 on the hydrophobic interior of isolated plasma membranes by steady-state anisotropy of diphenylhexatriene (DPH) fluorescence indicated a marked increase of membrane fluidity. The time-resolved analysis of decay of DPH fluorescence by the "wobble in cone" model of DPH motion in the membrane indicated that the exposure to the increasing concentrations of Brij-58 led to a decreased order and higher motional freedom of the dye. Limited perturbation of plasma membrane integrity by low concentrations of non-ionic detergent Brij-58 results in alteration of δ-OR-G protein coupling. Maximum G protein-response to agonist stimulation (efficacy) is increased; affinity of response (potency) is decreased. The total degradation plasma membrane structure at high detergent concentrations results in diminution of functional coupling between �

Effect of gastric anacidity on the intestinal absorption of liver bound 57Co-labelled cobalamins

International Nuclear Information System (INIS)

Kittang, E.

1987-01-01

57 Co-labelled cyanocobalamin injected in rabbit was transformed within the liver to 57 Co-labelled desoxyadenosylcobalamin and methylcovalamin. The absorption of 57 Co-labelled liver bound cobalamins could be determined with acceptable accuracy by the double isotope fecal excretion method. Treatment with the H 2-receptor antagonist, ranitidine, did not result in decreased absorption of 57 Co-labelled liver bound cobalamins in healthy individuals. R-protein and the R-proteincobalamin complex were determined by the FPLC Mono S chromatography method with a high degree of correlation to the charcoal method in saliva, gastric and duodenal juice, and with a high degree of reproducibility. Omeprazole markedly inhibited the gastric acid and pepsin secretion, but did nor inhibit the IF secretion. Omeprazole treatment resulted in anacidity in 14 of 17 individuals, but did not reduce the absorption of liver bound 57 Co-labelled cobalamins. The intrinsic factor concentration in gastric aspirates measured during the study was unchanged during omeprazole treatment. The release of cobalamins from liver homogenate was markedly inhibited by neutralized gastric juice in vitro, probably due to decreased pepsin mediated proteolysis. In vivo the cobalamin release from liver homogenate was modestly inhibited in the stomach but was unaffected in jejunum during omeprazole treatment. The major part of 57 Co-labelled liver cobalamins bound to R-protein in acid and neutral gastric juice in vitro, and omeprazole induced anacidity, did not influence the cobalamin binding either in gastric or jejunal juice in vivo

Myeloperoxidase-mediated protein lysine oxidation generates 2-aminoadipic acid and lysine nitrile in vivo.

Science.gov (United States)

Lin, Hongqiao; Levison, Bruce S; Buffa, Jennifer A; Huang, Ying; Fu, Xiaoming; Wang, Zeneng; Gogonea, Valentin; DiDonato, Joseph A; Hazen, Stanley L

2017-03-01

Recent studies reveal 2-aminoadipic acid (2-AAA) is both elevated in subjects at risk for diabetes and mechanistically linked to glucose homeostasis. Prior studies also suggest enrichment of protein-bound 2-AAA as an oxidative post-translational modification of lysyl residues in tissues associated with degenerative diseases of aging. While in vitro studies suggest redox active transition metals or myeloperoxidase (MPO) generated hypochlorous acid (HOCl) may produce protein-bound 2-AAA, the mechanism(s) responsible for generation of 2-AAA during inflammatory diseases are unknown. In initial studies we observed that traditional acid- or base-catalyzed protein hydrolysis methods previously employed to measure tissue 2-AAA can artificially generate protein-bound 2-AAA from an alternative potential lysine oxidative product, lysine nitrile (LysCN). Using a validated protease-based digestion method coupled with stable isotope dilution LC/MS/MS, we now report protein bound 2-AAA and LysCN are both formed by hypochlorous acid (HOCl) and the MPO/H 2 O 2 /Cl - system of leukocytes. At low molar ratio of oxidant to target protein N ε -lysine moiety, 2-AAA is formed via an initial N ε -monochloramine intermediate, which ultimately produces the more stable 2-AAA end-product via sequential generation of transient imine and semialdehyde intermediates. At higher oxidant to target protein N ε -lysine amine ratios, protein-bound LysCN is formed via initial generation of a lysine N ε -dichloramine intermediate. In studies employing MPO knockout mice and an acute inflammation model, we show that both free and protein-bound 2-AAA, and in lower yield, protein-bound LysCN, are formed by MPO in vivo during inflammation. Finally, both 2-AAA and to lesser extent LysCN are shown to be enriched in human aortic atherosclerotic plaque, a tissue known to harbor multiple MPO-catalyzed protein oxidation products. Collectively, these results show that MPO-mediated oxidation of protein lysyl

Scattering by bound nucleons

International Nuclear Information System (INIS)

Tezuka, Hirokazu.

1984-10-01

Scattering of a particle by bound nucleons is discussed. Effects of nucleons that are bound in a nucleus are taken as a structure function. The way how to calculate the structure function is given. (author)

Bounds for Asian basket options

Science.gov (United States)

Deelstra, Griselda; Diallo, Ibrahima; Vanmaele, Michèle

2008-09-01

In this paper we propose pricing bounds for European-style discrete arithmetic Asian basket options in a Black and Scholes framework. We start from methods used for basket options and Asian options. First, we use the general approach for deriving upper and lower bounds for stop-loss premia of sums of non-independent random variables as in Kaas et al. [Upper and lower bounds for sums of random variables, Insurance Math. Econom. 27 (2000) 151-168] or Dhaene et al. [The concept of comonotonicity in actuarial science and finance: theory, Insurance Math. Econom. 31(1) (2002) 3-33]. We generalize the methods in Deelstra et al. [Pricing of arithmetic basket options by conditioning, Insurance Math. Econom. 34 (2004) 55-57] and Vanmaele et al. [Bounds for the price of discrete sampled arithmetic Asian options, J. Comput. Appl. Math. 185(1) (2006) 51-90]. Afterwards we show how to derive an analytical closed-form expression for a lower bound in the non-comonotonic case. Finally, we derive upper bounds for Asian basket options by applying techniques as in Thompson [Fast narrow bounds on the value of Asian options, Working Paper, University of Cambridge, 1999] and Lord [Partially exact and bounded approximations for arithmetic Asian options, J. Comput. Finance 10 (2) (2006) 1-52]. Numerical results are included and on the basis of our numerical tests, we explain which method we recommend depending on moneyness and time-to-maturity.

Impact of cell type and epitope tagging on heterologous expression of G protein-coupled receptor: a systematic study on angiotensin type II receptor.

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Lili Jiang

Full Text Available Despite heterologous expression of epitope-tagged GPCR is widely adopted for functional characterization, there is lacking of systematic analysis of the impact of expression host and epitope tag on GPCR expression. Angiotensin type II (AT2 receptor displays agonist-dependent and -independent activities, coupling to a spectrum of signaling molecules. However, consensus has not been reached on the subcellular distributions, signaling cascades and receptor-mediated actions. To examine the contributions of host cell and epitope tag on receptor expression and activity, epitope-tagged AT2 receptor variants were transiently or stably expressed in HEK293, CHO-K1 and PC12 cells. The epitope-tagged AT2 receptor variants were detected both on the cell membrane and in the perinuclear region. In transiently transfected HEK293 cells, Myc-AT2 existed predominantly as monomer. Additionally, a ladder of ubiquitinated AT2 receptor proteins was detected. By contrast, stably expressed epitope-tagged AT2 receptor variants existed as both monomer and high molecular weight complexes, and the latter was enriched in cell surface. Glycosylation promoted cell surface expression of Myc-AT2 but had no effect on AT2-GFP in HEK293 cells. In cells that stably expressed Myc-AT2, serum starvation induced apoptosis in CHO-K1 cells but not in HEK293 or PC12 cells. Instead, HEK293 and PC12 cells stably expressing Myc-AT2 exhibited partial cell cycle arrest with cells accumulating at G1 and S phases, respectively. Taken together, these results suggest that expression levels, subcellular distributions and ligand-independent constitutive activities of AT2 receptor were cell type-dependent while posttranslational processing of nascent AT2 receptor protein was modulated by epitope tag and mode of expression.

Market access through bound tariffs

DEFF Research Database (Denmark)

Sala, Davide; Yalcin, Erdal; Schröder, Philipp

2010-01-01

on the risk that exporters face in destination markets. The present paper formalizes the underlying interaction of risk, fixed export costs and firms' market entry decisions based on techniques known from the real options literature; doing so we highlight the important role of bound tariffs at the extensive...... margin of trade. We find that bound tariffs are more effective with higher risk destination markets, that a large binding overhang may still command substantial market access, and that reductions in bound tariffs generate effective market access even when bound rates are above current and longterm...

Market Access through Bound Tariffs

DEFF Research Database (Denmark)

Sala, Davide; Schröder, Philipp J.H.; Yalcin, Erdal

on the risk that exporters face in destination markets. The present paper formalizes the underlying interaction of risk, fixed export costs and firms' market entry decisions based on techniques known from the real options literature; doing so we highlight the important role of bound tariffs at the extensive...... margin of trade. We find that bound tariffs are more effective with higher risk destination markets, that a large binding overhang may still command substantial market access, and that reductions in bound tariffs generate effective market access even when bound rates are above current and long...

Bound and rebound states

International Nuclear Information System (INIS)

Orzalesi, C.A.

1979-01-01

In relativistic quantum theory, bound states generate forces in the crossed channel; such forces can affect the binding and self-consistent solutions should be sought for the bound-state problem. The author investigates how self-consistency can be achieved by successive approximations, in a simple scalar model and with successive relativistic eikonal approximations (EAs). Within the generalized ladder approximation, some exact properties of the resulting ''first generation'' bound states are discussed. The binding energies in this approximation are rather small even for rather large values of the primary coupling constant. The coupling of the constituent particles to the first-generation reggeon is determined by a suitable EA and a new generalized ladder amplitude is constructed with rungs given either by the primary gluons or by the first-generation reggeons. The resulting new (second-generation) bound states are found in a reggeized EA. The size of the corrections to the binding energies due to the rebinding effects is surprisingly large. The procedure is then iterated, so as to find - again in an EA - the third-generation bound states. The procedure is found to be self-consistent already at this stage: the third-generation bound states coincide with those of second generation, and no further rebinding takes place in the higher iterations of the approximation method. Features - good and bad - of the model are discussed, as well as the possible relevance of rebinding mechanisms in hadron dynamics. (author)

Characteristics of stably expressed human dopamine D1a and D1b receptors: atypical behavior of the dopamine D1b receptor

DEFF Research Database (Denmark)

Pedersen, U B; Norby, B; Jensen, Anders A.

1994-01-01

Human dopamine D1a and D1b receptors were stably expressed in Baby Hamster Kidney (BHK) or Chinese Hamster Ovary (CHO) cells. [3H]SCH23390 saturation experiments indicated the presence of only a single binding site in the D1a expressing cell line with a Kd of 0.5 nM. In D1b expressing cell lines...

Tritium NMR spectroscopy of ligand binding to maltose-binding protein

International Nuclear Information System (INIS)

Gehring, K.; Williams, P.G.; Pelton, J.G.; Morimoto, H.; Wemmer, D.E.

1991-01-01

Tritium-labeled α- and β-maltodextrins have been used to study their complexes with maltose-binding protein (MBP), a 40-kDa bacterial protein. Five substrates, from maltose to maltohexaose, were labeled at their reducing ends and their binding studied. Tritium NMR specctroscopy of the labeled sugars showed large upfield chamical shift changes upon binding and strong anomeric specficity. At 10 degrees C, MBP bound α-maltose with 2.7 ± 0.5-fold higher affinity than β-maltose, and, for longer maltodextrins, the ratio of affinities was even larger. The maximum chemical shift change was 2.2 ppm, suggesting that the reducing end of bound α-maltodextrin makes close contact with an aromatic residue in the MBP-binding site. Experiments with maltotriose (and longer maltodextrins) also revealed the presence of two bound β-maltotriose resonances in rapid exchange. The authors interpret these two resonances as arising from two distinct sugar-protein complexes. In one complex, the β-maltodextrin is bound by its reducing end, and, in the other complex, the β-maltodextrin is bound by the middle glucose residue(s). This interpretation also suggests how MBP is able to bind both linear and circular maltodextrins

HTLV-1 Tax mediated downregulation of miRNAs associated with chromatin remodeling factors in T cells with stably integrated viral promoter.

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Saifur Rahman

Full Text Available RNA interference (RNAi is a natural cellular mechanism to silence gene expression and is predominantly mediated by microRNAs (miRNAs that target messenger RNA. Viruses can manipulate the cellular processes necessary for their replication by targeting the host RNAi machinery. This study explores the effect of human T-cell leukemia virus type 1 (HTLV-1 transactivating protein Tax on the RNAi pathway in the context of a chromosomally integrated viral long terminal repeat (LTR using a CD4(+ T-cell line, Jurkat. Transcription factor profiling of the HTLV-1 LTR stably integrated T-cell clone transfected with Tax demonstrates increased activation of substrates and factors associated with chromatin remodeling complexes. Using a miRNA microarray and bioinformatics experimental approach, Tax was also shown to downregulate the expression of miRNAs associated with the translational regulation of factors required for chromatin remodeling. These observations were validated with selected miRNAs and an HTLV-1 infected T cells line, MT-2. miR-149 and miR-873 were found to be capable of directly targeting p300 and p/CAF, chromatin remodeling factors known to play critical role in HTLV-1 pathogenesis. Overall, these results are first in line establishing HTLV-1/Tax-miRNA-chromatin concept and open new avenues toward understanding retroviral latency and/or replication in a given cell type.

The quantification of free Amadori compounds and amino acids allows to model the bound Maillard reaction products formation in soybean products.

Science.gov (United States)

Troise, Antonio Dario; Wiltafsky, Markus; Fogliano, Vincenzo; Vitaglione, Paola

2018-05-01

The quantification of protein bound Maillard reaction products (MRPs) is still a challenge in food chemistry. Protein hydrolysis is the bottleneck step: it is time consuming and the protein degradation is not always complete. In this study, the quantitation of free amino acids and Amadori products (APs) was compared to the percentage of blocked lysine by using chemometric tools. Eighty thermally treated soybean samples were analyzed by mass spectrometry to measure the concentration of free amino acids, free APs and the protein-bound markers of the Maillard reaction (furosine, Nε-(carboxymethyl)-l-lysine, Nε-(carboxyethyl)-l-lysine, total lysine). Results demonstrated that Discriminant Analysis (DA) and Correlated Component Regression (CCR) correctly estimated the percent of blocked lysine in a validation and prediction set. These findings indicate that the measure of free markers reflects the extent of protein damage in soybean samples and it suggests the possibility to obtain rapid information on the quality of the industrial processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

ER-bound protein tyrosine phosphatase PTP1B interacts with Src at the plasma membrane/substrate interface.

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Melisa C Monteleone

Full Text Available PTP1B is an endoplasmic reticulum (ER anchored enzyme whose access to substrates is partly dependent on the ER distribution and dynamics. One of these substrates, the protein tyrosine kinase Src, has been found in the cytosol, endosomes, and plasma membrane. Here we analyzed where PTP1B and Src physically interact in intact cells, by bimolecular fluorescence complementation (BiFC in combination with temporal and high resolution microscopy. We also determined the structural basis of this interaction. We found that BiFC signal is displayed as puncta scattered throughout the ER network, a feature that was enhanced when the substrate trapping mutant PTP1B-D181A was used. Time-lapse and co-localization analyses revealed that BiFC puncta did not correspond to vesicular carriers; instead they localized at the tip of dynamic ER tubules. BiFC puncta were retained in ventral membrane preparations after cell unroofing and were also detected within the evanescent field of total internal reflection fluorescent microscopy (TIRFM associated to the ventral membranes of whole cells. Furthermore, BiFC puncta often colocalized with dark spots seen by surface reflection interference contrast (SRIC. Removal of Src myristoylation and polybasic motifs abolished BiFC. In addition, PTP1B active site and negative regulatory tyrosine 529 on Src were primary determinants of BiFC occurrence, although the SH3 binding motif on PTP1B also played a role. Our results suggest that ER-bound PTP1B dynamically interacts with the negative regulatory site at the C-terminus of Src at random puncta in the plasma membrane/substrate interface, likely leading to Src activation and recruitment to adhesion complexes. We postulate that this functional ER/plasma membrane crosstalk could apply to a wide array of protein partners, opening an exciting field of research.

Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays.

Science.gov (United States)

Kojima, Takaaki; Mizoguchi, Takuro; Ota, Eri; Hata, Jumpei; Homma, Keisuke; Zhu, Bo; Hitomi, Kiyotaka; Nakano, Hideo

2016-02-01

A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding OR consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Identification of arsenite-and arsenic diglutathione-binding proteins in human hepatocarcinoma cells

Energy Technology Data Exchange (ETDEWEB)

Mizumura, Ayano; Watanabe, Takayuki [Graduate School of Pharmaceutical Sciences, Chiba University, Yayoi, Inage, Chiba 263-8522 (Japan); Kobayashi, Yayoi [Graduate School of Pharmaceutical Sciences, Chiba University, Yayoi, Inage, Chiba 263-8522 (Japan); Environmental Health Sciences Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506 (Japan); Hirano, Seishiro [Graduate School of Pharmaceutical Sciences, Chiba University, Yayoi, Inage, Chiba 263-8522 (Japan); Research Center for Environmental Risk, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506 (Japan)

2010-01-15

It is generally accepted that trivalent arsenicals are more toxic than the corresponding pentavalent arsenicals, since trivalent arsenicals bind the thiol groups of biomolecules, leading to a deterioration in cellular functions. In the present study, we prepared three different arsenic-bound sepharoses and investigated the binding of hepatic cytosolic proteins to pentavalent, trivalent, and glutathione-conjugated trivalent arsenicals. SDS-PAGE showed no proteins bound to pentavalent arsenic specifically. In contrast, we found a number of proteins that have specific and high affinity for trivalent arsenic. Two of those proteins were identified: protein disulfide isomerase-related protein 5 (PDSIRP5) and peroxiredoxin 1/enhancer protein (PRX1/EP). These proteins have vicinal cysteines, as previously reported. In contrast, one of the prominent proteins that did not bind to trivalent arsenic was identified as calreticulin precursor. Although there are 3 cysteines in calreticulin precursor, two of the cysteines are spaced more than 25 amino acids apart. Five synthetic peptides containing 2 vicinal cysteines were prepared to study whether they would inhibit the binding of PDSIRP5, PRX1/EP, and other arsenic-binding proteins to trivalent arsenicals. Only two of the five peptides effectively inhibited binding, suggesting that other amino acids besides the 2 vicinal cysteines may modulate the affinity of cysteine-rich proteins for trivalent arsenicals. We further investigated hepatic cytosolic proteins that bound specifically to glutathione-conjugated trivalent arsenic, which is the most abundant form of arsenical in bile fluid. Four proteins that bound specifically to glutathione-conjugated trivalent arsenic were identified; interestingly, these proteins were different from the trivalent arsenic-binding proteins. These results suggest that although glutathione-conjugation is an important process in the metabolism, excretion, and detoxification of arsenicals, glutathione

Metabolism of organically bound tritium

International Nuclear Information System (INIS)

Travis, C.C.

1984-01-01

The classic methodology for estimating dose to man from environmental tritium ignores the fact that organically bound tritium in foodstuffs may be directly assimilated in the bound compartment of tissues without previous oxidation. We propose a four-compartment model consisting of a free body water compartment, two organic compartments, and a small, rapidly metabolizing compartment. The utility of this model lies in the ability to input organically bound tritium in foodstuffs directly into the organic compartments of the model. We found that organically bound tritium in foodstuffs can increase cumulative total body dose by a factor of 1.7 to 4.5 times the free body water dose alone, depending on the bound-to-loose ratio of tritium in the diet. Model predictions are compared with empirical measurements of tritium in human urine and tissue samples, and appear to be in close agreement. 10 references, 4 figures, 3 tables

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

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Peer Schrapers

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

Bound states in string nets

Science.gov (United States)

Schulz, Marc Daniel; Dusuel, Sébastien; Vidal, Julien

2016-11-01

We discuss the emergence of bound states in the low-energy spectrum of the string-net Hamiltonian in the presence of a string tension. In the ladder geometry, we show that a single bound state arises either for a finite tension or in the zero-tension limit depending on the theory considered. In the latter case, we perturbatively compute the binding energy as a function of the total quantum dimension. We also address this issue in the honeycomb lattice where the number of bound states in the topological phase depends on the total quantum dimension. Finally, the internal structure of these bound states is analyzed in the zero-tension limit.

Detection of free and covalently bound microcystins in animal tissues by liquid chromatography-tandem mass spectrometry

International Nuclear Information System (INIS)

Neffling, Milla-Riina; Lance, Emilie; Meriluoto, Jussi

2010-01-01

Microcystins are cyanobacterial hepatotoxins capable of accumulation into animal tissues. The toxins act by inhibiting specific protein phosphatases and both non-covalent and covalent interactions occur. The 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) method determines the total, i.e. the sum of free and protein-bound microcystin in tissues. The aim of the method development in this paper was to tackle the problems with the MMPB methodology: the rather laborious workflow and the loss of material during different steps of the method. In the optimised workflow the oxidation recovery was of acceptable level (29-40%), the extraction efficiency good (62-97%), but the signal suppression effect from the matrix remained severe in our system (16-37% signal left). The extraction efficiency for the determination of the free, extractable microcystins, was found to be good, 52-100%, depending on the sample and the toxin variant and concentration. - The study concerns method development for the LC-MS-MS analysis of both free and protein-bound microcystin in tissue materials.

Detection of free and covalently bound microcystins in animal tissues by liquid chromatography-tandem mass spectrometry

Energy Technology Data Exchange (ETDEWEB)

Neffling, Milla-Riina, E-mail: mneffling@gmail.co [Department of Biochemistry and Pharmacy, Abo Akademi University, Tykistoekatu 6 A, Biocity 3rd floor, FI-20520, Turku (Finland); Lance, Emilie [UMR CNRS Ecobio 6553, University of Rennes 1, Avenue du General Leclerc, 35042, Rennes Cedex (France); Meriluoto, Jussi [Department of Biochemistry and Pharmacy, Abo Akademi University, Tykistoekatu 6 A, Biocity 3rd floor, FI-20520, Turku (Finland)

2010-03-15

Microcystins are cyanobacterial hepatotoxins capable of accumulation into animal tissues. The toxins act by inhibiting specific protein phosphatases and both non-covalent and covalent interactions occur. The 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) method determines the total, i.e. the sum of free and protein-bound microcystin in tissues. The aim of the method development in this paper was to tackle the problems with the MMPB methodology: the rather laborious workflow and the loss of material during different steps of the method. In the optimised workflow the oxidation recovery was of acceptable level (29-40%), the extraction efficiency good (62-97%), but the signal suppression effect from the matrix remained severe in our system (16-37% signal left). The extraction efficiency for the determination of the free, extractable microcystins, was found to be good, 52-100%, depending on the sample and the toxin variant and concentration. - The study concerns method development for the LC-MS-MS analysis of both free and protein-bound microcystin in tissue materials.

Protein sequences bound to mineral surfaces persist into deep time

DEFF Research Database (Denmark)

Demarchi, Beatrice; Hall, Shaun; Roncal-Herrero, Teresa

2016-01-01

of Laetoli (3.8 Ma) and Olduvai Gorge (1.3 Ma) in Tanzania. By tracking protein diagenesis back in time we find consistent patterns of preservation, demonstrating authenticity of the surviving sequences. Molecular dynamics simulations of struthiocalcin-1 and -2, the dominant proteins within the eggshell......, reveal that distinct domains bind to the mineral surface. It is the domain with the strongest calculated binding energy to the calcite surface that is selectively preserved. Thermal age calculations demonstrate that the Laetoli and Olduvai peptides are 50 times older than any previously authenticated...

Bounding approaches to system identification

CERN Document Server

Norton, John; Piet-Lahanier, Hélène; Walter, Éric

1996-01-01

In response to the growing interest in bounding error approaches, the editors of this volume offer the first collection of papers to describe advances in techniques and applications of bounding of the parameters, or state variables, of uncertain dynamical systems. Contributors explore the application of the bounding approach as an alternative to the probabilistic analysis of such systems, relating its importance to robust control-system design.

Structural analysis of DNA–protein complexes regulating the restriction–modification system Esp1396I

International Nuclear Information System (INIS)

Martin, Richard N. A.; McGeehan, John E.; Ball, Neil J.; Streeter, Simon D.; Thresh, Sarah-Jane; Kneale, G. G.

2013-01-01

Comparison of bound and unbound DNA in protein–DNA co-crystal complexes reveals insights into controller-protein binding and DNA distortion in transcriptional regulation. The controller protein of the type II restriction–modification (RM) system Esp1396I binds to three distinct DNA operator sequences upstream of the methyltransferase and endonuclease genes in order to regulate their expression. Previous biophysical and crystallographic studies have shown molecular details of how the controller protein binds to the operator sites with very different affinities. Here, two protein–DNA co-crystal structures containing portions of unbound DNA from native operator sites are reported. The DNA in both complexes shows significant distortion in the region between the conserved symmetric sequences, similar to that of a DNA duplex when bound by the controller protein (C-protein), indicating that the naked DNA has an intrinsic tendency to bend when not bound to the C-protein. Moreover, the width of the major groove of the DNA adjacent to a bound C-protein dimer is observed to be significantly increased, supporting the idea that this DNA distortion contributes to the substantial cooperativity found when a second C-protein dimer binds to the operator to form the tetrameric repression complex

Binding of MCM-interacting proteins to ATP-binding site in MCM6

Directory of Open Access Journals (Sweden)

Hosoi A

2016-03-01

Full Text Available Atsutoshi Hosoi, Taku Sakairi, Yukio Ishimi Graduate School of Science and Engineering, Ibaraki University, Mito, Ibaraki, Japan Abstract: The function of MCM2–7 complex that is a DNA helicase in DNA replication may be regulated by various MCM-interacting proteins, including CDC45, RPA, TIM, TIPIN, Claspin, MCM10, and MCM-BP. It has been shown by immunoprecipitation that human MCM6 interacts with all these proteins in coexpressed insect cells. To determine the region in MCM6 to interact with these proteins, we prepared various truncated forms of MCM6 and examined the interaction of these MCM6 fragments with the MCM-interacting proteins. All these proteins bound to C-terminal half of MCM6, and CDC45, RPA2, TIM, TIPIN, MCM-BP, and MCM10 bound to the fragments containing ATP-binding motifs. CDC45 and RPA2 bound to the smallest fragment containing Walker motif A. Only MCM-BP is bound to the N-terminal half of MCM6. Site-directed mutagenesis study suggests that hydrophobic interaction is involved in the interaction of MCM6 with CDC45 and TIM. These results suggest a possibility that MCM-interacting proteins regulate MCM2–7 function by modulating the ATP-binding ability of the MCM2–7. Keywords: DNA helicase, DNA replication, checkpoint, MCM2–7 proteins

Bounded Intention Planning Revisited

OpenAIRE

Sievers Silvan; Wehrle Martin; Helmert Malte

2014-01-01

Bounded intention planning provides a pruning technique for optimal planning that has been proposed several years ago. In addition partial order reduction techniques based on stubborn sets have recently been investigated for this purpose. In this paper we revisit bounded intention planning in the view of stubborn sets.

Bounded Gaussian process regression

DEFF Research Database (Denmark)

Jensen, Bjørn Sand; Nielsen, Jens Brehm; Larsen, Jan

2013-01-01

We extend the Gaussian process (GP) framework for bounded regression by introducing two bounded likelihood functions that model the noise on the dependent variable explicitly. This is fundamentally different from the implicit noise assumption in the previously suggested warped GP framework. We...... with the proposed explicit noise-model extension....

A symmetric Roos bound for linear codes

NARCIS (Netherlands)

Duursma, I.M.; Pellikaan, G.R.

2006-01-01

The van Lint–Wilson AB-method yields a short proof of the Roos bound for the minimum distance of a cyclic code. We use the AB-method to obtain a different bound for the weights of a linear code. In contrast to the Roos bound, the role of the codes A and B in our bound is symmetric. We use the bound

Bounded Tamper Resilience

DEFF Research Database (Denmark)

Damgård, Ivan Bjerre; Faust, Sebastian; Mukherjee, Pratyay

2013-01-01

Related key attacks (RKAs) are powerful cryptanalytic attacks where an adversary can change the secret key and observe the effect of such changes at the output. The state of the art in RKA security protects against an a-priori unbounded number of certain algebraic induced key relations, e.......g., affine functions or polynomials of bounded degree. In this work, we show that it is possible to go beyond the algebraic barrier and achieve security against arbitrary key relations, by restricting the number of tampering queries the adversary is allowed to ask for. The latter restriction is necessary......-protocols (including the Okamoto scheme, for instance) are secure even if the adversary can arbitrarily tamper with the prover’s state a bounded number of times and obtain some bounded amount of leakage. Interestingly, for the Okamoto scheme we can allow also independent tampering with the public parameters. We show...

P2-16: Dual-Bound Model and the Role of Time Bound in Perceptual Decision Making

Directory of Open Access Journals (Sweden)

Daeseob Lim

2012-10-01

Full Text Available The diffusion model (DM encapsulates the dynamics of perceptual decision within a ‘diffusion field’ that is defined by a basis with sensory-evidence (SE and time vectors. At the core of the DM, it assumes that a decision is not made until an evidence particle drifts in the diffusion field and eventually hits one of the two pre-fixed bounds defined in the SE axis. This assumption dictates when and which choice is made by referring to when and which bound will be hit by the evidence particle. What if urgency pressures the decision system to make a choice even when the evidence particle has yet hit the SE bound? Previous modeling attempts at coping with time pressure, despite differences in detail, all manipulated the coordinate of SE bounds. Here, we offer a novel solution by adopting another bound on the time axis. This ‘dual-bound’ model (DBM posits that decisions can also be made when the evidence particle hits a time bound, which is determined on a trial-by-trial basis by a ‘perceived time interval’ – how long the system can stay in the ‘diffusion’ field. The classic single-bound model (SBM exhibited systematic errors in predicting both the reaction time distributions and the time-varying bias in choice. Those errors were not corrected by previously proposed variants of the SBM until the time bound was introduced. The validity of the DBM was further supported by the strong across-individual correlation between observed precision of interval timing and the predicted trial-by-trial variability of the time bound.

Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins

International Nuclear Information System (INIS)

Sung, Uhna; Jennings, Jennifer L.; Link, Andrew J.; Blakely, Randy D.

2005-01-01

The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH 2 -terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking

Computational design of protein interactions: designing proteins that neutralize influenza by inhibiting its hemagglutinin surface protein

Science.gov (United States)

Fleishman, Sarel

2012-02-01

Molecular recognition underlies all life processes. Design of interactions not seen in nature is a test of our understanding of molecular recognition and could unlock the vast potential of subtle control over molecular interaction networks, allowing the design of novel diagnostics and therapeutics for basic and applied research. We developed the first general method for designing protein interactions. The method starts by computing a region of high affinity interactions between dismembered amino acid residues and the target surface and then identifying proteins that can harbor these residues. Designs are tested experimentally for binding the target surface and successful ones are affinity matured using yeast cell surface display. Applied to the conserved stem region of influenza hemagglutinin we designed two unrelated proteins that, following affinity maturation, bound hemagglutinin at subnanomolar dissociation constants. Co-crystal structures of hemagglutinin bound to the two designed binders were within 1Angstrom RMSd of their models, validating the accuracy of the design strategy. One of the designed proteins inhibits the conformational changes that underlie hemagglutinin's cell-invasion functions and blocks virus infectivity in cell culture, suggesting that such proteins may in future serve as diagnostics and antivirals against a wide range of pathogenic influenza strains. We have used this method to obtain experimentally validated binders of several other target proteins, demonstrating the generality of the approach. We discuss the combination of modeling and high-throughput characterization of design variants which has been key to the success of this approach, as well as how we have used the data obtained in this project to enhance our understanding of molecular recognition. References: Science 332:816 JMB, in press Protein Sci 20:753

Ligand Depot: a data warehouse for ligands bound to macromolecules.

Science.gov (United States)

Feng, Zukang; Chen, Li; Maddula, Himabindu; Akcan, Ozgur; Oughtred, Rose; Berman, Helen M; Westbrook, John

2004-09-01

Ligand Depot is an integrated data resource for finding information about small molecules bound to proteins and nucleic acids. The initial release (version 1.0, November, 2003) focuses on providing chemical and structural information for small molecules found as part of the structures deposited in the Protein Data Bank. Ligand Depot accepts keyword-based queries and also provides a graphical interface for performing chemical substructure searches. A wide variety of web resources that contain information on small molecules may also be accessed through Ligand Depot. Ligand Depot is available at http://ligand-depot.rutgers.edu/. Version 1.0 supports multiple operating systems including Windows, Unix, Linux and the Macintosh operating system. The current drawing tool works in Internet Explorer, Netscape and Mozilla on Windows, Unix and Linux.

Spectroscopic and functional characterization of iron-bound forms of Azotobacter vinelandii (Nif)IscA.

Science.gov (United States)

Mapolelo, Daphne T; Zhang, Bo; Naik, Sunil G; Huynh, Boi Hanh; Johnson, Michael K

2012-10-16

The ability of Azotobacter vinelandii(Nif)IscA to bind Fe has been investigated to assess the role of Fe-bound forms in NIF-specific Fe-S cluster biogenesis. (Nif)IscA is shown to bind one Fe(III) or one Fe(II) per homodimer and the spectroscopic and redox properties of both the Fe(III)- and Fe(II)-bound forms have been characterized using the UV-visible absorption, circular dichroism, and variable-temperature magnetic circular dichroism, electron paramagnetic resonance, Mössbauer and resonance Raman spectroscopies. The results reveal a rhombic intermediate-spin (S = 3/2) Fe(III) center (E/D = 0.33, D = 3.5 ± 1.5 cm(-1)) that is most likely 5-coordinate with two or three cysteinate ligands and a rhombic high spin (S = 2) Fe(II) center (E/D = 0.28, D = 7.6 cm(-1)) with properties similar to reduced rubredoxins or rubredoxin variants with three cysteinate and one or two oxygenic ligands. Iron-bound (Nif)IscA undergoes reversible redox cycling between the Fe(III)/Fe(II) forms with a midpoint potential of +36 ± 15 mV at pH 7.8 (versus NHE). l-Cysteine is effective in mediating release of free Fe(II) from both the Fe(II)- and Fe(III)-bound forms of (Nif)IscA. Fe(III)-bound (Nif)IscA was also shown to be a competent iron source for in vitro NifS-mediated [2Fe-2S] cluster assembly on the N-terminal domain of NifU, but the reaction occurs via cysteine-mediated release of free Fe(II) rather than direct iron transfer. The proposed roles of A-type proteins in storing Fe under aerobic growth conditions and serving as iron donors for cluster assembly on U-type scaffold proteins or maturation of biological [4Fe-4S] centers are discussed in light of these results.

Major membrane surface proteins of Mycoplasma hyopneumoniae selectively modified by covalently bound lipid

International Nuclear Information System (INIS)

Wise, K.S.; Kim, M.F.

1987-01-01

Surface protein antigens of Mycoplasma hyopneumoniae were identified by direct antibody-surface binding or by radioimmunoprecipitation of surface 125 I-labeled proteins with a series of monoclonal antibodies (MAbs). Radioimmunoprecipitation of TX-114-phase proteins from cells labeled with [ 35 S] methionine, 14 C-amino acids, or [ 3 H] palmitic acid showed that proteins p65, p50, and p44 were abundant and (with one other hydrophobic protein, p60) were selectively labeled with lipid. Alkaline hydroxylamine treatment of labeled proteins indicated linkage of lipids by amide or stable O-linked ester bonds. Proteins p65, p50, and p44 were highly immunogenic in the natural host as measured by immunoblots of TX-114-phase proteins with antisera from swine inoculated with whole organisms. These proteins were antigenically and structurally unrelated, since hyperimmune mouse antibodies to individual gel-purified proteins were monospecific and gave distinct proteolytic epitope maps. Intraspecies size variants of one surface antigen of M. hyopneumoniae were revealed by a MAb to p70 (defined in strain J, ATCC 25934), which recognized a large p73 component on strain VPP11 (ATCC 25617). In addition, MAb to internal, aqueous-phase protein p82 of strain J failed to bind an analogous antigen in strain VPP11

Major membrane surface proteins of Mycoplasma hyopneumoniae selectively modified by covalently bound lipid

Energy Technology Data Exchange (ETDEWEB)

Wise K.S.; Kim, M.F.

1987-12-01

Surface protein antigens of Mycoplasma hyopneumoniae were identified by direct antibody-surface binding or by radioimmunoprecipitation of surface /sup 125/I-labeled proteins with a series of monoclonal antibodies (MAbs). Radioimmunoprecipitation of TX-114-phase proteins from cells labeled with (/sup 35/S) methionine, /sup 14/C-amino acids, or (/sup 3/H) palmitic acid showed that proteins p65, p50, and p44 were abundant and (with one other hydrophobic protein, p60) were selectively labeled with lipid. Alkaline hydroxylamine treatment of labeled proteins indicated linkage of lipids by amide or stable O-linked ester bonds. Proteins p65, p50, and p44 were highly immunogenic in the natural host as measured by immunoblots of TX-114-phase proteins with antisera from swine inoculated with whole organisms. These proteins were antigenically and structurally unrelated, since hyperimmune mouse antibodies to individual gel-purified proteins were monospecific and gave distinct proteolytic epitope maps. Intraspecies size variants of one surface antigen of M. hyopneumoniae were revealed by a MAb to p70 (defined in strain J, ATCC 25934), which recognized a large p73 component on strain VPP11 (ATCC 25617). In addition, MAb to internal, aqueous-phase protein p82 of strain J failed to bind an analogous antigen in strain VPP11.

Quantifying drug-protein binding in vivo

International Nuclear Information System (INIS)

Buchholz, B; Bench, G; Keating III, G; Palmblad, M; Vogel, J; Grant, P G; Hillegonds, D

2004-01-01

Accelerator mass spectrometry (AMS) provides precise quantitation of isotope labeled compounds that are bound to biological macromolecules such as DNA or proteins. The sensitivity is high enough to allow for sub-pharmacological (''micro-'') dosing to determine macromolecular targets without inducing toxicities or altering the system under study, whether it is healthy or diseased. We demonstrated an application of AMS in quantifying the physiologic effects of one dosed chemical compound upon the binding level of another compound in vivo at sub-toxic doses [4].We are using tissues left from this study to develop protocols for quantifying specific binding to isolated and identified proteins. We also developed a new technique to quantify nanogram to milligram amounts of isolated protein at precisions that are comparable to those for quantifying the bound compound by AMS

Establishment of a novel immortalized human prostatic epithelial cell line stably expressing androgen receptor and its application for the functional screening of androgen receptor modulators

International Nuclear Information System (INIS)

Yu, Shan; Wang, Ming-Wei; Yao, Xiaoqiang; Chan, F.L.

2009-01-01

In this study, we developed a human prostatic epithelial cell line BPH-1-AR stably expressing AR by lentiviral transduction. Characterization by immunoblot and RT-PCR showed that AR was stably expressed in all representative BPH-1-AR clones. Androgen treatment induced a secretory differentiation phenotype in BPH-1-AR cells but suppressed their cell proliferation. Treatments with AR agonists induced transactivation of a transfected PSA-gene promoter reporter in BPH-1-AR cells, whereas this transactivation was suppressed by an AR antagonist flutamide, indicating that the transduced AR in BPH-1-AR cells was functional. Finally, we utilized BPH-1-AR cells to evaluate the androgenic activities and growth effects of five newly developed non-steroidal compounds. Results showed that these compounds showed androgenic activities and growth-inhibitory effects on BPH-1-AR cells. Our results showed that BPH-1-AR cell line would be a valuable in vitro model for the study of androgen-regulated processes in prostatic epithelial cells and identification of compounds with AR-modulating activities.

Singlet oxygen-mediated damage to proteins and its consequences

DEFF Research Database (Denmark)

Davies, Michael Jonathan

2003-01-01

by the transfer of energy to ground state (triplet) molecular oxygen by either protein-bound, or other, chromophores. Singlet oxygen can also be generated by a range of other enzymatic and non-enzymatic reactions including processes mediated by heme proteins, lipoxygenases, and activated leukocytes, as well...... the absorption of UV radiation by the protein, or bound chromophore groups, thereby generating excited states (singlet or triplets) or radicals via photo-ionisation. The second major process involves indirect oxidation of the protein via the formation and subsequent reactions of singlet oxygen generated...... as radical termination reactions. This paper reviews the data available on singlet oxygen-mediated protein oxidation and concentrates primarily on the mechanisms by which this excited state species brings about changes to both the side-chains and backbone of amino acids, peptides, and proteins. Recent work...

Cartilage Acidic Protein 2 a hyperthermostable, high affinity calcium-binding protein.

Science.gov (United States)

Anjos, Liliana; Gomes, Ana S; Melo, Eduardo P; Canário, Adelino V; Power, Deborah M

2013-03-01

Cartilage Acidic Protein 2 (CRTAC2) is a novel protein present from prokaryotes to vertebrates with abundant expression in the teleost fish pituitary gland and an isoform of CRTAC1, a chondrocyte marker in humans. The two proteins are non-integrins containing N-terminal integrin-like Ca(2+)-binding motifs and their structure and function remain to be assigned. Structural studies of recombinant sea bream (sb)CRTAC2 revealed it is composed of 8.8% α-helix, 33.4% β-sheet and 57.8% unordered protein. sbCRTAC2 bound Ca(2+) with high affinity (K(d)=1.46nM) and favourable Gibbs free energy (∆G=-12.4kcal/mol). The stoichiometry for Ca(2+) bound to sbCRTAC2 at saturation indicated six Ca(2+) ligand-binding sites exist per protein molecule. No conformational change in sbCRTAC2 occurred in the presence of Ca(2+). Fluorescence emission revealed that the tertiary structure of the protein is hyperthermostable between 25°C and 95°C and the fully unfolded state is only induced by chemical denaturing (4M GndCl). sbCRTAC has a widespread tissue distribution and is present as high molecular weight aggregates, although strong reducing conditions promote formation of the monomer. sbCRTAC2 promotes epithelial cell outgrowth in vitro suggesting it may share functional homology with mammalian CRTAC1, recently implicated in cell-cell and cell-matrix interactions. Copyright © 2012 Elsevier B.V. All rights reserved.

Crystallization and preliminary X-ray analysis of membrane-bound pyrophosphatases.

Science.gov (United States)

Kellosalo, Juho; Kajander, Tommi; Honkanen, Riina; Goldman, Adrian

2013-02-01

Membrane-bound pyrophosphatases (M-PPases) are enzymes that enhance the survival of plants, protozoans and prokaryotes in energy constraining stress conditions. These proteins use pyrophosphate, a waste product of cellular metabolism, as an energy source for sodium or proton pumping. To study the structure and function of these enzymes we have crystallized two membrane-bound pyrophosphatases recombinantly produced in Saccharomyces cerevisae: the sodium pumping enzyme of Thermotoga maritima (TmPPase) and the proton pumping enzyme of Pyrobaculum aerophilum (PaPPase). Extensive crystal optimization has allowed us to grow crystals of TmPPase that diffract to a resolution of 2.6 Å. The decisive step in this optimization was in-column detergent exchange during the two-step purification procedure. Dodecyl maltoside was used for high temperature solubilization of TmPPase and then exchanged to a series of different detergents. After extensive screening, the new detergent, octyl glucose neopentyl glycol, was found to be the optimal for TmPPase but not PaPPase.

Actin-interacting Protein 1 Promotes Disassembly of Actin-depolymerizing Factor/Cofilin-bound Actin Filaments in a pH-dependent Manner.

Science.gov (United States)

Nomura, Kazumi; Hayakawa, Kimihide; Tatsumi, Hitoshi; Ono, Shoichiro

2016-03-04

Actin-interacting protein 1 (AIP1) is a conserved WD repeat protein that promotes disassembly of actin filaments when actin-depolymerizing factor (ADF)/cofilin is present. Although AIP1 is known to be essential for a number of cellular events involving dynamic rearrangement of the actin cytoskeleton, the regulatory mechanism of the function of AIP1 is unknown. In this study, we report that two AIP1 isoforms from the nematode Caenorhabditis elegans, known as UNC-78 and AIPL-1, are pH-sensitive in enhancement of actin filament disassembly. Both AIP1 isoforms only weakly enhance disassembly of ADF/cofilin-bound actin filaments at an acidic pH but show stronger disassembly activity at neutral and basic pH values. However, a severing-defective mutant of UNC-78 shows pH-insensitive binding to ADF/cofilin-decorated actin filaments, suggesting that the process of filament severing or disassembly, but not filament binding, is pH-dependent. His-60 of AIP1 is located near the predicted binding surface for the ADF/cofilin-actin complex, and an H60K mutation of AIP1 partially impairs its pH sensitivity, suggesting that His-60 is involved in the pH sensor for AIP1. These biochemical results suggest that pH-dependent changes in AIP1 activity might be a novel regulatory mechanism of actin filament dynamics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Susceptibility to viral infection is enhanced by stable expression of 3A or 3AB proteins from foot-and-mouth disease virus

International Nuclear Information System (INIS)

Rosas, Maria F.; Vieira, Yuri A.; Postigo, Raul; Martin-Acebes, Miguel A.; Armas-Portela, Rosario; Martinez-Salas, Encarnacion; Sobrino, Francisco

2008-01-01

The foot-and-mouth disease virus (FMDV) 3A protein is involved in virulence and host range. A distinguishing feature of FMDV 3B among picornaviruses is that three non-identical copies are encoded in the viral RNA and required for optimal replication in cell culture. Here, we have studied the involvement of the 3AB region on viral infection using constitutive and transient expression systems. BHK-21 stably transformed clones expressed low levels of FMDV 3A or 3A(B) proteins in the cell cytoplasm. Transformed cells stably expressing these proteins did not exhibit inner cellular rearrangements detectable by electron microscope analysis. Upon FMDV infection, clones expressing either 3A alone or 3A(B) proteins showed a significant increase in the percentage of infected cells, the number of plaque forming units and the virus yield. The 3A-enhancing effect was specific for FMDV as no increase in viral multiplication was observed in transformed clones infected with another picornavirus, encephalomyocarditis virus, or the negative-strand RNA virus vesicular stomatitis virus. A potential role of 3A protein in viral RNA translation was discarded by the lack of effect on FMDV IRES-dependent translation. Increased viral susceptibility was not caused by a released factor; neither the supernatant of transformed clones nor the addition of purified 3A protein to the infection medium was responsible for this effect. Unlike stable expression, high levels of 3A or 3A(B) protein transient expression led to unspecific inhibition of viral infection. Therefore, the effect observed on viral yield, which inversely correlated with the intracellular levels of 3A protein, suggests a transacting role operating on the FMDV multiplication cycle

Instanton bound states in ABJM theory

Energy Technology Data Exchange (ETDEWEB)

Hatsuda, Yasuyuki [DESY Hamburg (Germany). Theory Group; Tokyo Institute of Technology (Japan). Dept. of Physics; Moriyama, Sanefumi [Nagoya Univ. (Japan). Kobayashi Maskawa Inst. and Graduate School of Mathematics; Okuyama, Kazumi [Shinshu Univ., Matsumoto, Nagano (Japan). Dept. of Physics

2013-06-15

The partition function of the ABJM theory receives non-perturbative corrections due to instanton effects. We study these non-perturbative corrections, including bound states of worldsheet instantons and membrane instantons, in the Fermi-gas approach. We require that the total non-perturbative correction should be always finite for arbitrary Chern-Simons level. This finiteness is realized quite non-trivially because each bound state contribution naively diverges at some levels. The poles of each contribution should be canceled out in total. We use this pole cancellation mechanism to find unknown bound state corrections from known ones. We conjecture a general expression of the bound state contribution. Summing up all the bound state contributions, we find that the effect of bound states is simply incorporated into the worldsheet instanton correction by a redefinition of the chemical potential in the Fermi-gas system. Analytic expressions of the 3- and 4-membrane instanton corrections are also proposed.

Radiolysis of DNA-protein complexes

Energy Technology Data Exchange (ETDEWEB)

Begusova, Marie [Department of Radiation Dosimetry, Nuclear Physics Institute, Na Truhlarce 39/64, CZ-18086, Prague 8 (Czech Republic)]. E-mail: begusova@ujf.cas.cz; Gillard, Nathalie [Centre de Biophysique Moleculaire, CNRS, rue Charles-Sadron, F-45071 Orleans Cedex 2 (France); Sy, Denise [Centre de Biophysique Moleculaire, CNRS, rue Charles-Sadron, F-45071 Orleans Cedex 2 (France); Castaing, Bertrand [Centre de Biophysique Moleculaire, CNRS, rue Charles-Sadron, F-45071 Orleans Cedex 2 (France); Charlier, Michel [Centre de Biophysique Moleculaire, CNRS, rue Charles-Sadron, F-45071 Orleans Cedex 2 (France); Spotheim-Maurizot, Melanie [Centre de Biophysique Moleculaire, CNRS, rue Charles-Sadron, F-45071 Orleans Cedex 2 (France)

2005-02-01

We discuss here modifications of DNA and protein radiolysis due to the interaction of these two partners in specific complexes. Experimental patterns of frank strand breaks (FSB) and alkali revealed breaks (ARB) obtained for DNA lac operator bound to the lac repressor and for a DNA containing an abasic site analog bound to the formamidopyrimidine-DNA glycosylase are reported. Experimental data are compared to predicted damage distribution obtained using the theoretical model RADACK.

Radiolysis of DNA-protein complexes

International Nuclear Information System (INIS)

Begusova, Marie; Gillard, Nathalie; Sy, Denise; Castaing, Bertrand; Charlier, Michel; Spotheim-Maurizot, Melanie

2005-01-01

We discuss here modifications of DNA and protein radiolysis due to the interaction of these two partners in specific complexes. Experimental patterns of frank strand breaks (FSB) and alkali revealed breaks (ARB) obtained for DNA lac operator bound to the lac repressor and for a DNA containing an abasic site analog bound to the formamidopyrimidine-DNA glycosylase are reported. Experimental data are compared to predicted damage distribution obtained using the theoretical model RADACK

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection

Directory of Open Access Journals (Sweden)

Chinestra Patrick

2008-03-01

Full Text Available Abstract Background The Rho GTPases A, B and C proteins, members of the Rho family whose activity is regulated by GDP/GTP cycling, function in many cellular pathways controlling proliferation and have recently been implicated in tumorigenesis. Although overexpression of Rho GTPases has been correlated with tumorigenesis, only their GTP-bound forms are able to activate the signalling pathways implicated in tumorigenesis. Thus, the focus of much recent research has been to identify biological tools capable of quantifying the level of cellular GTP-bound Rho, or determining the subcellular location of activation. However useful, these tools used to study the mechanism of Rho activation still have limitations. The aim of the present work was to employ phage display to identify a conformationally-specific single chain fragment variable (scFv that recognizes the active, GTP-bound, form of Rho GTPases and is able to discriminate it from the inactive, GDP-bound, Rho in endogenous settings. Results After five rounds of phage selection using a constitutively activated mutant of RhoB (RhoBQ63L, three scFvs (A8, C1 and D11 were selected for subsequent analysis. Further biochemical characterization was pursued for the single clone, C1, exhibiting an scFv structure. C1 was selective for the GTP-bound form of RhoA, RhoB, as well as RhoC, and failed to recognize GTP-loaded Rac1 or Cdc42, two other members of the Rho family. To enhance its production, soluble C1 was expressed in fusion with the N-terminal domain of phage protein pIII (scFv C1-N1N2, it appeared specifically associated with GTP-loaded recombinant RhoA and RhoB via immunoprecipitation, and endogenous activated Rho in HeLa cells as determined by immunofluorescence. Conclusion We identified an antibody, C1-N1N2, specific for the GTP-bound form of RhoB from a phage library, and confirmed its specificity towards GTP-bound RhoA and RhoC, as well as RhoB. The success of C1-N1N2 in discriminating activated

Career Development and Personal Functioning Differences between Work-Bound and Non-Work Bound Students

Science.gov (United States)

Creed, Peter A.; Patton, Wendy; Hood, Michelle

2010-01-01

We surveyed 506 Australian high school students on career development (exploration, planning, job-knowledge, decision-making, indecision), personal functioning (well-being, self-esteem, life satisfaction, school satisfaction) and control variables (parent education, school achievement), and tested differences among work-bound, college-bound and…

Pharmacological and functional characterisation of the wild-type and site-directed mutants of the human H1 histamine receptor stably expressed in CHO cells.

Science.gov (United States)

Moguilevsky, N; Varsalona, F; Guillaume, J P; Noyer, M; Gillard, M; Daliers, J; Henichart, J P; Bollen, A

1995-01-01

A cDNA clone for the human histamine H1 receptor was isolated from a lung cDNA library and stably expressed in CHO cells. The recombinant receptor protein present in the cell membranes, displayed the functional and binding characteristics of histamine H1 receptors. Mutation of Ser155 to Ala in the fourth transmembrane domain did not significantly change the affinity of the receptor for histamine and H1 antagonists. However, mutation of the fifth transmembrane Asn198 to Ala resulted in a dramatic decrease of the affinity for histamine binding, and for the histamine-induced polyphosphoinositides breakdown, whereas the affinity towards antagonists was not significantly modified. In addition, mutation of another fifth transmembrane amino acid, Thr194 to Ala also diminished, but to a lesser extent, the affinity for histamine. These data led us to propose a molecular model for histamine interaction with the human H1 receptor. In this model, the amide moiety of Asn198 and the hydroxyl group of Thr194 are involved in hydrogen bonding with the nitrogen atoms of the imidazole ring of histamine. Moreover, mutation of Thr194 to Ala demonstrated that this residue is responsible for the discrimination between enantiomers of cetirizine.

SwarmDock and the Use of Normal Modes in Protein-Protein Docking

Directory of Open Access Journals (Sweden)

Paul A. Bates

2010-09-01

Full Text Available Here is presented an investigation of the use of normal modes in protein-protein docking, both in theory and in practice. Upper limits of the ability of normal modes to capture the unbound to bound conformational change are calculated on a large test set, with particular focus on the binding interface, the subset of residues from which the binding energy is calculated. Further, the SwarmDock algorithm is presented, to demonstrate that the modelling of conformational change as a linear combination of normal modes is an effective method of modelling flexibility in protein-protein docking.

Structural and dynamical insights into the membrane-bound α-synuclein.

Directory of Open Access Journals (Sweden)

Neha Jain

Full Text Available Membrane-induced disorder-to-helix transition of α-synuclein, a presynaptic protein, has been implicated in a number of important neuronal functions as well as in the etiology of Parkinson's disease. In order to obtain structural insights of membrane-bound α-synuclein at the residue-specific resolution, we took advantage of the fact that the protein is devoid of tryptophan and incorporated single tryptophan at various residue positions along the sequence. These tryptophans were used as site-specific markers to characterize the structural and dynamical aspects of α-synuclein on the negatively charged small unilamellar lipid vesicles. An array of site-specific fluorescence readouts, such as the spectral-shift, quenching efficiency and anisotropy, allowed us to discern various features of the conformational rearrangements occurring at different locations of α-synuclein on the lipid membrane. In order to define the spatial localization of various regions of the protein near the membrane surface, we utilized a unique and sensitive indicator, namely, red-edge excitation shift (REES, which originates when a fluorophore is located in a highly ordered micro-environment. The extent of REES observed at different residue positions allowed us to directly identify the residues that are localized at the membrane-water interface comprising a thin (∼ 15 Å layer of motionally restrained water molecules and enabled us to construct a dynamic hydration map of the protein. The combination of site-specific fluorescence readouts allowed us to unravel the intriguing molecular details of α-synuclein on the lipid membrane in a direct model-free fashion. Additionally, the combination of methodologies described here are capable of distinguishing subtle but important structural alterations of α-synuclein bound to different negatively charged lipids with varied head-group chemistry. We believe that the structural modulations of α-synuclein on the membrane could

Mitochondrial Protein Synthesis, Import, and Assembly

Science.gov (United States)

Fox, Thomas D.

2012-01-01

The mitochondrion is arguably the most complex organelle in the budding yeast cell cytoplasm. It is essential for viability as well as respiratory growth. Its innermost aqueous compartment, the matrix, is bounded by the highly structured inner membrane, which in turn is bounded by the intermembrane space and the outer membrane. Approximately 1000 proteins are present in these organelles, of which eight major constituents are coded and synthesized in the matrix. The import of mitochondrial proteins synthesized in the cytoplasm, and their direction to the correct soluble compartments, correct membranes, and correct membrane surfaces/topologies, involves multiple pathways and macromolecular machines. The targeting of some, but not all, cytoplasmically synthesized mitochondrial proteins begins with translation of messenger RNAs localized to the organelle. Most proteins then pass through the translocase of the outer membrane to the intermembrane space, where divergent pathways sort them to the outer membrane, inner membrane, and matrix or trap them in the intermembrane space. Roughly 25% of mitochondrial proteins participate in maintenance or expression of the organellar genome at the inner surface of the inner membrane, providing 7 membrane proteins whose synthesis nucleates the assembly of three respiratory complexes. PMID:23212899

Determining Normal-Distribution Tolerance Bounds Graphically

Science.gov (United States)

Mezzacappa, M. A.

1983-01-01

Graphical method requires calculations and table lookup. Distribution established from only three points: mean upper and lower confidence bounds and lower confidence bound of standard deviation. Method requires only few calculations with simple equations. Graphical procedure establishes best-fit line for measured data and bounds for selected confidence level and any distribution percentile.

Binding energies of two deltas bound states

International Nuclear Information System (INIS)

Sato, Hiroshi; Saito, Koichi.

1982-06-01

Bound states of the two-deltas system are investigated by employing the realistic one boson exchange potential. It is found that there exist many bound states in each isospin channel and also found that the tensor interaction plays important role in producing these bound states. Relationship between these bound states and dibaryon resonances is discussed. (J.P.N.)

p56Lck and p59Fyn Regulate CD28 Binding to Phosphatidylinositol 3-Kinase, Growth Factor Receptor-Bound Protein GRB-2, and T Cell-Specific Protein-Tyrosine Kinase ITK: Implications for T-Cell Costimulation

Science.gov (United States)

Raab, Monika; Cai, Yun-Cai; Bunnell, Stephen C.; Heyeck, Stephanie D.; Berg, Leslie J.; Rudd, Christopher E.

1995-09-01

T-cell activation requires cooperative signals generated by the T-cell antigen receptor ξ-chain complex (TCRξ-CD3) and the costimulatory antigen CD28. CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS). PI 3-kinase and GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains. The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28. Candidate kinases include p56Lck, p59Fyn, ξ-chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56Lck and p59Fyn phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28. Phosphatase digestion of CD28 eliminated binding. In contrast to Src kinases, ZAP-70 and ITK failed to induce these events. Further, ITK binding to CD28 was dependent on the presence of p56Lck and is thus likely to act downstream of p56Lck/p59Fyn in a signaling cascade. p56Lck is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.

[Protein-energy malnutrition in clinical practice].

Science.gov (United States)

Necheva, G I; Druk, I V; Lialiukova, E A

2013-01-01

The analysis of the reasons and mechanisms of development of an protein-energy malnutrition, communication of fetal pathology and development of an protein-energy malnutrition at mature age is submitted. Systemic character of a syndrome is marked out. Importance of a problem of an protein-energy malnutrition at patients with a dysplasia of a connecting tissue is bound to high prevalence of a syndrome at this pathology.

Variational lower bound on the scattering length

International Nuclear Information System (INIS)

Rosenberg, L.; Spruch, L.

1975-01-01

The scattering length A characterizes the zero-energy scattering of one system by another. It was shown some time ago that a variational upper bound on A could be obtained using methods, of the Rayleigh-Ritz type, which are commonly employed to obtain upper bounds on energy eigenvalues. Here we formulate a method for obtaining a variational lower bound on A. Once again the essential idea is to express the scattering length as a variational estimate plus an error term and then to reduce the problem of bounding the error term to one involving bounds on energy eigenvalues. In particular, the variational lower bound on A is rigorously established provided a certin modified Hamiltonian can be shown to have no discrete states lying below the level of the continuum threshold. It is unfortunately true that necessary conditions for the existence of bound states are not available for multiparticle systems in general. However, in the case of positron-atom scattering the adiabatic approximation can be introduced as an (essentially) solvable comparison problem to rigorously establish the nonexistence of bound states of the modified Hamiltonian. It has recently been shown how the validity of the variational upper bound on A can be maintained when the target ground-state wave function is imprecisely known. Similar methods can be used to maintain the variational lower bound on A. Since the bound is variational, the error in the calculated scattering length will be of second order in the error in the wave function. The use of the adiabatic approximation in the present context places no limitation in principle on the accuracy achievable

Milk proteins interact with goat Binder of SPerm (BSP) proteins and decrease their binding to sperm.

Science.gov (United States)

de Menezes, Erika Bezerra; van Tilburg, Mauricio; Plante, Geneviève; de Oliveira, Rodrigo V; Moura, Arlindo A; Manjunath, Puttaswamy

2016-11-01

Seminal plasma Binder of SPerm (BSP) proteins bind to sperm at ejaculation and promote capacitation. When in excess, however, BSP proteins damage the sperm membrane. It has been suggested that milk components of semen extenders associate with BSP proteins, potentially protecting sperm. Thus, this study was conducted to investigate if milk proteins interact with BSP proteins and reduce BSP binding to goat sperm. Using gel filtration chromatography, milk was incubated with goat seminal plasma proteins and loaded onto columns with and without calcium. Milk was also fractionated into parts containing mostly whey proteins or mostly caseins, incubated with seminal plasma proteins and subjected to gel filtration. Eluted fractions were evaluated by immunoblot using anti-goat BSP antibodies, confirming milk protein-BSP protein interactions. As determined by ELISA, milk proteins coated on polystyrene wells bound to increasing of goat BSP proteins. Far-western dot blots confirmed that BSP proteins bound to caseins and β-lactoglobulin in a concentration-dependent manner. Then, cauda epididymal sperm from five goats was incubated with seminal plasma; seminal plasma followed by milk; and milk followed by seminal plasma. Sperm membrane proteins were extracted and evaluated by immunoblotting. The pattern of BSP binding to sperm membrane proteins was reduced by 59.3 % when epididymal sperm were incubated with seminal plasma and then with skimmed milk (p  0.05). In conclusion, goat BSP proteins have an affinity for caseins and whey proteins. Milk reduces BSP binding to goat sperm, depending whether or not sperm had been previously exposed to seminal plasma. Such events may explain the protective effect of milk during goat sperm preservation.

A Review on Structures and Functions of Bcl-2 Family Proteins from Homo sapiens.

Science.gov (United States)

Sivakumar, Dakshinamurthy; Sivaraman, Thirunavukkarasu

2016-01-01

Cancer cells evade apoptosis, which is regulated by proteins of Bcl-2 family in the intrinsic pathways. Numerous experimental three-dimensional (3D) structures of the apoptotic proteins and the proteins bound with small chemical molecules/peptides/proteins have been reported in the literature. In this review article, the 3D structures of the Bcl-2 family proteins from Homo sapiens and as well complex structures of the anti-apoptotic proteins bound with small molecular inhibitors reported in the literature to date have been comprehensively listed out and described in detail. Moreover, the molecular mechanisms by which the Bcl-2 family proteins modulate the apoptotic processes and strategies for designing antagonists to anti-apoptotic proteins have been concisely discussed.

Capacity Bounds for Parallel Optical Wireless Channels

KAUST Repository

Chaaban, Anas; Rezki, Zouheir; Alouini, Mohamed-Slim

2016-01-01

A system consisting of parallel optical wireless channels with a total average intensity constraint is studied. Capacity upper and lower bounds for this system are derived. Under perfect channel-state information at the transmitter (CSIT), the bounds have to be optimized with respect to the power allocation over the parallel channels. The optimization of the lower bound is non-convex, however, the KKT conditions can be used to find a list of possible solutions one of which is optimal. The optimal solution can then be found by an exhaustive search algorithm, which is computationally expensive. To overcome this, we propose low-complexity power allocation algorithms which are nearly optimal. The optimized capacity lower bound nearly coincides with the capacity at high SNR. Without CSIT, our capacity bounds lead to upper and lower bounds on the outage probability. The outage probability bounds meet at high SNR. The system with average and peak intensity constraints is also discussed.

A library of 7TM receptor C-terminal tails - Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP)

DEFF Research Database (Denmark)

Heydorn, A.; Sondergaard, B.P.; Ersbøll, Bjarne Kjær

2004-01-01

Adaptor and scaffolding proteins determine the cellular targeting, the spatial, and thereby the functional association of G protein-coupled seven-transmembrane receptors with co-receptors, transducers, and downstream effectors and the adaptors determine post-signaling events such as receptor...... only a single receptor tail, i.e. the beta(2)-adrenergic receptor, whereas N-ethylmaleimide-sensitive factor bound 11 of the tail-fusion proteins. Of the two proteins proposed to target receptors for lysosomal degradation, sorting nexin 1 (SNX1) bound 10 and the C-terminal domain of G protein...... the expected nanomolar affinities for interaction with SNX1. Truncations of the NK1 receptor revealed that an extended binding epitope is responsible for the interaction with both SNX1 and G protein-coupled receptor-associated sorting protein as well as with N-ethylmaleimide-sensitive factor. It is concluded...

A library of 7TM receptor C-terminal tails. Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP)

DEFF Research Database (Denmark)

Heydorn, Arne; Søndergaard, Birgitte P; Ersbøll, Bjarne

2004-01-01

Adaptor and scaffolding proteins determine the cellular targeting, the spatial, and thereby the functional association of G protein-coupled seven-transmembrane receptors with co-receptors, transducers, and downstream effectors and the adaptors determine post-signaling events such as receptor...... only a single receptor tail, i.e. the beta(2)-adrenergic receptor, whereas N-ethylmaleimide-sensitive factor bound 11 of the tail-fusion proteins. Of the two proteins proposed to target receptors for lysosomal degradation, sorting nexin 1 (SNX1) bound 10 and the C-terminal domain of G protein...... the expected nanomolar affinities for interaction with SNX1. Truncations of the NK(1) receptor revealed that an extended binding epitope is responsible for the interaction with both SNX1 and G protein-coupled receptor-associated sorting protein as well as with N-ethylmaleimide-sensitive factor. It is concluded...

Label-free quantitative proteome analysis of the surface-bound salivary pellicle.

Science.gov (United States)

Delius, Judith; Trautmann, Simone; Médard, Guillaume; Kuster, Bernhard; Hannig, Matthias; Hofmann, Thomas

2017-04-01

The salivary pellicle, covering natural as well as restored tooth surfaces in the oral cavity as an immobilized protein-rich layer, acts as an important physico-chemical and biological mediator at the tooth-saliva-interface. For the first time, the pellicle's proteome of individual volunteers were analyzed separately on three consecutive days and the relative protein abundance determined by a label-free quantitative nano-LC-MS/MS approach. A total of 72 major proteins were identified in the initial pellicles formed intraorally on dental ceramic specimens already after 3min with high inter-individual and inter-day consistency. In comparison, significant differences in protein abundance were evident between subjects, thus indicating unique individual pellicle profiles. Furthermore, the relative protein abundance in pellicles was compared to the proteome pattern in the corresponding saliva samples of the same individuals to provide first data on significantly enriched and depleted salivary proteins (p <0.05) within the surface-bound salivary pellicle. Our findings reveal the initial adsorption of salivary proteins at the solid-liquid interface to be a rapid, highly selective, and reproducible process leading to the immobilization of a broad range of protective proteins and enzymes on the substratum surface within a few minutes. This provides evidence that the pellicle layer might be physiologically functional even without further maturation. Copyright © 2017 Elsevier B.V. All rights reserved.

Product differentiation under bounded rationality

NARCIS (Netherlands)

Vermeulen, B.; Poutré, La J.A.; Kok, de A.G.; Pyka, A.; Handa, H.; Ishibuchi, H.; Ong, Y.-S.; Tan, K.-C.

2015-01-01

We study product differentiation equilibria and dynamics on the Salop circle under bounded rationality. Due to bounded rationality, firms tend to agglomerate in pairs. Upon adding a second tier of component suppliers, downstream assemblers may escape pairwise horizontal agglomeration. Moreover, we

Characterisation of bioactive protein-bound polysaccharides from Amanita ponderosa cultures

OpenAIRE

Salvador, C; Martins, M R; Arteiro, J M; Caldeira, A T

2015-01-01

The aim of this study was to evaluate the production of protein-polysaccharide complexes obtained from A. ponderosa cultures using a new microanalytical approach to monitoring quickly and easily the production process.

Protein docking prediction using predicted protein-protein interface

Directory of Open Access Journals (Sweden)

Li Bin

2012-01-01

Full Text Available Abstract Background Many important cellular processes are carried out by protein complexes. To provide physical pictures of interacting proteins, many computational protein-protein prediction methods have been developed in the past. However, it is still difficult to identify the correct docking complex structure within top ranks among alternative conformations. Results We present a novel protein docking algorithm that utilizes imperfect protein-protein binding interface prediction for guiding protein docking. Since the accuracy of protein binding site prediction varies depending on cases, the challenge is to develop a method which does not deteriorate but improves docking results by using a binding site prediction which may not be 100% accurate. The algorithm, named PI-LZerD (using Predicted Interface with Local 3D Zernike descriptor-based Docking algorithm, is based on a pair wise protein docking prediction algorithm, LZerD, which we have developed earlier. PI-LZerD starts from performing docking prediction using the provided protein-protein binding interface prediction as constraints, which is followed by the second round of docking with updated docking interface information to further improve docking conformation. Benchmark results on bound and unbound cases show that PI-LZerD consistently improves the docking prediction accuracy as compared with docking without using binding site prediction or using the binding site prediction as post-filtering. Conclusion We have developed PI-LZerD, a pairwise docking algorithm, which uses imperfect protein-protein binding interface prediction to improve docking accuracy. PI-LZerD consistently showed better prediction accuracy over alternative methods in the series of benchmark experiments including docking using actual docking interface site predictions as well as unbound docking cases.

Protein docking prediction using predicted protein-protein interface.

Science.gov (United States)

Li, Bin; Kihara, Daisuke

2012-01-10

Many important cellular processes are carried out by protein complexes. To provide physical pictures of interacting proteins, many computational protein-protein prediction methods have been developed in the past. However, it is still difficult to identify the correct docking complex structure within top ranks among alternative conformations. We present a novel protein docking algorithm that utilizes imperfect protein-protein binding interface prediction for guiding protein docking. Since the accuracy of protein binding site prediction varies depending on cases, the challenge is to develop a method which does not deteriorate but improves docking results by using a binding site prediction which may not be 100% accurate. The algorithm, named PI-LZerD (using Predicted Interface with Local 3D Zernike descriptor-based Docking algorithm), is based on a pair wise protein docking prediction algorithm, LZerD, which we have developed earlier. PI-LZerD starts from performing docking prediction using the provided protein-protein binding interface prediction as constraints, which is followed by the second round of docking with updated docking interface information to further improve docking conformation. Benchmark results on bound and unbound cases show that PI-LZerD consistently improves the docking prediction accuracy as compared with docking without using binding site prediction or using the binding site prediction as post-filtering. We have developed PI-LZerD, a pairwise docking algorithm, which uses imperfect protein-protein binding interface prediction to improve docking accuracy. PI-LZerD consistently showed better prediction accuracy over alternative methods in the series of benchmark experiments including docking using actual docking interface site predictions as well as unbound docking cases.

Massive Galileon positivity bounds

Science.gov (United States)

de Rham, Claudia; Melville, Scott; Tolley, Andrew J.; Zhou, Shuang-Yong

2017-09-01

The EFT coefficients in any gapped, scalar, Lorentz invariant field theory must satisfy positivity requirements if there is to exist a local, analytic Wilsonian UV completion. We apply these bounds to the tree level scattering amplitudes for a massive Galileon. The addition of a mass term, which does not spoil the non-renormalization theorem of the Galileon and preserves the Galileon symmetry at loop level, is necessary to satisfy the lowest order positivity bound. We further show that a careful choice of successively higher derivative corrections are necessary to satisfy the higher order positivity bounds. There is then no obstruction to a local UV completion from considerations of tree level 2-to-2 scattering alone. To demonstrate this we give an explicit example of such a UV completion.

A combinatorial perspective of the protein inference problem.

Science.gov (United States)

Yang, Chao; He, Zengyou; Yu, Weichuan

2013-01-01

In a shotgun proteomics experiment, proteins are the most biologically meaningful output. The success of proteomics studies depends on the ability to accurately and efficiently identify proteins. Many methods have been proposed to facilitate the identification of proteins from peptide identification results. However, the relationship between protein identification and peptide identification has not been thoroughly explained before. In this paper, we devote ourselves to a combinatorial perspective of the protein inference problem. We employ combinatorial mathematics to calculate the conditional protein probabilities (protein probability means the probability that a protein is correctly identified) under three assumptions, which lead to a lower bound, an upper bound, and an empirical estimation of protein probabilities, respectively. The combinatorial perspective enables us to obtain an analytical expression for protein inference. Our method achieves comparable results with ProteinProphet in a more efficient manner in experiments on two data sets of standard protein mixtures and two data sets of real samples. Based on our model, we study the impact of unique peptides and degenerate peptides (degenerate peptides are peptides shared by at least two proteins) on protein probabilities. Meanwhile, we also study the relationship between our model and ProteinProphet. We name our program ProteinInfer. Its Java source code, our supplementary document and experimental results are available at: >http://bioinformatics.ust.hk/proteininfer.

Crystal Structure of the Dimeric Oct6 (Pou3fl) POU Domain Bound to Palindromic MORE DNA

Energy Technology Data Exchange (ETDEWEB)

R Jauch; S Choo; C Ng; P Kolatkar

2011-12-31

POU domains (named after their identification in Pit1, Oct1 unc86) are found in around 15 transcription factors encoded in mammalian genomes many of which feature prominently as key regulators at development bifurcations. For example, the POU III class Octamer binding protein 6 (Oct6) is expressed in embryonic stem cells and during neural development and drives the differentia5tion of myelinated cells in the central and peripheral nervous system. Defects in oct6 expression levels are linked to neurological disorders such as schizophrenia. POU proteins contain a bi-partite DNA binding domain that assembles on various DNA motifs with differentially configured subdomains. Intriguingly, alternative configurations of POU domains on different DNA sites were shown to affect the subsequent recruitment of transcriptional coactivators. Namely, binding of Oct1 to a Palindromic Oct-factor Recognition Element (PORE) was shown to facilitate the recruitment of the OBF1 coactivator whereas More of PORE (MORE) bound Oct1 does not. Moreover, Pit1 was shown to recruit the corepressor N-CoR only when bound to a variant MORE motif with a 2 bp half-site spacing. Therefore, POU proteins are seen as a paradigm for DNA induced allosteric effects on transcription factors modulating their regulatory potential. However, a big unresolved conundrum for the POU class and for most if not all other transcription factor classes is how highly similar proteins regulate different sets of genes causing fundamentally different biological responses. Ultimately, there must be subtle features enabling those factors to engage in contrasting molecular interactions in the cell. Thus, the dissection of the molecular details of the transcription-DNA recognition in general, and the formation of multimeric regulatory complexes, in particular, is highly desirable. To contribute to these efforts they solved the 2.05 {angstrom} crystal structure of Oct6 bound as a symmetrical homodimer to palindromic MORE DNA.

The Development of Protein Microarrays and Their Applications in DNA-Protein and Protein-Protein Interaction Analyses of Arabidopsis Transcription Factors

Science.gov (United States)

Gong, Wei; He, Kun; Covington, Mike; Dinesh-Kumar, S. P.; Snyder, Michael; Harmer, Stacey L.; Zhu, Yu-Xian; Deng, Xing Wang

2009-01-01

We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to construct protein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and protein-protein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale. PMID:19802365

LOCALIZATION OF POLYSOME-BOUND ALBUMIN AND SERINE DEHYDRATASE IN RAT LIVER CELL FRACTIONS

Science.gov (United States)

Ikehara, Yukio; Pitot, Henry C.

1973-01-01

The polysomes involved in albumin and serine dehydratase synthesis were identified and localized by the binding to rat liver polysomes of anti-rat serum albumin and anti-serine dehydratase [125I]Fab dimer and monomer. Techniques were developed for the isolation of undegraded free and membrane-bound polysomes and for the preparation of [125I]Fab monomers and dimers from the IgG obtained from the antisera to the two proteins, rat serum albumin and serine dehydratase. The distribution of anti-rat serum albumin [125I]Fab dimer in the polysome profile is in accordance with the size of polysomes that are expected to be synthesizing albumin. By direct precipitation, it has been demonstrated that nascent chains isolated from the membrane-bound polysomes by puromycin were precipitated by anti-rat serum albumin-IgG at a level of 5–6 times those released from free polysomes. Anti-rat serum albumin-[125I]Fab dimer reacted with membrane-bound polysomes almost exclusively compared to the binding of nonimmune, control [125I]Fab dimer; a significant degree of binding of anti-rat serum albumin-[125I]Fab to free polysomes was also obtained. The [125I]Fab dimer made from normal control rabbit serum does not react with polysomes from liver at all and this preparation will not interact with polysomes extracted from tissues that do not synthesize rat serum albumin. Both anti-serine dehydratase-[125I]Fab monomer and dimer react with free and bound polysomes from livers of animals fed a chow diet or those fed a high 90% protein diet and given glucagon. In the latter instance, however, it is clear that the majority of the binding occurs to the bound polysomes. Furthermore, the specificity of this reaction may be further shown by the use of kidney polysomes that do not normally synthesize serine dehydratase. When these latter polysomes are isolated, even after the addition of crude and purified serine dehydratase, no reaction with anti-serine dehydratase-Fab fragments could be

New bounds on isotropic Lorentz violation

International Nuclear Information System (INIS)

Carone, Christopher D.; Sher, Marc; Vanderhaeghen, Marc

2006-01-01

Violations of Lorentz invariance that appear via operators of dimension four or less are completely parametrized in the Standard Model Extension (SME). In the pure photonic sector of the SME, there are 19 dimensionless, Lorentz-violating parameters. Eighteen of these have experimental upper bounds ranging between 10 -11 and 10 -32 ; the remaining parameter, k-tilde tr , is isotropic and has a much weaker bound of order 10 -4 . In this Brief Report, we point out that k-tilde tr gives a significant contribution to the anomalous magnetic moment of the electron and find a new upper bound of order 10 -8 . With reasonable assumptions, we further show that this bound may be improved to 10 -14 by considering the renormalization of other Lorentz-violating parameters that are more tightly constrained. Using similar renormalization arguments, we also estimate bounds on Lorentz-violating parameters in the pure gluonic sector of QCD

Effect of fullerenol surface chemistry on nanoparticle binding-induced protein misfolding

Science.gov (United States)

Radic, Slaven; Nedumpully-Govindan, Praveen; Chen, Ran; Salonen, Emppu; Brown, Jared M.; Ke, Pu Chun; Ding, Feng

2014-06-01

Fullerene and its derivatives with different surface chemistry have great potential in biomedical applications. Accordingly, it is important to delineate the impact of these carbon-based nanoparticles on protein structure, dynamics, and subsequently function. Here, we focused on the effect of hydroxylation -- a common strategy for solubilizing and functionalizing fullerene -- on protein-nanoparticle interactions using a model protein, ubiquitin. We applied a set of complementary computational modeling methods, including docking and molecular dynamics simulations with both explicit and implicit solvent, to illustrate the impact of hydroxylated fullerenes on the structure and dynamics of ubiquitin. We found that all derivatives bound to the model protein. Specifically, the more hydrophilic nanoparticles with a higher number of hydroxyl groups bound to the surface of the protein via hydrogen bonds, which stabilized the protein without inducing large conformational changes in the protein structure. In contrast, fullerene derivatives with a smaller number of hydroxyl groups buried their hydrophobic surface inside the protein, thereby causing protein denaturation. Overall, our results revealed a distinct role of surface chemistry on nanoparticle-protein binding and binding-induced protein misfolding.Fullerene and its derivatives with different surface chemistry have great potential in biomedical applications. Accordingly, it is important to delineate the impact of these carbon-based nanoparticles on protein structure, dynamics, and subsequently function. Here, we focused on the effect of hydroxylation -- a common strategy for solubilizing and functionalizing fullerene -- on protein-nanoparticle interactions using a model protein, ubiquitin. We applied a set of complementary computational modeling methods, including docking and molecular dynamics simulations with both explicit and implicit solvent, to illustrate the impact of hydroxylated fullerenes on the structure and

Tight Bounds for Distributed Functional Monitoring

DEFF Research Database (Denmark)

Woodruff, David P.; Zhang, Qin

2011-01-01

$, our bound resolves their main open question. Our lower bounds are based on new direct sum theorems for approximate majority, and yield significant improvements to problems in the data stream model, improving the bound for estimating $F_p, p > 2,$ in $t$ passes from $\\tilde{\\Omega}(n^{1-2/p}/(\\eps^{2/p......} t))$ to $\\tilde{\\Omega}(n^{1-2/p}/(\\eps^{4/p} t))$, giving the first bound for estimating $F_0$ in $t$ passes of $\\Omega(1/(\\eps^2 t))$ bits of space that does not use the gap-hamming problem, and showing a distribution for the gap-hamming problem with high external information cost or super...

MOLECULAR MODELING INDICATES THAT HOMOCYSTEINE INDUCES CONFORMATIONAL CHANGES IN THE STRUCTURE OF PUTATIVE TARGET PROTEINS

Directory of Open Access Journals (Sweden)

Yumnam Silla

2015-09-01

Full Text Available An elevated level of homocysteine, a reactive thiol containing amino acid is associated with a multitude of complex diseases. A majority (>80% of homocysteine in circulation is bound to protein cysteine residues. Although, till date only 21 proteins have been experimentally shown to bind with homocysteine, using an insilico approach we had earlier identified several potential target proteins that could bind with homocysteine. Shomocysteinylation of proteins could potentially alter the structure and/or function of the protein. Earlier studies have shown that binding of homocysteine to protein alters its function. However, the effect of homocysteine on the target protein structure has not yet been documented. In the present work, we assess conformational or structural changes if any due to protein homocysteinylation using two proteins, granzyme B (GRAB and junctional adhesion molecule 1 (JAM1, which could potentially bind to homocysteine. We, for the first time, constructed computational models of homocysteine bound to target proteins and monitored their structural changes using explicit solvent molecular dynamic (MD simulation. Analysis of homocysteine bound trajectories revealed higher flexibility of the active site residues and local structural perturbations compared to the unbound native structure’s simulation, which could affect the stability of the protein. In addition, secondary structure analysis of homocysteine bound trajectories also revealed disappearance of â-helix within the G-helix and linker region that connects between the domain regions (as defined in the crystal structure. Our study thus captures the conformational transitions induced by homocysteine and we suggest these structural alterations might have implications for hyperhomocysteinemia induced pathologies.

Combining Alphas via Bounded Regression

Directory of Open Access Journals (Sweden)

Zura Kakushadze

2015-11-01

Full Text Available We give an explicit algorithm and source code for combining alpha streams via bounded regression. In practical applications, typically, there is insufficient history to compute a sample covariance matrix (SCM for a large number of alphas. To compute alpha allocation weights, one then resorts to (weighted regression over SCM principal components. Regression often produces alpha weights with insufficient diversification and/or skewed distribution against, e.g., turnover. This can be rectified by imposing bounds on alpha weights within the regression procedure. Bounded regression can also be applied to stock and other asset portfolio construction. We discuss illustrative examples.

Quasi-bound states in continuum

International Nuclear Information System (INIS)

Nakamura, Hiroaki; Hatano, Naomichi; Garmon, Sterling; Petrosky, Tomio

2007-08-01

We report the prediction of quasi-bound states (resonant states with very long lifetimes) that occur in the eigenvalue continuum of propagating states for a wide region of parameter space. These quasi-bound states are generated in a quantum wire with two channels and an adatom, when the energy bands of the two channels overlap. A would-be bound state that lays just below the upper energy band is slightly destabilized by the lower energy band and thereby becomes a resonant state with a very long lifetime (a second QBIC lays above the lower energy band). (author)

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

Science.gov (United States)

Einfinger, Katrin; Badrnya, Sigrun; Furtmüller, Margareta; Handschuh, Daniela; Lindner, Herbert; Geiger, Margarethe

2015-01-01

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. PMID:26580551

Selenolate complexes of CYP101 and the heme-bound hHO-1/H25A proximal cavity mutant.

Science.gov (United States)

Jiang, Yongying; Ortiz de Montellano, Paul R

2008-05-05

Thiolate and selenolate complexes of CYP101 (P450cam) and the H25A proximal cavity mutant of heme-bound human heme oxygenase-1 (hHO-1) have been examined by UV-vis spectroscopy. Both thiolate and selenolate ligands bound to the heme distal side in CYP101 and gave rise to characteristic hyperporphyrin spectra. Thiolate ligands also bound to the proximal side of the heme in the cavity created by the H25A mutation in hHO-1, giving a Soret absorption similar to that of the H25C hHO-1 mutant. Selenolate ligands also bound to this cavity mutant under anaerobic conditions but reduced the heme iron to the ferrous state, as shown by the formation of a ferrous CO complex. Under aerobic conditions, the selenolate ligand but not the thiolate ligand was rapidly oxidized. These results indicate that selenocysteine-coordinated heme proteins will not be stable species in the absence of a redox potential stabilizing effect.

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

International Nuclear Information System (INIS)

Kornhauser, A.

1976-01-01

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.)

P-aminobenzoic acid and tritiated cyanoborohydride for the detection of pyruvoyl residues in proteins

International Nuclear Information System (INIS)

van Poelje, P.D.; Snell, E.E.

1987-01-01

A procedure for the detection of covalently bound pyruvic acid in purified proteins or in crude extracts is described. The dialyzed sample is first treated with sodium cyanoborohydride to reduce any Schiff bases present and then incubated with p-aminobenzoic acid and sodium [ 3 H]cyanoborohydride. Derivatized proteins are visualized by fluorography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel slices containing the labeled proteins are hydrolyzed, and, after removal of polyacrylic acid, the hydrolysate is subjected to ion-exchange high-performance liquid chromatography. The presence of pyruvic acid is established by the detection of a tritiated, 280-nm absorbing compound with a retention time corresponding to that of synthetic N-(p-carboxyphenyl)alanine. The procedure is capable of detecting protein-bound pyruvic acid in the picomolar range and is easily modified to screen for other covalently bound keto acids

Bounded Rationality and Budgeting

OpenAIRE

Ibrahim, Mukdad

2016-01-01

This article discusses the theory of bounded rationality which had been introduced by Herbert Simon in the 1950s. Simon introduced the notion of bounded rationality stating that while decision-makers strive for rationality, they are limited by the effect of the environment, their information process capacity and by the constraints on their information storage and retrieval capabilities. Moreover, this article tries to specifically blend this notion into budgeting, using the foundations of inc...

Asporin stably expressed in the surface layer of mandibular condylar cartilage and augmented in the deeper layer with age.

Science.gov (United States)

Miyamoto, Yutaka; Kanzaki, Hiroyuki; Wada, Satoshi; Tsuruoka, Sari; Itohiya, Kanako; Kumagai, Kenichi; Hamada, Yoshiki; Nakamura, Yoshiki

2017-12-01

Mandibular condylar cartilage (MCC) exhibits dual roles both articular cartilage and growth center. Of many growth factors, TGF-β has been implicated in the growth of articular cartilage including MCC. Recently, Asporin, decoy to TGF-β, was discovered and it blocks TGF-β signaling. Asporin is expressed in a variety of tissues including osteoarthritic articular cartilage, though there was no report of Asporin expression in MCC. In the present study, we investigated the temporal and spatial expression of Asporin in MCC. Gene expression profile of MCC and epiphyseal cartilage in tibia of 5 weeks old ICR mice were firstly compared with microarray analysis using the laser capture microdissected samples. Variance of gene expression was further confirmed by real-time RT-PCR and immunohistochemical staining at 1,3,10, and 20 weeks old. TGF-β and its signaling molecule, phosphorylated Smad-2/3 (p-Smad2/3), were also examined by immunohistochemical staining. Microarray analysis revealed that Asporin was highly expressed in MCC. Real-time RT-PCR analysis confirmed that the fibrous layer of MCC exhibited stable higher Asporin expression at any time points as compared to epiphyseal cartilage. This was also observed in immunohistochemical staining. Deeper layer in MCC augmented Asporin expression with age. Whereas, TGF-β was stably highly observed in the layer. The fibrous layer of MCC exhibited weak staining of p-Smad2/3, though the proliferating layer of MCC was strongly stained as compared to epiphyseal cartilage of tibia at early time point. Consistent with the increase of Asporin expression in the deeper layer of MCC, the intensity of p-Smad-2/3 staining was decreased with age. In conclusion, we discovered that Asporin was stably expressed at the fibrous layer of MCC, which makes it possible to manage both articular cartilage and growth center at the same time.

Fuzzy upper bounds and their applications

Energy Technology Data Exchange (ETDEWEB)

Soleimani-damaneh, M. [Department of Mathematics, Faculty of Mathematical Science and Computer Engineering, Teacher Training University, 599 Taleghani Avenue, Tehran 15618 (Iran, Islamic Republic of)], E-mail: soleimani_d@yahoo.com

2008-04-15

This paper considers the concept of fuzzy upper bounds and provides some relevant applications. Considering a fuzzy DEA model, the existence of a fuzzy upper bound for the objective function of the model is shown and an effective approach to solve that model is introduced. Some dual interpretations are provided, which are useful for practical purposes. Applications of the concept of fuzzy upper bounds in two physical problems are pointed out.

A Lagrangian lower bound for the container transshipment problem at a railway hub for a fast branch-and-bound algorithm

OpenAIRE

M Barketau; H Kopfer; E Pesch

2013-01-01

In this paper, we consider the container transshipment problem at a railway hub. A simple lower bound known for this problem will be improved by a new Lagrangian relaxation lower bound. Computational tests show that this lower bound outperforms the simple one and decreases substantially the run time of the branch-and-bound algorithm.

Glove powder's carrying capacity for latex protein: analysis using the ASTM ELISA test.

Science.gov (United States)

Beezhold, D; Horton, K; Hickey, V; Daddona, J; Kostyal, D

2003-01-01

Glove donning powders carry latex proteins and disperse them into the workplace environment. We have used the ASTM D6499 ELISA to quantify the amount of latex antigen bound to and carried by glove powders. We could differentiate between a small amount of protein actually bound to the powders and a larger amount carried by the powder. Enhanced binding of a major allergen, Hev b 5, to the starch powders was demonstrated by Western blot. The D6499 ELISA is able to measure total latex antigen, soluble and powder bound, simultaneously without the need to centrifuge the samples.

Photoaffinity labeling of serum vitamin D binding protein by 3-deoxy-3-azido-25-hydroxyvitamin D3

International Nuclear Information System (INIS)

Link, R.P.; Kutner, A.; Schnoes, H.K.; DeLuca, H.F.

1987-01-01

3-Deoxy-3-azido-25-hydroxyvitamin D3 was covalently incorporated in the 25-hydroxyvitamin D3 binding site of purified human plasma vitamin D binding protein. Competition experiments showed that 3-deoxy-3-azido-25-hydroxyvitamin D3 and 25-hydroxyvitamin D3 bind at the same site on the protein. Tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was synthesized from tritiated 25-hydroxyvitamin D3, retaining the high specific activity of the parent compound. The tritiated azido label bound reversibly to human vitamin D binding protein in the dark and covalently to human vitamin D binding protein after exposure to ultraviolet light. Reversible binding of tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was compared to tritiated 25-hydroxyvitamin D3 binding to human vitamin D binding protein. Scatchard analysis of the data indicated equivalent maximum density binding sites with a KD,app of 0.21 nM for 25-hydroxyvitamin D3 and a KD,app of 1.3 nM for the azido derivative. Covalent binding was observed only after exposure to ultraviolet irradiation, with an average of 3% of the reversibly bound label becoming covalently bound to vitamin D binding protein. The covalent binding was reduced 70-80% when 25-hydroxyvitamin D3 was present, indicating strong covalent binding at the vitamin D binding site of the protein. When tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was incubated with human plasma in the absence and presence of 25-hydroxyvitamin D3, 12% of the azido derivative was reversibly bound to vitamin D binding protein. After ultraviolet irradiation, four plasma proteins covalently bound the azido label, but vitamin D binding protein was the only protein of the four that was unlabeled in the presence of 25-hydroxyvitamin D3

Prediction of protein–protein interactions: unifying evolution and structure at protein interfaces

International Nuclear Information System (INIS)

Tuncbag, Nurcan; Gursoy, Attila; Keskin, Ozlem

2011-01-01

The vast majority of the chores in the living cell involve protein–protein interactions. Providing details of protein interactions at the residue level and incorporating them into protein interaction networks are crucial toward the elucidation of a dynamic picture of cells. Despite the rapid increase in the number of structurally known protein complexes, we are still far away from a complete network. Given experimental limitations, computational modeling of protein interactions is a prerequisite to proceed on the way to complete structural networks. In this work, we focus on the question 'how do proteins interact?' rather than 'which proteins interact?' and we review structure-based protein–protein interaction prediction approaches. As a sample approach for modeling protein interactions, PRISM is detailed which combines structural similarity and evolutionary conservation in protein interfaces to infer structures of complexes in the protein interaction network. This will ultimately help us to understand the role of protein interfaces in predicting bound conformations

Regulation of SUMO2 Target Proteins by the Proteasome in Human Cells Exposed to Replication Stress

DEFF Research Database (Denmark)

Bursomanno, Sara; McGouran, Joanna F; Kessler, Benedikt M

2015-01-01

In human cells, SUMO2 is predominantly conjugated to target proteins in response to cellular stress. Previous studies suggested that proteins conjugated to SUMO2, but not to SUMO1, could be regulated by the ubiquitin-mediated proteasome system. Hence, we set out to understand the role...... of the proteasome in determining the fate of proteins conjugated to SUMO2 when cells are treated with DNA replication stress conditions. We conducted a quantitative proteomic analysis in a U2OS cell line stably expressing SUMO2(Q87R) tagged with StrepHA in the presence or absence of epoxomicin (EPOX), a proteasome...... inhibitor. We identified subgroups of putative SUMO2 targets that were either degraded or stabilized by EPOX upon SUMO2 conjugation in response to replication stress. Interestingly, the subgroup of proteins degraded upon SUMO2 conjugation was enriched in proteins playing roles in DNA damage repair...

Isolation, lactoperoxidase catalyzed radioiodination, and recovery of proteins bound to insoluble immunoadsorbents

International Nuclear Information System (INIS)

Cort, S.; McDougall, J.S.

1977-01-01

A method for the direct radioiodination and recovery of proteins specifically adsorbed to an insoluble immunoadsorbent is described. The optimal conditions for adsorption, washing, radiolabelling by lactoperoxidase-catalyzed iodination, and elution of radio-labelled proteins from the immunoadsorbent have been determined. The technique is a rapid and efficient means of isolating and radioiodinating specific proteins present in biological fluids and has been applied to the detection of immunoglobulin and histocompatibility antigens in mouse cell culture supernates. This method should be particularly applicable in research situations in which the specific antisera are available but the antigen concentration is low or the volume of material to be analyzed is limited

Chemical species of iodine in some seaweeds. Pt. 2. Iodine-bound biological macromolecules

International Nuclear Information System (INIS)

Xiaolin Hou; Chifang Chai; Xiaojun Yan

2000-01-01

The distribution of iodine in various biological macromolecules in Sargassum kjellmanianum was studied using neutron activation analysis combined with chemical and biochemical separation techniques. The results indicate that iodine is mainly bound with protein, part of iodine with pigment and polyphenol, and little with polysaccharides, such as algin, fucoidan and cellulose. This result is significant for the mechanism of enriching iodine of algae and utilization of alga iodine. (author)

Improved Range Searching Lower Bounds

DEFF Research Database (Denmark)

Larsen, Kasper Green; Nguyen, Huy L.

2012-01-01

by constructing a hard input set and query set, and then invoking Chazelle and Rosenberg's [CGTA'96] general theorem on the complexity of navigation in the pointer machine. For the group model, we show that input sets and query sets that are hard for range reporting in the pointer machine (i.e. by Chazelle...... and Rosenberg's theorem), are also hard for dynamic range searching in the group model. This theorem allows us to reuse decades of research on range reporting lower bounds to immediately obtain a range of new group model lower bounds. Amongst others, this includes an improved lower bound for the fundamental...

In vivo assessment of a novel dacron surface with covalently bound recombinant hirudin.

Science.gov (United States)

Wyers, M C; Phaneuf, M D; Rzucidlo, E M; Contreras, M A; LoGerfo, F W; Quist, W C

1999-01-01

Prosthetic arterial graft surfaces are relatively thrombogenic and fail to heal with a cellular neointima. The goal of this study was to characterize the in vivo antithrombin properties of a novel Dacron surface with covalently linked recombinant hirudin (rHir) implanted in a canine thoracic aorta with high flow and shear rates. rHir was bound to a knitted Dacron patch using crosslinker-modified bovine serum albumin (BSA) as a basecoat protein. BSA was first reacted with the heterobifunctional crosslinker, sulfo-SMCC. This BSA-SMCC complex was then bound to the carboxylic acid groups of hydrolyzed Dacron patches using the carbodiimide crosslinker, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. Iodinated, Traut's-modified rHir (125I-rHir-SH) was then reacted with the Dacron-BSA-SMCC surface, thereby covalently binding 125I-rHir. Graft segments were washed and sonicated to remove any nonspecifically bound 125I-rHir. Dacron-BSA-SMCC-S-125I-rHir patches (n = 5) and control Dacron-BSA patches (n = 5) were implanted in series in the thoracic aortas of canines. These patches were exposed to nonheparinized, arterial blood flow for 2 hours. Patches were explanted and assessed for 125I-rHir loss. Antithrombin activity of explanted 1-cm2 patch segments was evaluated using a chromogenic assay with 1, 5, 10, 15 units of added thrombin. Light microscopy was performed to qualitatively examine the pseudointima. Two animals were excluded from the study owing to excessive bleeding through the knitted 125I-rHir patch. Comparison of preoperative and postoperative 125I-rHir gamma counts revealed an overall decrease of 20+/-5.4% over the period studied. Explanted 125I-rHir patch segments were able to inhibit 1, 5, and 7 NIHU of thrombin, demonstrating retained antithrombin activity. Gross and microscopic examination of the control and test Dacron surfaces showed marked differences. Dacron surfaces with covalently bound 125I-rHir had no gross thrombus and a thin

Determination of steady-state protein breakdown rate in vivo by the disappearance of protein-bound tracer-labeled amino acids

DEFF Research Database (Denmark)

Holm, Lars; O'Rourke, Bruce; Ebenstein, David

2013-01-01

A method to determine the rate of protein breakdown in individual proteins was developed and tested in rats and confirmed in humans, using administration of deuterium oxide and incorporation of the deuterium into alanine that was subsequently incorporated into body proteins. Measurement of the fr...

Two-phonon bound states in imperfect crystals

International Nuclear Information System (INIS)

Behera, S.N.; Samsur, Sk.

1980-01-01

The question of the occurrence of two-phonon bound states in imperfect crystals is investigated. It is shown that the anharmonicity mediated two-phonon bound state which is present in perfect crystals gets modified due to the presence of impurities. Moreover, the possibility of the occurrence of a purely impurity mediated two-phonon bound state is demonstrated. The bound state frequencies are calculated using the simple Einstein oscillator model for the host phonons. The two-phonon density of states for the imperfect crystal thus obtained has peaks at the combination and difference frequencies of two host phonons besides the peaks at the bound state frequencies. For a perfect crystal the theory predicts a single peak at the two-phonon bound state frequency in conformity with experimental observations and other theoretical calculations. Experimental data on the two-phonon infrared absorption and Raman scattering from mixed crystals of Gasub(1-c)Alsub(c)P and Gesub(1-c)Sisub(c) are analysed to provide evidence in support of impurity-mediated two-phonon bound states. The relevance of the zero frequency (difference spectrum) peak to the central peak, observed in structural phase transitions, is conjectured. (author)

Receptor tyrosine phosphatase R-PTP-alpha is tyrosine-phosphorylated and associated with the adaptor protein Grb2

DEFF Research Database (Denmark)

Su, J; Batzer, A; Sap, J

1994-01-01

Receptor tyrosine phosphatases (R-PTPases) have generated interest because of their suspected involvement in cellular signal transduction. The adaptor protein Grb2 has been implicated in coupling receptor tyrosine kinases to Ras. We report that a ubiquitous R-PTPase, R-PTP-alpha, is tyrosine......-phosphorylated and associated in vivo with the Grb2 protein. This association can be reproduced in stably and transiently transfected cells, as well as in vitro using recombinant Grb2 protein. Association requires the presence of an intact SH2 domain in Grb2, as well as tyrosine phosphorylation of R-PTP-alpha. This observation...... links a receptor tyrosine phosphatase with a key component of a central cellular signalling pathway and provides a basis for addressing R-PTP-alpha function....

Nuclear Overhauser effect studies on the conformation of magnesium adenosine 5'-triphosphate bound to rabbit muscle creatine kinase

International Nuclear Information System (INIS)

Rosevear, P.R.; Powers, V.M.; Dowhan, D.; Mildvan, A.S.; Kenyon, G.L.

1987-01-01

Nuclear Overhauser effects were used to determine interproton distances on MgATP bound to rabbit muscle creatine kinase. The internuclear distances were used in a distance geometry program that objectively determines both the conformation of the bound MgATP and its uniqueness. Two classes of structures were found that satisfied the measured interproton distances. Both classes had the same anti glycosidic torsional angle (X = 78 +/- 10 0 ) but differed in their ribose ring puckers (O1'-endo or C4'-exo). The uniqueness of the glycosidic torsional angle is consistent with the preference of creatine kinase for adenine nucleotides. One of these conformations of MgATP bound to creatine kinase is indistinguishable from the conformation found for Co(NH 3 ) 4 ATP bound to the catalytic subunit of protein kinase, which also has a high specificity for adenine nucleotides. Distance geometry calculations also suggest that upper limit distances, when low enough (≤ 3.4 A), can be used instead of measured distances to define, within experimental error, the glycosidic torsional angle of bound nucleotides. However, this approach does not permit an evaluation of the ribose ring pucker

On semidefinite programming bounds for graph bandwidth

NARCIS (Netherlands)

de Klerk, E.; Nagy, M.; Sotirov, R.

2013-01-01

In this paper, we propose two new lower bounds on graph bandwidth and cyclic bandwidth based on semidefinite programming (SDP) relaxations of the quadratic assignment problem. We compare the new bounds with two other SDP bounds reported in [A. Blum, G. Konjevod, R. Ravi, and S. Vempala,

Interaction of the Sliding Clamp β-Subunit and Hda, a DnaA-Related Protein

OpenAIRE

Kurz, Mareike; Dalrymple, Brian; Wijffels, Gene; Kongsuwan, Kritaya

2004-01-01

In Escherichia coli, interactions between the replication initiation protein DnaA, the β subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation...

Small GTP-binding proteins in human endothelial cells

NARCIS (Netherlands)

de Leeuw, H. P.; Koster, P. M.; Calafat, J.; Janssen, H.; van Zonneveld, A. J.; van Mourik, J. A.; Voorberg, J.

1998-01-01

Small GTP-binding proteins of the Ras superfamily control an extensive number of intracellular events by alternating between GDP- and GTP-bound conformation. The presence of members of this protein family was examined in human umbilical vein endothelial cells employing RT-PCR. Sequence analysis of

Receptor-like proteins involved in plant disease resistance

NARCIS (Netherlands)

Kruijt, M.; Kock, de M.J.D.; Wit, de P.J.G.M.

2005-01-01

Race-specific resistance in plants against microbial pathogens is governed by several distinct classes of resistance (R) genes. This review focuses on the class that consists of the plasma membrane-bound leucine-rich repeat proteins known as receptor-like proteins (RLPs). The first isolated

Silicon homo-heterojunction solar cells: A promising candidate to realize high performance more stably

Directory of Open Access Journals (Sweden)

Miao Tan

2017-08-01

Full Text Available We have investigated the influences of diverse physical parameters on the performances of a silicon homo-heterojunction (H-H solar cell, which encompasses both homojunction and heterojunction, together with their underlying mechanisms by the aid of AFORS-HET simulation. It is found that the performances of H-H solar cell are less sensitive to (i the work function of the transparent conductive oxide layer, (ii the interfacial density of states at the front hydrogenated amorphous silicon/crystalline silicon (a-Si:H/c-Si interface, (iii the peak dangling bond defect densities within the p-type a-Si:H (p-a-Si:H layer, and (iv the doping concentration of the p-a-Si:H layer, when compared to that of the conventional heterojunction with intrinsic thin layer (HIT counterparts. These advantages are due to the fact that the interfacial recombination and the recombination within the a-Si:H region are less affected by all the above parameters, which fundamentally benefit from the field-effect passivation of the homojunction. Therefore, the design of H-H structure can provide an opportunity to produce high-efficiency solar cells more stably.

Silicon homo-heterojunction solar cells: A promising candidate to realize high performance more stably

Science.gov (United States)

Tan, Miao; Zhong, Sihua; Wang, Wenjie; Shen, Wenzhong

2017-08-01

We have investigated the influences of diverse physical parameters on the performances of a silicon homo-heterojunction (H-H) solar cell, which encompasses both homojunction and heterojunction, together with their underlying mechanisms by the aid of AFORS-HET simulation. It is found that the performances of H-H solar cell are less sensitive to (i) the work function of the transparent conductive oxide layer, (ii) the interfacial density of states at the front hydrogenated amorphous silicon/crystalline silicon (a-Si:H/c-Si) interface, (iii) the peak dangling bond defect densities within the p-type a-Si:H (p-a-Si:H) layer, and (iv) the doping concentration of the p-a-Si:H layer, when compared to that of the conventional heterojunction with intrinsic thin layer (HIT) counterparts. These advantages are due to the fact that the interfacial recombination and the recombination within the a-Si:H region are less affected by all the above parameters, which fundamentally benefit from the field-effect passivation of the homojunction. Therefore, the design of H-H structure can provide an opportunity to produce high-efficiency solar cells more stably.

Bounding the electrostatic free energies associated with linear continuum models of molecular solvation.

Science.gov (United States)

Bardhan, Jaydeep P; Knepley, Matthew G; Anitescu, Mihai

2009-03-14

The importance of electrostatic interactions in molecular biology has driven extensive research toward the development of accurate and efficient theoretical and computational models. Linear continuum electrostatic theory has been surprisingly successful, but the computational costs associated with solving the associated partial differential equations (PDEs) preclude the theory's use in most dynamical simulations. Modern generalized-Born models for electrostatics can reproduce PDE-based calculations to within a few percent and are extremely computationally efficient but do not always faithfully reproduce interactions between chemical groups. Recent work has shown that a boundary-integral-equation formulation of the PDE problem leads naturally to a new approach called boundary-integral-based electrostatics estimation (BIBEE) to approximate electrostatic interactions. In the present paper, we prove that the BIBEE method can be used to rigorously bound the actual continuum-theory electrostatic free energy. The bounds are validated using a set of more than 600 proteins. Detailed numerical results are presented for structures of the peptide met-enkephalin taken from a molecular-dynamics simulation. These bounds, in combination with our demonstration that the BIBEE methods accurately reproduce pairwise interactions, suggest a new approach toward building a highly accurate yet computationally tractable electrostatic model.

Bounding the electrostatic free energies associated with linear continuum models of molecular solvation.

Energy Technology Data Exchange (ETDEWEB)

Bardhan, J. P.; Knepley, M. G.; Anitescu, M. (Biosciences Division); ( MCS); (Rush Univ.)

2009-03-01

The importance of electrostatic interactions in molecular biology has driven extensive research toward the development of accurate and efficient theoretical and computational models. Linear continuum electrostatic theory has been surprisingly successful, but the computational costs associated with solving the associated partial differential equations (PDEs) preclude the theory's use in most dynamical simulations. Modern generalized-Born models for electrostatics can reproduce PDE-based calculations to within a few percent and are extremely computationally efficient but do not always faithfully reproduce interactions between chemical groups. Recent work has shown that a boundary-integral-equation formulation of the PDE problem leads naturally to a new approach called boundary-integral-based electrostatics estimation (BIBEE) to approximate electrostatic interactions. In the present paper, we prove that the BIBEE method can be used to rigorously bound the actual continuum-theory electrostatic free energy. The bounds are validated using a set of more than 600 proteins. Detailed numerical results are presented for structures of the peptide met-enkephalin taken from a molecular-dynamics simulation. These bounds, in combination with our demonstration that the BIBEE methods accurately reproduce pairwise interactions, suggest a new approach toward building a highly accurate yet computationally tractable electrostatic model.

Semi-supervised Learning Predicts Approximately One Third of the Alternative Splicing Isoforms as Functional Proteins

Directory of Open Access Journals (Sweden)

Yanqi Hao

2015-07-01

Full Text Available Alternative splicing acts on transcripts from almost all human multi-exon genes. Notwithstanding its ubiquity, fundamental ramifications of splicing on protein expression remain unresolved. The number and identity of spliced transcripts that form stably folded proteins remain the sources of considerable debate, due largely to low coverage of experimental methods and the resulting absence of negative data. We circumvent this issue by developing a semi-supervised learning algorithm, positive unlabeled learning for splicing elucidation (PULSE; http://www.kimlab.org/software/pulse, which uses 48 features spanning various categories. We validated its accuracy on sets of bona fide protein isoforms and directly on mass spectrometry (MS spectra for an overall AU-ROC of 0.85. We predict that around 32% of “exon skipping” alternative splicing events produce stable proteins, suggesting that the process engenders a significant number of previously uncharacterized proteins. We also provide insights into the distribution of positive isoforms in various functional classes and into the structural effects of alternative splicing.

Bounded elements in Locally C*-algebras

International Nuclear Information System (INIS)

El Harti, Rachid

2001-09-01

In order to get more useful information about Locally C*-algebras, we introduce in this paper the notion of bounded elements. First, we study the connection between bounded elements and spectrally bounded elements. Some structural results of Locally C*-algebras are established in Theorems 1 , 2 and 3. As an immediate consequence of Theorem 3, we give a characterization of the connected component of the identity in the group of unitary elements for a Locally C*-algebra. (author)

Covariant entropy bound and loop quantum cosmology

International Nuclear Information System (INIS)

Ashtekar, Abhay; Wilson-Ewing, Edward

2008-01-01

We examine Bousso's covariant entropy bound conjecture in the context of radiation filled, spatially flat, Friedmann-Robertson-Walker models. The bound is violated near the big bang. However, the hope has been that quantum gravity effects would intervene and protect it. Loop quantum cosmology provides a near ideal setting for investigating this issue. For, on the one hand, quantum geometry effects resolve the singularity and, on the other hand, the wave function is sharply peaked at a quantum corrected but smooth geometry, which can supply the structure needed to test the bound. We find that the bound is respected. We suggest that the bound need not be an essential ingredient for a quantum gravity theory but may emerge from it under suitable circumstances.

Enhanced photoprotection by protein-bound vs free xanthophyll pools: a comparative analysis of chlorophyll b and xanthophyll biosynthesis mutants.

Science.gov (United States)

Dall'Osto, Luca; Cazzaniga, Stefano; Havaux, Michel; Bassi, Roberto

2010-05-01

When light absorbed by plants exceeds the capacity of photosynthesis, the xanthophyll violaxanthin is reversibly de-epoxidized to zeaxanthin in the so-called xanthophyll cycle. Zeaxanthin plays a key role in the protection of photosynthetic organisms against excess light, by promoting rapidly reversible (qE) and long-term (qI) quenching of excited chlorophylls, and preventing lipid oxidation. The photoprotective role of zeaxanthin, either free or bound to light-harvesting complexes (Lhcs), has been investigated by using mutants lacking Chl b (ch1) and/or specific xanthophyll species (npq, lut2). The ch1 mutation causes (1) the absence of Lhcb proteins; (2) strong reduction of the feedback de-excitation (qE); and (3) accumulation of xanthophylls as free pigments into thylakoids. Ch1 mutants showed extreme sensitivity to photo-oxidative stress in high light, due to higher singlet oxygen (¹O₂) release. The double mutant ch1npq1 was more sensitive to photo-oxidation than ch1, showing that zeaxanthin does protect lipids even when free in the membrane. Nevertheless, lack of zeaxanthin had a much stronger impact on the level of lipid peroxidation in Lhcs-containing plants (WT vs npq1) with respect to Lhc-less plants (ch1 vs ch1npq1), implying that its protective effect is enhanced by interaction with antenna proteins. It is proposed that the antioxidant capacity of zeaxanthin is empowered in the presence of PSII-LHCs-Zea complexes, while its effect on enhancement of qE only provides a minor contribution. Comparison of the sensitivity of WT vs npq1 plants to exogenous ¹O₂ suggests that besides the scavenging of ¹O₂, at least one additional mechanism is involved in chloroplast photoprotection.

Tiamulin inhibits human CYP3A4 activity in an NIH/3T3 cell line stably expressing CYP3A4 cDNA.

Science.gov (United States)

De Groene, E M; Nijmeijer, S M; Horbach, G J; Witkamp, R F

1995-09-07

Tiamulin is an antibiotic frequently used in veterinary medicine. The drug has been shown to produce clinically important interactions with other compounds that are administered simultaneously. An NIH/3T3 cell line, stably expressing human cytochrome P450 (EC 1.14.14.1) cDNA (CYP3A4), was used to study the effect of tiamulin on CYP3A4 activity. The 6 beta-hydroxylation activity of testosterone, which is increased in CYP3A4-expressing cells compared to vector-transfected cells, showed reduced activity after incubation with 1 microM tiamulin and was completely reduced to background level after incubation with 2, 5 and 10 microM tiamulin. The CYP3A4-expressing cell line was used in combination with a shuttle vector containing the bacterial lacZ' gene to study the effect of tiamulin on CYP3A4-mediated mutagenicity of aflatoxin B1. The mutation frequency of aflatoxin B1 could be completely inhibited by tiamulin in CYP3A4-expressing cells, but no effect was observed on the mutation frequency of the direct mutagen ethylmethanesulphonate. Western blotting of homogenates of the CYP3A4-expressing cell line showed stabilization of CYP3A4 protein after incubation with tiamulin, supporting the hypothesis that the mechanism of inhibition is by binding of tiamulin to the cytochrome.

Conformational sampling in template-free protein loop structure modeling: an overview.

Science.gov (United States)

Li, Yaohang

2013-01-01

Accurately modeling protein loops is an important step to predict three-dimensional structures as well as to understand functions of many proteins. Because of their high flexibility, modeling the three-dimensional structures of loops is difficult and is usually treated as a "mini protein folding problem" under geometric constraints. In the past decade, there has been remarkable progress in template-free loop structure modeling due to advances of computational methods as well as stably increasing number of known structures available in PDB. This mini review provides an overview on the recent computational approaches for loop structure modeling. In particular, we focus on the approaches of sampling loop conformation space, which is a critical step to obtain high resolution models in template-free methods. We review the potential energy functions for loop modeling, loop buildup mechanisms to satisfy geometric constraints, and loop conformation sampling algorithms. The recent loop modeling results are also summarized.

CONFORMATIONAL SAMPLING IN TEMPLATE-FREE PROTEIN LOOP STRUCTURE MODELING: AN OVERVIEW

Directory of Open Access Journals (Sweden)

Yaohang Li

2013-02-01

Full Text Available Accurately modeling protein loops is an important step to predict three-dimensional structures as well as to understand functions of many proteins. Because of their high flexibility, modeling the three-dimensional structures of loops is difficult and is usually treated as a “mini protein folding problem” under geometric constraints. In the past decade, there has been remarkable progress in template-free loop structure modeling due to advances of computational methods as well as stably increasing number of known structures available in PDB. This mini review provides an overview on the recent computational approaches for loop structure modeling. In particular, we focus on the approaches of sampling loop conformation space, which is a critical step to obtain high resolution models in template-free methods. We review the potential energy functions for loop modeling, loop buildup mechanisms to satisfy geometric constraints, and loop conformation sampling algorithms. The recent loop modeling results are also summarized.

Proteomic Identification of Dengue Virus Binding Proteins in Aedes aegypti Mosquitoes and Aedes albopictus Cells

Directory of Open Access Journals (Sweden)

Maria de Lourdes Muñoz

2013-01-01

Full Text Available The main vector of dengue in America is the mosquito Aedes aegypti, which is infected by dengue virus (DENV through receptors of midgut epithelial cells. The envelope protein (E of dengue virus binds to receptors present on the host cells through its domain III that has been primarily recognized to bind cell receptors. In order to identify potential receptors, proteins from mosquito midgut tissue and C6/36 cells were purified by affinity using columns with the recombinant E protein domain III (rE-DIII or DENV particles bound covalently to Sepharose 4B to compare and evaluate their performance to bind proteins including putative receptors from female mosquitoes of Ae. aegypti. To determine their identity mass spectrometric analysis of purified proteins separated by polyacrylamide gel electrophoresis was performed. Our results indicate that both viral particles and rE-DIII bound proteins with the same apparent molecular weights of 57 and 67 kDa. In addition, viral particles bound high molecular weight proteins. Purified proteins identified were enolase, beta-adrenergic receptor kinase (beta-ARK, translation elongation factor EF-1 alpha/Tu, and cadherin.

Degenerate quantum codes and the quantum Hamming bound

International Nuclear Information System (INIS)

Sarvepalli, Pradeep; Klappenecker, Andreas

2010-01-01

The parameters of a nondegenerate quantum code must obey the Hamming bound. An important open problem in quantum coding theory is whether the parameters of a degenerate quantum code can violate this bound for nondegenerate quantum codes. In this article we show that Calderbank-Shor-Steane (CSS) codes, over a prime power alphabet q≥5, cannot beat the quantum Hamming bound. We prove a quantum version of the Griesmer bound for the CSS codes, which allows us to strengthen the Rains' bound that an [[n,k,d

Some Improved Nonperturbative Bounds for Fermionic Expansions

Energy Technology Data Exchange (ETDEWEB)

Lohmann, Martin, E-mail: marlohmann@gmail.com [Universita di Roma Tre, Dipartimento di Matematica (Italy)

2016-06-15

We reconsider the Gram-Hadamard bound as it is used in constructive quantum field theory and many body physics to prove convergence of Fermionic perturbative expansions. Our approach uses a recursion for the amplitudes of the expansion, discovered in a model problem by Djokic (2013). It explains the standard way to bound the expansion from a new point of view, and for some of the amplitudes provides new bounds, which avoid the use of Fourier transform, and are therefore superior to the standard bounds for models like the cold interacting Fermi gas.

Grass cell wall feruloylation: distribution of bound ferulate and candidate gene expression in Brachypodium distachyon

Directory of Open Access Journals (Sweden)

Hugo Bruno Correa Molinari

2013-03-01

Full Text Available The cell walls of grasses such as wheat, maize, rice and sugar cane, contain large amounts of ferulate that is ester-linked to the cell wall polysaccharide glucuronoarabinoxylan (GAX. This ferulate is considered to limit the digestibility of polysaccharide in grass biomass as it forms covalent linkages between polysaccharide and lignin components. Candidate genes within a grass-specific clade of the BAHD acyl-coA transferase superfamily have been identified as being responsible for the ester linkage of ferulate to GAX. Manipulation of these BAHD genes may therefore be a biotechnological target for increasing efficiency of conversion of grass biomass into biofuel. Here, we describe the expression of these candidate genes and amounts of bound ferulate from various tissues and developmental stages of the model grass Brachypodium distachyon. BAHD candidate transcripts and significant amounts of bound ferulate were present in every tissue and developmental stage. We hypothesise that BAHD candidate genes similar to the recently described rice OsPMT gene (PMT sub-clade are principally responsible for the bound coumaric acid (pCA, and that other BAHD candidates (non-PMT sub-clade are responsible for bound ferulic acid (FA. There were some similarities with between the ratio of expression non-PMT / PMT genes and the ratio of bound FA / pCA between tissue types, compatible with this hypothesis. However, much further work to modify BAHD genes in grasses and to characterise the heterologously expressed proteins is required to demonstrate their function.

Membrane-bound transcription factors: regulated release by RIP or RUP.

Science.gov (United States)

Hoppe, T; Rape, M; Jentsch, S

2001-06-01

Regulated nuclear transport of transcription factors from cytoplasmic pools is a major route by which eukaryotes control gene expression. Exquisite examples are transcription factors that are kept in a dormant state in the cytosol by membrane anchors; such proteins are released from membranes by proteolytic cleavage, which enables these transcription factors to enter the nucleus. Cleavage can be mediated either by regulated intramembrane proteolysis (RIP) catalysed by specific membrane-bound proteases or by regulated ubiquitin/proteasome-dependent processing (RUP). In both cases processing can be controlled by cues that originate at or in the vicinity of the membrane.

Sharp Bounds for Symmetric and Asymmetric Diophantine Approximation

Institute of Scientific and Technical Information of China (English)

Cornelis KRAAIKAMP; Ionica SMEETS

2011-01-01

In 2004,Tong found bounds for the approximation quality of a regular continued fraction convergent to a rational number,expressed in bounds for both the previous and next approximation.The authors sharpen his results with a geometric method and give both sharp upper and lower bounds.The asymptotic frequencies that these bounds occur are also calculated.

Structural insights into calcium-bound S100P and the V domain of the RAGE complex.

Directory of Open Access Journals (Sweden)

Srinivasa R Penumutchu

Full Text Available The S100P protein is a member of the S100 family of calcium-binding proteins and possesses both intracellular and extracellular functions. Extracellular S100P binds to the cell surface receptor for advanced glycation end products (RAGE and activates its downstream signaling cascade to meditate tumor growth, drug resistance and metastasis. Preventing the formation of this S100P-RAGE complex is an effective strategy to treat various disease conditions. Despite its importance, the detailed structural characterization of the S100P-RAGE complex has not yet been reported. In this study, we report that S100P preferentially binds to the V domain of RAGE. Furthermore, we characterized the interactions between the RAGE V domain and Ca(2+-bound S100P using various biophysical techniques, including isothermal titration calorimetry (ITC, fluorescence spectroscopy, multidimensional NMR spectroscopy, functional assays and site-directed mutagenesis. The entropy-driven binding between the V domain of RAGE and Ca(+2-bound S100P was found to lie in the micromolar range (Kd of ∼ 6 µM. NMR data-driven HADDOCK modeling revealed the putative sites that interact to yield a proposed heterotetrameric model of the S100P-RAGE V domain complex. Our study on the spatial structural information of the proposed protein-protein complex has pharmaceutical relevance and will significantly contribute toward drug development for the prevention of RAGE-related multifarious diseases.

Integration of G protein α (Gα) signaling by the regulator of G protein signaling 14 (RGS14).

Science.gov (United States)

Brown, Nicole E; Goswami, Devrishi; Branch, Mary Rose; Ramineni, Suneela; Ortlund, Eric A; Griffin, Patrick R; Hepler, John R

2015-04-03

RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gαi/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gαi1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gαi1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gαo-AlF4(-). Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gαi1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gαo-AlF4(-) and an AlF4(-)-insensitive mutant (G42R) of Gαi1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gαi1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gαo-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Lack of induction of tissue transglutaminase but activation of the preexisting enzyme in c-Myc-induced apoptosis of CHO cells.

Science.gov (United States)

Balajthy, Z; Kedei, N; Nagy, L; Davies, P J; Fésüs, L

1997-07-18

The intracellular activity and expression of tissue transglutaminase, which crosslinks proteins through epsilon(gamma-glutamyl)lysine isodipeptide bond, was investigated in CHO cells and those stably transfected with either inducible c-Myc (which leads to apoptosis) or with c-myc and the apoptosis inhibitor Bcl-2. Protein-bound cross-link content was significantly higher when apoptosis was induced by c-Myc while the concomitant presence of Bcl-2 markedly reduced both apoptosis and enzymatic protein cross-linking. The expression of tissue transglutaminase did not change following the initiation of apoptosis by c-Myc or when it was blocked by Bcl-2. Studying transiently co-transfected elements of the mouse tissue transglutaminase promoter linked to a reporter enzyme revealed their overall repression in cells expressing c-Myc. This repression was partially suspended in cells also carrying Bcl-2. Our data suggest that tissue transglutaminase is not induced when c-Myc initiates apoptosis but the pre-existing endogenous enzyme is activated.

Phenomenology of two-dimensional stably stratified turbulence under large-scale forcing

KAUST Repository

Kumar, Abhishek; Verma, Mahendra K.; Sukhatme, Jai

2017-01-01

In this paper, we characterise the scaling of energy spectra, and the interscale transfer of energy and enstrophy, for strongly, moderately and weakly stably stratified two-dimensional (2D) turbulence, restricted in a vertical plane, under large-scale random forcing. In the strongly stratified case, a large-scale vertically sheared horizontal flow (VSHF) coexists with small scale turbulence. The VSHF consists of internal gravity waves and the turbulent flow has a kinetic energy (KE) spectrum that follows an approximate k−3 scaling with zero KE flux and a robust positive enstrophy flux. The spectrum of the turbulent potential energy (PE) also approximately follows a k−3 power-law and its flux is directed to small scales. For moderate stratification, there is no VSHF and the KE of the turbulent flow exhibits Bolgiano–Obukhov scaling that transitions from a shallow k−11/5 form at large scales, to a steeper approximate k−3 scaling at small scales. The entire range of scales shows a strong forward enstrophy flux, and interestingly, large (small) scales show an inverse (forward) KE flux. The PE flux in this regime is directed to small scales, and the PE spectrum is characterised by an approximate k−1.64 scaling. Finally, for weak stratification, KE is transferred upscale and its spectrum closely follows a k−2.5 scaling, while PE exhibits a forward transfer and its spectrum shows an approximate k−1.6 power-law. For all stratification strengths, the total energy always flows from large to small scales and almost all the spectral indicies are well explained by accounting for the scale-dependent nature of the corresponding flux.

Phenomenology of two-dimensional stably stratified turbulence under large-scale forcing

KAUST Repository

Kumar, Abhishek

2017-01-11

In this paper, we characterise the scaling of energy spectra, and the interscale transfer of energy and enstrophy, for strongly, moderately and weakly stably stratified two-dimensional (2D) turbulence, restricted in a vertical plane, under large-scale random forcing. In the strongly stratified case, a large-scale vertically sheared horizontal flow (VSHF) coexists with small scale turbulence. The VSHF consists of internal gravity waves and the turbulent flow has a kinetic energy (KE) spectrum that follows an approximate k−3 scaling with zero KE flux and a robust positive enstrophy flux. The spectrum of the turbulent potential energy (PE) also approximately follows a k−3 power-law and its flux is directed to small scales. For moderate stratification, there is no VSHF and the KE of the turbulent flow exhibits Bolgiano–Obukhov scaling that transitions from a shallow k−11/5 form at large scales, to a steeper approximate k−3 scaling at small scales. The entire range of scales shows a strong forward enstrophy flux, and interestingly, large (small) scales show an inverse (forward) KE flux. The PE flux in this regime is directed to small scales, and the PE spectrum is characterised by an approximate k−1.64 scaling. Finally, for weak stratification, KE is transferred upscale and its spectrum closely follows a k−2.5 scaling, while PE exhibits a forward transfer and its spectrum shows an approximate k−1.6 power-law. For all stratification strengths, the total energy always flows from large to small scales and almost all the spectral indicies are well explained by accounting for the scale-dependent nature of the corresponding flux.

SHARP ENTRYWISE PERTURBATION BOUNDS FOR MARKOV CHAINS.

Science.gov (United States)

Thiede, Erik; VAN Koten, Brian; Weare, Jonathan

For many Markov chains of practical interest, the invariant distribution is extremely sensitive to perturbations of some entries of the transition matrix, but insensitive to others; we give an example of such a chain, motivated by a problem in computational statistical physics. We have derived perturbation bounds on the relative error of the invariant distribution that reveal these variations in sensitivity. Our bounds are sharp, we do not impose any structural assumptions on the transition matrix or on the perturbation, and computing the bounds has the same complexity as computing the invariant distribution or computing other bounds in the literature. Moreover, our bounds have a simple interpretation in terms of hitting times, which can be used to draw intuitive but rigorous conclusions about the sensitivity of a chain to various types of perturbations.

Ubiquilin overexpression reduces GFP-polyalanine-induced protein aggregates and toxicity

International Nuclear Information System (INIS)

Wang Hongmin; Monteiro, Mervyn J.

2007-01-01

Several human disorders are associated with an increase in a continuous stretch of alanine amino acids in proteins. These so-called polyalanine expansion diseases share many similarities with polyglutamine-related disorders, including a length-dependent reiteration of amino acid induction of protein aggregation and cytotoxicity. We previously reported that overexpression of ubiquilin reduces protein aggregates and toxicity of expanded polyglutamine proteins. Here, we demonstrate a similar role for ubiquilin toward expanded polyalanine proteins. Overexpression of ubiquilin-1 in HeLa cells reduced protein aggregates and the cytotoxicity associated with expression of a transfected nuclear-targeted GFP-fusion protein containing 37-alanine repeats (GFP-A37), in a dose dependent manner. Ubiquilin coimmunoprecipitated more with GFP proteins containing a 37-polyalanine tract compared to either 7 (GFP-A7), or no alanine tract (GFP). Moreover, overexpression of ubiquilin suppressed the increased vulnerability of HeLa cell lines stably expressing the GFP-A37 fusion protein to oxidative stress-induced cell death compared to cell lines expressing GFP or GFP-A7 proteins. By contrast, siRNA knockdown of ubiquilin expression in the GFP-A37 cell line was associated with decreased cellular proliferation, and increases in GFP protein aggregates, nuclear fragmentation, and cell death. Our results suggest that boosting ubiquilin levels in cells might provide a universal and attractive strategy to prevent toxicity of proteins containing reiterative expansions of amino acids involved in many human diseases

Covalent modification of platelet proteins by palmitate

International Nuclear Information System (INIS)

Muszbek, L.; Laposata, M.

1989-01-01

Covalent attachment of fatty acid to proteins plays an important role in association of certain proteins with hydrophobic membrane structures. In platelets, the structure of many membrane glycoproteins (GPs) has been examined in detail, but the question of fatty acid acylation of platelet proteins has not been addressed. In this study, we wished to determine (a) whether platelet proteins could be fatty acid acylated; and, if so, (b) whether these modified proteins were present in isolated platelet membranes and cytoskeletal fractions; and (c) if the pattern of fatty acid acylated proteins changed on stimulation of the platelets with the agonist thrombin. We observed that in platelets allowed to incorporate 3H-palmitate, a small percentage (1.37%) of radioactivity incorporated into the cells became covalently bound to protein. Selective cleavage of thioester, thioester plus O-ester, and amide-linked 3H-fatty acids from proteins, and their subsequent analysis by high-performance liquid chromatography (HPLC) indicated that the greatest part of 3H-fatty acid covalently bound to protein was thioester-linked 3H-palmitate. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography, at least ten major radiolabeled proteins were detected. Activation of platelets by thrombin greatly increased the quantity of 3H-palmitoylated proteins associated with the cytoskeleton. Nearly all radiolabeled proteins were recovered in the membrane fraction, indicating that these proteins are either integral or peripheral membrane proteins or proteins tightly associated to membrane constituents. Components of the GPIIb-IIIa complex were not palmitoylated. Thus, platelet proteins are significantly modified posttranslationally by 3H-palmitate, and incorporation of palmitoylated proteins into the cytoskeleton is a prominent component of the platelet response to thrombin stimulation

Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography.

Science.gov (United States)

Mandal, Kalyaneswar; Uppalapati, Maruti; Ault-Riché, Dana; Kenney, John; Lowitz, Joshua; Sidhu, Sachdev S; Kent, Stephen B H

2012-09-11

Total chemical synthesis was used to prepare the mirror image (D-protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D-VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D- and L- forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L-protein ligand bound only to D-VEGF-A, whereas the D-protein ligand bound only to L-VEGF-A. The D-protein ligand, but not the L-protein ligand, inhibited the binding of natural VEGF(165) to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of {D-protein antagonist + L-protein form of VEGF-A}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 Å. Detailed analysis of the interaction between the D-protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800 Å(2) in accord with our design objectives, and that the D-protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2.

Relativistic bound state wave functions

International Nuclear Information System (INIS)

Micu, L.

2005-01-01

A particular method of writing the bound state wave functions in relativistic form is applied to the solutions of the Dirac equation with confining potentials in order to obtain a relativistic description of a quark antiquark bound system representing a given meson. Concerning the role of the effective constituent in the present approach we first observe that without this additional constituent we couldn't expand the bound state wave function in terms of products of free states. Indeed, we notice that if the wave function depends on the relative coordinates only, all the expansion coefficients would be infinite. Secondly we remark that the effective constituent enabled us to give a Lorentz covariant meaning to the potential energy of the bound system which is now seen as the 4th component of a 4-momentum. On the other side, by relating the effective constituent to the quantum fluctuations of the background field which generate the binding, we provided a justification for the existence of some spatial degrees of freedom accompanying the interaction potential. These ones, which are quite unusual in quantum mechanics, in our model are the natural consequence of the the independence of the quarks and can be seen as the effect of the imperfect cancellation of the vector momenta during the quantum fluctuations. Related with all these we remark that the adequate representation for the relativistic description of a bound system is the momentum representation, because of the transparent and easy way of writing the conservation laws and the transformation properties of the wave functions. The only condition to be fulfilled is to find a suitable way to take into account the potential energy of the bound system. A particular feature of the present approach is that the confining forces are due to a kind of glue where both quarks are embedded. This recalls other bound state models where the wave function is factorized in terms of constituent wave functions and the confinement is

Bounds on poloidal kinetic energy in plane layer convection

Science.gov (United States)

Tilgner, A.

2017-12-01

A numerical method is presented that conveniently computes upper bounds on heat transport and poloidal energy in plane layer convection for infinite and finite Prandtl numbers. The bounds obtained for the heat transport coincide with earlier results. These bounds imply upper bounds for the poloidal energy, which follow directly from the definitions of dissipation and energy. The same constraints used for computing upper bounds on the heat transport lead to improved bounds for the poloidal energy.

Structure of alanine racemase from Oenococcus oeni with bound pyridoxal 5′-phosphate

International Nuclear Information System (INIS)

Palani, Kandavelu; Burley, Stephen K.; Swaminathan, Subramanyam

2012-01-01

Alanine racemase from O. oeni exists as a dimer in the crystal structure. Both monomers contribute to the two active sites present, one for each monomer. The crystal structure of alanine racemase from Oenococcus oeni has been determined at 1.7 Å resolution using the single-wavelength anomalous dispersion (SAD) method and selenium-labelled protein. The protein exists as a symmetric dimer in the crystal, with both protomers contributing to the two active sites. Pyridoxal 5′-phosphate, a cofactor, is bound to each monomer and forms a Schiff base with Lys39. Structural comparison of alanine racemase from O. oeni (Alr) with homologous family members revealed similar domain organization and cofactor binding

Persistence-Based Branch Misprediction Bounds for WCET Analysis

DEFF Research Database (Denmark)

Puffitsch, Wolfgang

2015-01-01

Branch prediction is an important feature of pipelined processors to achieve high performance. However, it can lead to overly pessimistic worst-case execution time (WCET) bounds when being modeled too conservatively. This paper presents bounds on the number of branch mispredictions for local...... dynamic branch predictors. To handle interferences between branch instructions we use the notion of persistence, a concept that is also found in cache analyses. The bounds apply to branches in general, not only to branches that close a loop. Furthermore, the bounds can be easily integrated into integer...... linear programming formulations of the WCET problem. An evaluation on a number of benchmarks shows that with these bounds, dynamic branch prediction does not necessarily lead to higher WCET bounds than static prediction schemes....

Positivity bounds on double parton distributions

International Nuclear Information System (INIS)

Diehl, Markus; Kasemets, Tomas

2013-03-01

Double hard scattering in proton-proton collisions is described in terms of double parton distributions. We derive bounds on these distributions that follow from their interpretation as probability densities, taking into account all possible spin correlations between two partons in an unpolarized proton. These bounds constrain the size of the polarized distributions and can for instance be used to set upper limits on the effects of spin correlations in double hard scattering. We show that the bounds are stable under leading-order DGLAP evolution to higher scales.

Gamma irradiation effect on soy protein modification, protein - phenolic interaction and antioxidant activity in soybean

International Nuclear Information System (INIS)

Kumari, Sweta; Dahuja, Anil; Vinutha, T.; Singh, Bhupinder

2014-01-01

Soy protein is one of the most important sources of protein to feed the world population in the future. Consumption of soybean quality protein and their texture is dependent on the protein modification. In the present study, four soybean genotypes PL5039 (black), EC 472143 (black), Pusa 9814 (yellow) and SL525 (yellow), differing in their seed coat colour were gamma irradiated at 0.5,1.0, 2.0 and 5.0 kGy and the extent of protein modification and parameters affecting it viz. free phenolics, bound phenolics, lip oxygenase and antioxidant activity were analysed. Modifications of soybean proteins were investigated by chemical analysis and electrophoresis. The irradiation dose of 1.0 kGy showed decreased turbidity, protein oxidation, surface hydrophobicity but increased solubility and sulfhydryl and disulfide contents in all the genotypes. Further, SDS PAGE profile of treated soybean seeds revealed remarkable difference in electrophoretic bands as compared to the untreated seeds. Lipoxygense activity in all the genotypes decreased with increased exposure of gamma irradiation, which produced peroxide products that changes the structural characteristics of soy protein. Free phenolics, bound phenolics and total antioxidant activity measured in terms of FRAP in all the genotypes increased significantly at a dose of 2.0 kGy and it declined at a dose of 5.0 kGy. Antioxidant potential measured in terms of 1,1-diphenyl-2- picrylhydrazyl (DPPH) scavenging activity showed an increasing trend with dose, indicating that radiation processing as a method of food preservation has a positive nutritional implication. Hence, it is suggested that, mild gamma irradiation upto 2.0 kGy may reduce the protein oxidation, enhance the antioxidant activity and improve the soybean protein quality compared to higher dose 5.0 kGy, which reduced the protein quality. (author)

Comparison of Lasserre's Measure-based Bounds for Polynomial Optimization to Bounds Obtained by Simulated Annealing

NARCIS (Netherlands)

de Klerk, Etienne; Laurent, Monique

We consider the problem of minimizing a continuous function f over a compact set K. We compare the hierarchy of upper bounds proposed by Lasserre in [SIAM J. Optim. 21(3) (2011), pp. 864-885] to bounds that may be obtained from simulated annealing. We show that, when f is a polynomial and K a convex

Exosomal proteins as prognostic biomarkers in non-small cell lung cancer

DEFF Research Database (Denmark)

Sandfeld-Paulsen, B; Aggerholm-Pedersen, N; Bæk, R

2016-01-01

BACKGROUND: Use of exosomes as biomarkers in non-small cell lung cancer (NSCLC) is an intriguing approach in the liquid-biopsy era. Exosomes are nano-sized vesicles with membrane-bound proteins that reflect their originating cell. Prognostic biomarkers are needed to improve patient selection...... Bonferroni correction. Results were adjusted for clinico-pathological characteristics, stage, histology, age, sex and performance status. CONCLUSION: We illustrate the promising aspects associated with the use of exosomal membrane-bound proteins as a biomarker and demonstrate that they are a strong...

Regulation of Cellular and Molecular Functions by Protein ...

Indian Academy of Sciences (India)

... a high-energy linkage. The free energy of hydrolysis 1 of protein bound tyrosine phosphate ... protein kinases, cdc2 kinase (which regulates cell division cycle) and related cdc ... residues in response to extracellular signals such as hormones or growth factors. ... involved in regulating glycogen metabolism. The activity of.

Crystal Structure of a Eukaryotic GEN1 Resolving Enzyme Bound to DNA

Directory of Open Access Journals (Sweden)

Yijin Liu

2015-12-01

Full Text Available We present the crystal structure of the junction-resolving enzyme GEN1 bound to DNA at 2.5 Å resolution. The structure of the GEN1 protein reveals it to have an elaborated FEN-XPG family fold that is modified for its role in four-way junction resolution. The functional unit in the crystal is a monomer of active GEN1 bound to the product of resolution cleavage, with an extensive DNA binding interface for both helical arms. Within the crystal lattice, a GEN1 dimer interface juxtaposes two products, whereby they can be reconnected into a four-way junction, the structure of which agrees with that determined in solution. The reconnection requires some opening of the DNA structure at the center, in agreement with permanganate probing and 2-aminopurine fluorescence. The structure shows that a relaxation of the DNA structure accompanies cleavage, suggesting how second-strand cleavage is accelerated to ensure productive resolution of the junction.

Spontaneous reverse movement of mRNA-bound tRNA through the ribosome.

Science.gov (United States)

Konevega, Andrey L; Fischer, Niels; Semenkov, Yuri P; Stark, Holger; Wintermeyer, Wolfgang; Rodnina, Marina V

2007-04-01

During the translocation step of protein synthesis, a complex of two transfer RNAs bound to messenger RNA (tRNA-mRNA) moves through the ribosome. The reaction is promoted by an elongation factor, called EF-G in bacteria, which, powered by GTP hydrolysis, induces an open, unlocked conformation of the ribosome that allows for spontaneous tRNA-mRNA movement. Here we show that, in the absence of EF-G, there is spontaneous backward movement, or retrotranslocation, of two tRNAs bound to mRNA. Retrotranslocation is driven by the gain in affinity when a cognate E-site tRNA moves into the P site, which compensates the affinity loss accompanying the movement of peptidyl-tRNA from the P to the A site. These results lend support to the diffusion model of tRNA movement during translocation. In the cell, tRNA movement is biased in the forward direction by EF-G, which acts as a Brownian ratchet and prevents backward movement.

A conserved NAD+ binding pocket that regulates protein-protein interactions during aging.

Science.gov (United States)

Li, Jun; Bonkowski, Michael S; Moniot, Sébastien; Zhang, Dapeng; Hubbard, Basil P; Ling, Alvin J Y; Rajman, Luis A; Qin, Bo; Lou, Zhenkun; Gorbunova, Vera; Aravind, L; Steegborn, Clemens; Sinclair, David A

2017-03-24

DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD + (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD + to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate-ribose) polymerase], a critical DNA repair protein. As mice age and NAD + concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD + Thus, NAD + directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging. Copyright © 2017, American Association for the Advancement of Science.

Universal bounds in even-spin CFTs

Energy Technology Data Exchange (ETDEWEB)

Qualls, Joshua D. [Department of Physics, National Taiwan University,Taipei, Taiwan (China)

2015-12-01

We prove using invariance under the modular S− and ST−transformations that every unitary two-dimensional conformal field theory (CFT) having only even-spin primary operators (with no extended chiral algebra and with right- and left-central charges c,c̃>1) contains a primary operator with dimension Δ{sub 1} satisfying 0<Δ{sub 1}<((c+c̃)/24)+0.09280…. After deriving both analytical and numerical bounds, we discuss how to extend our methods to bound higher conformal dimensions before deriving lower and upper bounds on the number of primary operators in a given energy range. Using the AdS{sub 3}/CFT{sub 2} dictionary, the bound on Δ{sub 1} proves the lightest massive excitation in appropriate theories of 3D matter and gravity with cosmological constant Λ<0 can be no heavier than 1/8G{sub N}+O(√(−Λ)); the bounds on the number of operators are related via AdS/CFT to the entropy of states in the dual gravitational theory. In the flat-space approximation, the limiting mass is exactly that of the lightest BTZ black hole.

Targeting to cells of fluorescent liposomes covalently coupled with monoclonal antibody or protein A

Science.gov (United States)

Leserman, Lee D.; Barbet, Jacques; Kourilsky, François; Weinstein, John N.

1980-12-01

Many applications envisioned for liposomes in cell biology and chemotherapy require their direction to specific cellular targets1-3. The ability to use antibody as a means of conferring specificity to liposomes would markedly increase their usefulness. We report here a method for covalently coupling soluble proteins, including monoclonal antibody and Staphylococcus aureus protein A (ref. 4), to small sonicated liposomes, by using the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl 3-(2-pyridyldithio)propionate (SPDP, Pharmacia). Liposomes bearing covalently coupled mouse monoclonal antibody against human β2-microglobulin [antibody B1.1G6 (IgG2a, κ) (B. Malissen et al., in preparation)] bound specifically to human, but not to mouse cells. Liposomes bearing protein A became bound to human cells previously incubated with the B1.1G6 antibody, but not to cells incubated without antibody. The coupling method results in efficient binding of protein to the liposomes without aggregation and without denaturation of the coupled ligand; at least 60% of liposomes bound functional protein. Further, liposomes did not leak encapsulated carboxyfluorescein (CF) as a consequence of the reaction.

Aquaporin Protein-Protein Interactions

Directory of Open Access Journals (Sweden)

Jennifer Virginia Roche

2017-10-01

Full Text Available Aquaporins are tetrameric membrane-bound channels that facilitate transport of water and other small solutes across cell membranes. In eukaryotes, they are frequently regulated by gating or trafficking, allowing for the cell to control membrane permeability in a specific manner. Protein–protein interactions play crucial roles in both regulatory processes and also mediate alternative functions such as cell adhesion. In this review, we summarize recent knowledge about aquaporin protein–protein interactions; dividing the interactions into three types: (1 interactions between aquaporin tetramers; (2 interactions between aquaporin monomers within a tetramer (hetero-tetramerization; and (3 transient interactions with regulatory proteins. We particularly focus on the structural aspects of the interactions, discussing the small differences within a conserved overall fold that allow for aquaporins to be differentially regulated in an organism-, tissue- and trigger-specific manner. A deep knowledge about these differences is needed to fully understand aquaporin function and regulation in many physiological processes, and may enable design of compounds targeting specific aquaporins for treatment of human disease.

Functional advantages of dynamic protein disorder.

Science.gov (United States)

Berlow, Rebecca B; Dyson, H Jane; Wright, Peter E

2015-09-14

Intrinsically disordered proteins participate in many important cellular regulatory processes. The absence of a well-defined structure in the free state of a disordered domain, and even on occasion when it is bound to physiological partners, is fundamental to its function. Disordered domains are frequently the location of multiple sites for post-translational modification, the key element of metabolic control in the cell. When a disordered domain folds upon binding to a partner, the resulting complex buries a far greater surface area than in an interaction of comparably-sized folded proteins, thus maximizing specificity at modest protein size. Disorder also maintains accessibility of sites for post-translational modification. Because of their inherent plasticity, disordered domains frequently adopt entirely different structures when bound to different partners, increasing the repertoire of available interactions without the necessity for expression of many different proteins. This feature also adds to the faithfulness of cellular regulation, as the availability of a given disordered domain depends on competition between various partners relevant to different cellular processes. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Potential biological process of X-linked inhibitor of apoptosis protein in renal cell carcinoma based upon differential protein expression analysis.

Science.gov (United States)

Chen, Chao; Zhao, Si Cong; Yang, Wen Zheng; Chen, Zong Ping; Yan, Yong

2018-01-01

The X-linked inhibitor of apoptosis protein (XIAP) is the best characterized member of the IAP family and is a potent inhibitor of the caspase/apoptosis pathway. It has also been revealed that XIAP has additional biological functions that rely on its direct inhibition of apoptosis. In the present study, stably transfected Caki-1 cells with XIAP-knockdown were generated, and an isobaric tag for relative and absolute quantitation-based proteomics approach was employed to investigate the regulatory mechanism of XIAP in renal cell carcinoma (RCC). The results demonstrate that the sensitivity of the RCC cell line to apoptotic stimulation increased markedly with XIAP-knockdown. A number of differentially expressed proteins were detected between the original Caki-1 cell line and the XIAP-knockdown Caki-1 cell line; 87 at 0 h (prior to etoposide treatment), 178 at 0.5 h and 169 at 3 h, while no differentially expressed proteins were detected (ratio >1.5 or <0.5; P<0.05) at 12 h after etoposide treatment. Through analysis of the differentially expressed proteins, it was revealed that XIAP may participate in the tumor protein p53 pathway, the Wnt signaling pathway, glucose metabolism, endoplasmic reticulum stress, cytoskeletal regulation and DNA repair. These results indicate that XIAP may have a number of biological functions and may provide an insight into the biomedical significance of XIAP overexpression in RCC.

Characteristics of the formation of enzyme-bound ATP from medium inorganic phosphate by mitochondrial F1 adenosinetriphosphatase in the presence of dimethyl sulfoxide

International Nuclear Information System (INIS)

Kandpal, R.P.; Stempel, K.E.; Boyer, P.D.

1987-01-01

Addition of dimethyl sulfoxide promotes the formation of enzyme-bound ATP from medium P/sub i/ by mitochondrial F 1 adenosinetriphosphatase that has tightly bound ADP present. Measurements are reported of medium P/sub i/ ↔ H 18 OH exchange and of the dependence of formation of enzyme-bound ATP on P/sub i/ concentration. Attainment of an apparent equilibrium between medium P/sub i/ and bound ATP requires longer than 30 min, even though the rates of P/sub i/ binding and release after apparent equilibrium is reached would suffice for a faster approach to equilibrium. Slow protein conformational changes or other unknown modulating factors may be responsible for the slow rate of bound ATP formation. After apparent equilibrium is reached, each P/sub i/ that binds to the enzyme reversibly forms ATP about 50 times before being released to the medium. The rate of interconversion of bound ATP to bound ADP and P/sub i/ is much slower than that in the absence of dimethyl sulfoxide as measured with sufficient low ATP concentrations so that single-site catalysis is favored. Although the interconversion rate is slowed, the equilibrium constant for bound ATP formation from bound ADP and P/sub i/ is not far from unity. Dimethyl sulfoxide favors the formation of enzyme-bound ATP by promoting the competent binding of P/sub i/ to enzyme with ADP bound at a catalytic site rather than by promoting formation of bound ATP from bound ADP and P/sub i/

REDOR NMR Reveals Multiple Conformers for a Protein Kinase C Ligand in a Membrane Environment

Directory of Open Access Journals (Sweden)

Hao Yang

2018-01-01

Full Text Available Bryostatin 1 (henceforth bryostatin is in clinical trials for the treatment of Alzheimer’s disease and for HIV/AIDS eradication. It is also a preclinical lead for cancer immunotherapy and other therapeutic indications. Yet nothing is known about the conformation of bryostatin bound to its protein kinase C (PKC target in a membrane microenvironment. As a result, efforts to design more efficacious, better tolerated, or more synthetically accessible ligands have been limited to structures that do not include PKC or membrane effects known to influence PKC–ligand binding. This problem extends more generally to many membrane-associated proteins in the human proteome. Here, we use rotational-echo double-resonance (REDOR solid-state NMR to determine the conformations of PKC modulators bound to the PKCδ-C1b domain in the presence of phospholipid vesicles. The conformationally limited PKC modulator phorbol diacetate (PDAc is used as an initial test substrate. While unanticipated partitioning of PDAc between an immobilized protein-bound state and a mobile state in the phospholipid assembly was observed, a single conformation in the bound state was identified. In striking contrast, a bryostatin analogue (bryolog was found to exist exclusively in a protein-bound state, but adopts a distribution of conformations as defined by three independent distance measurements. The detection of multiple PKCδ-C1b-bound bryolog conformers in a functionally relevant phospholipid complex reveals the inherent dynamic nature of cellular systems that is not captured with single-conformation static structures. These results indicate that binding, selectivity, and function of PKC modulators, as well as the design of new modulators, are best addressed using a dynamic multistate model, an analysis potentially applicable to other membrane-associated proteins.

Lower bounds for the minimum distance of algebraic geometry codes

DEFF Research Database (Denmark)

Beelen, Peter

, such as the Goppa bound, the Feng-Rao bound and the Kirfel-Pellikaan bound. I will finish my talk by giving several examples. Especially for two-point codes, the generalized order bound is fairly easy to compute. As an illustration, I will indicate how a lower bound can be obtained for the minimum distance of some...... description of these codes in terms of order domains has been found. In my talk I will indicate how one can use the ideas behind the order bound to obtain a lower bound for the minimum distance of any AG-code. After this I will compare this generalized order bound with other known lower bounds...

Thermal unfolding of a Ca- and Lanthanide-binding protein

Energy Technology Data Exchange (ETDEWEB)

Fahmy, Karim [Helmholtz-Zentrum Dresden-Rossendorf e.V., Dresden (Germany). Biophysics; Goettfert, M. [Technische Univ. Dresden (Germany); Knoeppel, J.

2017-06-01

The MIIA (metal ion-induced autocleavage)-domain of the protein Vic001052 from the pathogen Vibrio coralliilyticus, comprises 173 amino acids and exhibits Ca-dependent autoproteolytic activity. It shows homology to nodulation proteins which are secreted by Rhizobiacea into plant host cells where they exert Ca-dependent functions. We have studied the structural and energetic aspects of metal protein interactions of the MIIA domain which appear attractive for engineering metal-binding synthetic peptides. Using a non-cleavable MIIA domain construct, we detected very similar structural changes upon binding to Ca{sup 2+} and Eu{sup 3+}. The thermal denaturation of the Ca-bound state was studied by circular dichroism spectroscopy. The metal-bound folded state unfolds reversibly into an unstructured metal-free state similar to the metal-free state at room temperature.

Aqueous Oxidative Heck Reaction as a Protein-Labeling Strategy

NARCIS (Netherlands)

Ourailidou, Marilena; van der Meer, Jan-Ytzen; Baas, Bert-Jan; Jeronimus-Stratingh, Catherine; Gottumukkala, Aditya L.; Poelarends, Gerrit J.; Minnaard, Adriaan J.; Dekker, Frans

2014-01-01

An increasing number of chemical reactions are being employed for bio-orthogonal ligation of detection labels to protein-bound functional groups. Several of these strategies, however, are limited in their application to pure proteins and are ineffective in complex biological samples such as cell

Polyamine binding to proteins in oat and Petunia protoplasts

Science.gov (United States)

Mizrahi, Y.; Applewhite, P. B.; Galston, A. W.

1989-01-01

Previous work (A Apelbaum et al. [1988] Plant Physiol 88: 996-998) has demonstrated binding of labeled spermidine (Spd) to a developmentally regulated 18 kilodalton protein in tobacco tissue cultures derived from thin surface layer explants. To assess the general importance of such Spd-protein complexes, we attempted bulk isolation from protoplasts of Petunia and oat (Avena sativa). In Petunia, as in tobacco, fed radioactive Spd is bound to protein, but in oat, Spd is first converted to 1,3,-diaminopropane (DAP), probably by polyamine oxidase action. In oat, binding of DAP to protein depends on age of donor leaf and conditions of illumination and temperature, and the extraction of the DAP-protein complex depends upon buffer and pH. The yield of the DAP-protein complex was maximized by extraction of frozen-thawed protoplasts with a pH 8.8 carbonate buffer containing SDS. Its molecular size, based on Sephacryl column fractionation of ammonium sulfate precipitated material, exceeded 45 kilodaltons. Bound Spd or DAP can be released from their complexes by the action of Pronase, but not DNAse, RNAse, or strong salt solutions, indicating covalent attachment to protein.

Dimerization in the Grb7 Protein

OpenAIRE

Peterson, Tabitha A.; Benallie, Renee L.; Bradford, Andrew M.; Pias, Sally C.; Yazzie, Jaron.; Lor, Siamee N.; Haulsee, Zachary M.; Park, Chad K.; Johnson, Dennis L.; Rohrschneider, Larry R.; Spuches, Anne.; Lyons, Barbara A.

2012-01-01

In previous studies, we showed that the tyrosine phosphorylation state of growth factor receptor–bound protein 7 (Grb7) affects its ability to bind to the transcription regulator FHL2 and the cortactin-interacting protein, human HS-1-associated protein-1. Here, we present results describing the importance of dimerization in the Grb7–Src homology 2 (SH2) domain in terms of its structural integrity and the ability to bind phosphorylated tyrosine peptide ligands. A tyrosine phosphorylation-mimic...

Absolute Lower Bound on the Bounce Action

Science.gov (United States)

Sato, Ryosuke; Takimoto, Masahiro

2018-03-01

The decay rate of a false vacuum is determined by the minimal action solution of the tunneling field: bounce. In this Letter, we focus on models with scalar fields which have a canonical kinetic term in N (>2 ) dimensional Euclidean space, and derive an absolute lower bound on the bounce action. In the case of four-dimensional space, we show the bounce action is generically larger than 24 /λcr, where λcr≡max [-4 V (ϕ )/|ϕ |4] with the false vacuum being at ϕ =0 and V (0 )=0 . We derive this bound on the bounce action without solving the equation of motion explicitly. Our bound is derived by a quite simple discussion, and it provides useful information even if it is difficult to obtain the explicit form of the bounce solution. Our bound offers a sufficient condition for the stability of a false vacuum, and it is useful as a quick check on the vacuum stability for given models. Our bound can be applied to a broad class of scalar potential with any number of scalar fields. We also discuss a necessary condition for the bounce action taking a value close to this lower bound.

Evacuation of Bed-bound Patients-STEPS Simulations

DEFF Research Database (Denmark)

Madsen, Anne; Dederichs, Anne Simone

2016-01-01

Fires in hospitals occur, and evacuation of bed-bound patients might be necessary in case of emergency. The current study concerns the evacuation of bed-bound patients from a fire section in a hospital using hospital porters. The simulations are performed using the STEPS program. The aim...... of the study is to investigate the evacuation time of bed-bound hospital patients using different walking speeds from the literature, and the influence of the number of hospital porters on the total evacuation times of bed-bound patients. Different scenarios were carried out with varying staff......-to-patient ratios that simulate the horizontal evacuation of 40 bed-bound patients into a different fire section. It was found that the staff-to-patient-ratio affects the total evacuation times. However, the total evacuation times do not decrease linearly and a saturation effect is seen at a staff-to-patient ratio...

Can infrared spectroscopy provide information on protein-protein interactions?

Science.gov (United States)

Haris, Parvez I

2010-08-01

For most biophysical techniques, characterization of protein-protein interactions is challenging; this is especially true with methods that rely on a physical phenomenon that is common to both of the interacting proteins. Thus, for example, in IR spectroscopy, the carbonyl vibration (1600-1700 cm(-1)) associated with the amide bonds from both of the interacting proteins will overlap extensively, making the interpretation of spectral changes very complicated. Isotope-edited infrared spectroscopy, where one of the interacting proteins is uniformly labelled with (13)C or (13)C,(15)N has been introduced as a solution to this problem, enabling the study of protein-protein interactions using IR spectroscopy. The large shift of the amide I band (approx. 45 cm(-1) towards lower frequency) upon (13)C labelling of one of the proteins reveals the amide I band of the unlabelled protein, enabling it to be used as a probe for monitoring conformational changes. With site-specific isotopic labelling, structural resolution at the level of individual amino acid residues can be achieved. Furthermore, the ability to record IR spectra of proteins in diverse environments means that isotope-edited IR spectroscopy can be used to structurally characterize difficult systems such as protein-protein complexes bound to membranes or large insoluble peptide/protein aggregates. In the present article, examples of application of isotope-edited IR spectroscopy for studying protein-protein interactions are provided.

Protein kinase C interaction with calcium: a phospholipid-dependent process.

LENUS (Irish Health Repository)

Bazzi, M D

1990-08-21

The calcium-binding properties of calcium- and phospholipid-dependent protein kinase C (PKC) were investigated by equilibrium dialysis in the presence and the absence of phospholipids. Calcium binding to PKC displayed striking and unexpected behavior; the free proteins bound virtually no calcium at intracellular calcium concentrations and bound limited calcium (about 1 mol\\/mol of PKC) at 200 microM calcium. However, in the presence of membranes containing acidic phospholipids, PKC bound at least eight calcium ions per protein. The presence of 1 microM phorbol dibutyrate (PDBu) in the dialysis buffer had little effect on these calcium-binding properties. Analysis of PKC-calcium binding by gel filtration under equilibrium conditions gave similar results; only membrane-associated PKC bound significant amounts of calcium. Consequently, PKC is a member of what may be a large group of proteins that bind calcium in a phospholipid-dependent manner. The calcium concentrations needed to induce PKC-membrane binding were similar to those needed for calcium binding (about 40 microM calcium at the midpoint). However, the calcium concentration required for PKC-membrane binding was strongly influenced by the phosphatidylserine composition of the membranes. Membranes with higher percentages of phosphatidylserine required lower concentrations of calcium. These properties suggested that the calcium sites may be generated at the interface between PKC and the membrane. Calcium may function as a bridge between PKC and phospholipids. These studies also suggested that calcium-dependent PKC-membrane binding and PKC function could be regulated by a number of factors in addition to calcium levels and diacylglycerol content of the membrane.

Bounds in the location-allocation problem

DEFF Research Database (Denmark)

Juel, Henrik

1981-01-01

Develops a family of stronger lower bounds on the objective function value of the location-allocation problem. Solution methods proposed to solve problems in location-allocation; Efforts to develop a more efficient bound solution procedure; Determination of the locations of the sources....

No-arbitrage bounds for financial scenarios

DEFF Research Database (Denmark)

Geyer, Alois; Hanke, Michael; Weissensteiner, Alex

2014-01-01

We derive no-arbitrage bounds for expected excess returns to generate scenarios used in financial applications. The bounds allow to distinguish three regions: one where arbitrage opportunities will never exist, a second where arbitrage may be present, and a third, where arbitrage opportunities...

Establishment of a protein frequency library and its application in the reliable identification of specific protein interaction partners.

Science.gov (United States)

Boulon, Séverine; Ahmad, Yasmeen; Trinkle-Mulcahy, Laura; Verheggen, Céline; Cobley, Andy; Gregor, Peter; Bertrand, Edouard; Whitehorn, Mark; Lamond, Angus I

2010-05-01

The reliable identification of protein interaction partners and how such interactions change in response to physiological or pathological perturbations is a key goal in most areas of cell biology. Stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry has been shown to provide a powerful strategy for characterizing protein complexes and identifying specific interactions. Here, we show how SILAC can be combined with computational methods drawn from the business intelligence field for multidimensional data analysis to improve the discrimination between specific and nonspecific protein associations and to analyze dynamic protein complexes. A strategy is shown for developing a protein frequency library (PFL) that improves on previous use of static "bead proteomes." The PFL annotates the frequency of detection in co-immunoprecipitation and pulldown experiments for all proteins in the human proteome. It can provide a flexible and objective filter for discriminating between contaminants and specifically bound proteins and can be used to normalize data values and facilitate comparisons between data obtained in separate experiments. The PFL is a dynamic tool that can be filtered for specific experimental parameters to generate a customized library. It will be continuously updated as data from each new experiment are added to the library, thereby progressively enhancing its utility. The application of the PFL to pulldown experiments is especially helpful in identifying either lower abundance or less tightly bound specific components of protein complexes that are otherwise lost among the large, nonspecific background.

Architecture-independent power bound for vibration energy harvesters

International Nuclear Information System (INIS)

Halvorsen, E; Le, C P; Mitcheson, P D; Yeatman, E M

2013-01-01

The maximum output power of energy harvesters driven by harmonic vibrations is well known for a range of specific harvester architectures. An architecture-independent bound based on the mechanical input-power also exists and gives a strict limit on achievable power with one mechanical degree of freedom, but is a least upper bound only for lossless devices. We report a new theoretical bound on the output power of vibration energy harvesters that includes parasitic, linear mechanical damping while still being architecture independent. This bound greatly improves the previous bound at moderate force amplitudes and is compared to the performance of established harvester architectures which are shown to agree with it in limiting cases. The bound is a hard limit on achievable power with one mechanical degree of freedom and can not be circumvented by transducer or power-electronic-interface design

Adjustable chain trees for proteins

DEFF Research Database (Denmark)

Winter, Pawel; Fonseca, Rasmus

2012-01-01

A chain tree is a data structure for changing protein conformations. It enables very fast detection of clashes and free energy potential calculations. A modified version of chain trees that adjust themselves to the changing conformations of folding proteins is introduced. This results in much...... tighter bounding volume hierarchies and therefore fewer intersection checks. Computational results indicate that the efficiency of the adjustable chain trees is significantly improved compared to the traditional chain trees....

Uniform bounds for Black--Scholes implied volatility

OpenAIRE

Tehranchi, Michael R.

2015-01-01

In this note, Black--Scholes implied volatility is expressed in terms of various optimisation problems. From these representations, upper and lower bounds are derived which hold uniformly across moneyness and call price. Various symmetries of the Black--Scholes formula are exploited to derive new bounds from old. These bounds are used to reprove asymptotic formulae for implied volatility at extreme strikes and/or maturities.

Simulation bounds for system availability

International Nuclear Information System (INIS)

Tietjen, G.L.; Waller, R.A.

1976-01-01

System availability is a dominant factor in the practicality of nuclear power electrical generating plants. A proposed model for obtaining either lower bounds or interval estimates on availability uses observed data on ''n'' failure-to-repair cycles of the system to estimate the parameters in the time-to-failure and time-to-repair models. These estimates are then used in simulating failure/repair cycles of the system. The availability estimate is obtained for each of 5000 samples of ''n'' failure/repair cycles to form a distribution of estimates. Specific percentile points of those simulated distributions are selected as lower simulation bounds or simulation interval bounds for the system availability. The method is illustrated with operational data from two nuclear plants for which an exponential time-to-failure and a lognormal time-to-repair are assumed

Identification of Proteins Bound to Dengue Viral RNA In Vivo Reveals New Host Proteins Important for Virus Replication

Directory of Open Access Journals (Sweden)

Stacia L. Phillips

2016-01-01

Full Text Available Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Due to the limited coding capacity of the viral genome and the complexity of the viral life cycle, host cell proteins play essential roles throughout the course of viral infection. Host RNA-binding proteins mediate various aspects of virus replication through their physical interactions with viral RNA. Here we describe a technique designed to identify such interactions in the context of infected cells using UV cross-linking followed by antisense-mediated affinity purification and mass spectrometry. Using this approach, we identified interactions, several of them novel, between host proteins and dengue viral RNA in infected Huh7 cells. Most of these interactions were subsequently validated using RNA immunoprecipitation. Using small interfering RNA (siRNA-mediated gene silencing, we showed that more than half of these host proteins are likely involved in regulating virus replication, demonstrating the utility of this method in identifying biologically relevant interactions that may not be identified using traditional in vitro approaches.

Complement factor H-related proteins CFHR2 and CFHR5 represent novel ligands for the infection-associated CRASP proteins of Borrelia burgdorferi.

Directory of Open Access Journals (Sweden)

Corinna Siegel

2010-10-01

Full Text Available One virulence property of Borrelia burgdorferi is its resistance to innate immunity, in particular to complement-mediated killing. Serum-resistant B. burgdorferi express up to five distinct complement regulator-acquiring surface proteins (CRASP which interact with complement regulator factor H (CFH and factor H-like protein 1 (FHL1 or factor H-related protein 1 (CFHR1. In the present study we elucidate the role of the infection-associated CRASP-3 and CRASP-5 protein to serve as ligands for additional complement regulatory proteins as well as for complement resistance of B. burgdorferi.To elucidate whether CRASP-5 and CRASP-3 interact with various human proteins, both borrelial proteins were immobilized on magnetic beads. Following incubation with human serum, bound proteins were eluted and separated by Glycine-SDS-PAGE. In addition to CFH and CFHR1, complement regulators CFHR2 and CFHR5 were identified as novel ligands for both borrelial proteins by employing MALDI-TOF. To further assess the contributions of CRASP-3 and CRASP-5 to complement resistance, a serum-sensitive B. garinii strain G1 which lacks all CFH-binding proteins was used as a valuable model for functional analyses. Both CRASPs expressed on the B. garinii outer surface bound CFH as well as CFHR1 and CFHR2 in ELISA. In contrast, live B. garinii bound CFHR1, CFHR2, and CFHR5 and only miniscute amounts of CFH as demonstrated by serum adsorption assays and FACS analyses. Further functional analysis revealed that upon NHS incubation, CRASP-3 or CRASP-5 expressing borreliae were killed by complement.In the absence of CFH and the presence of CFHR1, CFHR2 and CFHR5, assembly and integration of the membrane attack complex was not efficiently inhibited indicating that CFH in co-operation with CFHR1, CFHR2 and CFHR5 supports complement evasion of B. burgdorferi.

Role of protein disulfide isomerase and other thiol-reactive proteins in HIV-1 envelope protein-mediated fusion

International Nuclear Information System (INIS)

Ou Wu; Silver, Jonathan

2006-01-01

Cell-surface protein disulfide isomerase (PDI) has been proposed to promote disulfide bond rearrangements in HIV-1 envelope protein (Env) that accompany Env-mediated fusion. We evaluated the role of PDI in ways that have not been previously tested by downregulating PDI with siRNA and by overexpressing wild-type or variant forms of PDI in transiently and stably transfected cells. These manipulations, as well as treatment with anti-PDI antibodies, had only small effects on infection or cell fusion mediated by NL4-3 or AD8 strains of HIV-1. However, the cell-surface thiol-reactive reagent 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) had a much stronger inhibitory effect in our system, suggesting that cell-surface thiol-containing molecules other than PDI, acting alone or in concert, have a greater effect than PDI on HIV-1 Env-mediated fusion. We evaluated one such candidate, thioredoxin, a PDI family member reported to reduce a labile disulfide bond in CD4. We found that the ability of thioredoxin to reduce the disulfide bond in CD4 is enhanced in the presence of HIV-1 Env gp120 and that thioredoxin also reduces disulfide bonds in gp120 directly in the absence of CD4. We discuss the implications of these observations for identification of molecules involved in disulfide rearrangements in Env during fusion

Rigid Residue Scan Simulations Systematically Reveal Residue Entropic Roles in Protein Allostery.

Directory of Open Access Journals (Sweden)

Robert Kalescky

2016-04-01

Full Text Available Intra-protein information is transmitted over distances via allosteric processes. This ubiquitous protein process allows for protein function changes due to ligand binding events. Understanding protein allostery is essential to understanding protein functions. In this study, allostery in the second PDZ domain (PDZ2 in the human PTP1E protein is examined as model system to advance a recently developed rigid residue scan method combining with configurational entropy calculation and principal component analysis. The contributions from individual residues to whole-protein dynamics and allostery were systematically assessed via rigid body simulations of both unbound and ligand-bound states of the protein. The entropic contributions of individual residues to whole-protein dynamics were evaluated based on covariance-based correlation analysis of all simulations. The changes of overall protein entropy when individual residues being held rigid support that the rigidity/flexibility equilibrium in protein structure is governed by the La Châtelier's principle of chemical equilibrium. Key residues of PDZ2 allostery were identified with good agreement with NMR studies of the same protein bound to the same peptide. On the other hand, the change of entropic contribution from each residue upon perturbation revealed intrinsic differences among all the residues. The quasi-harmonic and principal component analyses of simulations without rigid residue perturbation showed a coherent allosteric mode from unbound and bound states, respectively. The projection of simulations with rigid residue perturbation onto coherent allosteric modes demonstrated the intrinsic shifting of ensemble distributions supporting the population-shift theory of protein allostery. Overall, the study presented here provides a robust and systematic approach to estimate the contribution of individual residue internal motion to overall protein dynamics and allostery.

Bounds on fluid permeability for viscous flow through porous media

International Nuclear Information System (INIS)

Berryman, J.G.

1985-01-01

General properties of variational bounds on Darcy's constant for slow viscous flow through porous media are studied. The bounds are also evaluated numerically for the penetrable sphere model. The bound of Doi depending on two-point correlations and the analytical bound of Weissberg and Prager give comparable results in the low density limit but the analytical bound is superior for higher densities. Prager's bound depending on three-point correlation functions is worse than the analytical bound at low densities but better (although comparable to it) at high densities. A procedure for methodically improving Prager's three point bound is presented. By introducing a Gaussian trial function, the three-point bound is improved by an order of magnitude for moderate values of porosity. The new bounds are comparable in magnitude to the Kozeny--Carman empirical relation for porous materials

Structure and DNA-binding of meiosis-specific protein Hop2

Science.gov (United States)

Zhou, Donghua; Moktan, Hem; Pezza, Roberto

2014-03-01

Here we report structure elucidation of the DNA binding domain of homologous pairing protein 2 (Hop2), which is important to gene diversity when sperms and eggs are produced. Together with another protein Mnd1, Hop2 enhances the strand invasion activity of recombinase Dmc1 by over 30 times, facilitating proper synapsis of homologous chromosomes. However, the structural and biochemical bases for the function of Hop2 and Mnd1 have not been well understood. As a first step toward such understanding, we recently solved the structure for the N-terminus of Hop2 (1-84) using solution NMR. This fragment shows a typical winged-head conformation with recognized DNA binding activity. DNA interacting sites were then investigated by chemical shift perturbations in a titration experiment. Information of these sites was used to guide protein-DNA docking with MD simulation, revealing that helix 3 is stably lodged in the DNA major groove and that wing 1 (connecting strands 2 and 3) transiently comes in contact with the minor groove in nanosecond time scale. Mutagenesis analysis further confirmed the DNA binding sites in this fragment of the protein.

Lower bound on inconclusive probability of unambiguous discrimination

International Nuclear Information System (INIS)

Feng Yuan; Zhang Shengyu; Duan Runyao; Ying Mingsheng

2002-01-01

We derive a lower bound on the inconclusive probability of unambiguous discrimination among n linearly independent quantum states by using the constraint of no signaling. It improves the bound presented in the paper of Zhang, Feng, Sun, and Ying [Phys. Rev. A 64, 062103 (2001)], and when the optimal discrimination can be reached, these two bounds coincide with each other. An alternative method of constructing an appropriate measurement to prove the lower bound is also presented

Models and Techniques for Proving Data Structure Lower Bounds

DEFF Research Database (Denmark)

Larsen, Kasper Green

In this dissertation, we present a number of new techniques and tools for proving lower bounds on the operational time of data structures. These techniques provide new lines of attack for proving lower bounds in both the cell probe model, the group model, the pointer machine model and the I...... bound of tutq = (lgd􀀀1 n). For ball range searching, we get a lower bound of tutq = (n1􀀀1=d). The highest previous lower bound proved in the group model does not exceed ((lg n= lg lg n)2) on the maximum of tu and tq. Finally, we present a new technique for proving lower bounds....../O-model. In all cases, we push the frontiers further by proving lower bounds higher than what could possibly be proved using previously known techniques. For the cell probe model, our results have the following consequences: The rst (lg n) query time lower bound for linear space static data structures...

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

Energy Technology Data Exchange (ETDEWEB)

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

1989-09-01

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

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

International Nuclear Information System (INIS)

Johnson, J.L.; Rajagopalan, K.V.; London, R.E.

1989-01-01

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

Novel Biosensor of Membrane Protein Proximity Based on Fluorogen Activated Proteins.

Science.gov (United States)

Vasilev, Kalin V; Gallo, Eugenio; Shank, Nathaniel; Jarvik, Jonathan W

2016-01-01

We describe a novel biosensor system for reporting proximity between cell surface proteins in live cultured cells. The biosensor takes advantage of recently developed fluorogen-activating proteins (FAPs) that display fluorescence only when bound to otherwise-nonfluorescent fluorogen molecules. To demonstrate feasibility for the approach, two recombinant rapamycin-binding proteins were expressed as single-pass plasma membrane proteins in HeLa cells; one of the proteins (scAvd- FRB) carried an extracellular avidin tag; the other (HL1-TO1-FKBP) carried an extracellular FAP. Cells were incubated with a membrane-impermeable bivalent ligand (biotin-PEG2000-DIR) consisting of biotin joined to a dimethyl-indole red (DIR) fluorogen by a polyethylene glycol linker, thus tethering the fluorogen to the scAvd-FRB fusion protein. Addition of rapamycin, which promotes FKBP-FRB dimerization and thereby brings the FAP in close proximity to the tethered fluorogen, led to a significant increase in DIR fluorescence. We call the new proximity assay TEFLA, for tethered fluorogen assay.

The predominant circular form of avocado sunblotch viroid accumulates in planta as a free RNA adopting a rod-shaped secondary structure unprotected by tightly bound host proteins.

Science.gov (United States)

López-Carrasco, Amparo; Flores, Ricardo

2017-07-01

Avocado sunblotch viroid (ASBVd), the type member of the family Avsunviroidae, replicates and accumulates in chloroplasts. Whether this minimal non-protein-coding circular RNA of 246-250 nt exists in vivo as a free nucleic acid or closely associated with host proteins remains unknown. To tackle this issue, the secondary structures of the monomeric circular (mc) (+) and (-) strands of ASBVd have been examined in silico by searching those of minimal free energy, and in vitro at single-nucleotide resolution by selective 2'-hydroxyl acylation analysed by primer extension (SHAPE). Both approaches resulted in predominant rod-like secondary structures without tertiary interactions, with the mc (+) RNA being more compact than its (-) counterpart as revealed by non-denaturing polyacryamide gel electrophoresis. Moreover, in vivo SHAPE showed that the mc ASBVd (+) form accumulates in avocado leaves as a free RNA adopting a similar rod-shaped conformation unprotected by tightly bound host proteins. Hence, the mc ASBVd (+) RNA behaves in planta like the previously studied mc (+) RNA of potato spindle tuber viroid, the type member of nuclear viroids (family Pospiviroidae), indicating that two different viroids replicating and accumulating in distinct subcellular compartments, have converged into a common structural solution. Circularity and compact secondary structures confer to these RNAs, and probably to all viroids, the intrinsic stability needed to survive in their natural habitats. However, in vivo SHAPE has not revealed the (possibly transient or loose) interactions of the mc ASBVd (+) RNA with two host proteins observed previously by UV irradiation of infected avocado leaves.

Stacked spheres and lower bound theorem

Indian Academy of Sciences (India)

BASUDEB DATTA

2011-11-20

Nov 20, 2011 ... Preliminaries. Lower bound theorem. On going work. Definitions. An n-simplex is a convex hull of n + 1 affinely independent points. (called vertices) in some Euclidean space R. N . Stacked spheres and lower bound theorem. Basudeb Datta. Indian Institute of Science. 2 / 27 ...

Exponential Lower Bounds For Policy Iteration

OpenAIRE

Fearnley, John

2010-01-01

We study policy iteration for infinite-horizon Markov decision processes. It has recently been shown policy iteration style algorithms have exponential lower bounds in a two player game setting. We extend these lower bounds to Markov decision processes with the total reward and average-reward optimality criteria.

An upper bound on Q-star masses

International Nuclear Information System (INIS)

Hochron, D.R.; Selipsky, S.B.

1992-06-01

Q-stars (the gravitational generalization of Q-balls, strongly bound bulk matter that an appear in field theories of strongly interacting hadrons) are the only known impact objects consistent with the known bulk structure of nuclei and chiral symmetry that evade the Rhoades-Ruffini upper bound of 3.2M circle-dot . Generic bounds are quite weak: M Q-star circle-dot . If, however, we assume that the 1.558 ms pulsar is a Q-star, equilibrium. A stability criteria of rotating fluids place a much stronger upper bound of M c ≤ 5.3M circle-dot on such models under certain special assumptions. This has important implications for heavy compact objects such as Cygnus X-1

Preference of small molecules for local minimum conformations when binding to proteins.

Directory of Open Access Journals (Sweden)

Qi Wang

2007-09-01

Full Text Available It is well known that small molecules (ligands do not necessarily adopt their lowest potential energy conformations when binding to proteins. Analyses of protein-bound ligand crystal structures have reportedly shown that many of them do not even adopt the conformations at local minima of their potential energy surfaces (local minimum conformations. The results of these analyses raise a concern regarding the validity of virtual screening methods that use ligands in local minimum conformations. Here we report a normal-mode-analysis (NMA study of 100 crystal structures of protein-bound ligands. Our data show that the energy minimization of a ligand alone does not automatically stop at a local minimum conformation if the minimum of the potential energy surface is shallow, thus leading to the folding of the ligand. Furthermore, our data show that all 100 ligand conformations in their protein-bound ligand crystal structures are nearly identical to their local minimum conformations obtained from NMA-monitored energy minimization, suggesting that ligands prefer to adopt local minimum conformations when binding to proteins. These results both support virtual screening methods that use ligands in local minimum conformations and caution about possible adverse effect of excessive energy minimization when generating a database of ligand conformations for virtual screening.

Upper bounds for reversible circuits based on Young subgroups

DEFF Research Database (Denmark)

Abdessaied, Nabila; Soeken, Mathias; Thomsen, Michael Kirkedal

2014-01-01

We present tighter upper bounds on the number of Toffoli gates needed in reversible circuits. Both multiple controlled Toffoli gates and mixed polarity Toffoli gates have been considered for this purpose. The calculation of the bounds is based on a synthesis approach based on Young subgroups...... that results in circuits using a more generalized gate library. Starting from an upper bound for this library we derive new bounds which improve the existing bound by around 77%....

Differential plasma protein binding to metal oxide nanoparticles

International Nuclear Information System (INIS)

Deng, Zhou J; Mortimer, Gysell; Minchin, Rodney F; Schiller, Tara; Musumeci, Anthony; Martin, Darren

2009-01-01

Nanoparticles rapidly interact with the proteins present in biological fluids, such as blood. The proteins that are adsorbed onto the surface potentially dictate the biokinetics of the nanomaterials and their fate in vivo. Using nanoparticles with different sizes and surface characteristics, studies have reported the effects of physicochemical properties on the composition of adsorbed plasma proteins. However, to date, few studies have been conducted focusing on the nanoparticles that are commonly exposed to the general public, such as the metal oxides. Using previously established ultracentrifugation approaches, two-dimensional gel electrophoresis and mass spectrometry, the current study investigated the binding of human plasma proteins to commercially available titanium dioxide, silicon dioxide and zinc oxide nanoparticles. We found that, despite these particles having similar surface charges in buffer, they bound different plasma proteins. For TiO 2 , the shape of the nanoparticles was also an important determinant of protein binding. Agglomeration in water was observed for all of the nanoparticles and both TiO 2 and ZnO further agglomerated in biological media. This led to an increase in the amount and number of different proteins bound to these nanoparticles. Proteins with important biological functions were identified, including immunoglobulins, lipoproteins, acute-phase proteins and proteins involved in complement pathways and coagulation. These results provide important insights into which human plasma proteins bind to particular metal oxide nanoparticles. Because protein absorption to nanoparticles may determine their interaction with cells and tissues in vivo, understanding how and why plasma proteins are adsorbed to these particles may be important for understanding their biological responses.

Experimental investigation on a diode-pumped cesium-vapor laser stably operated at continuous-wave and pulse regime.

Science.gov (United States)

Chen, Fei; Xu, Dongdong; Gao, Fei; Zheng, Changbin; Zhang, Kuo; He, Yang; Wang, Chunrui; Guo, Jin

2015-05-04

Employing a fiber-coupled diode-laser with a center wavelength of 852.25 nm and a line width of 0.17 nm, experimental investigation on diode-end-pumped cesium (Cs) vapor laser stably operated at continuous-wave (CW) and pulse regime is carried out. A 5 mm long cesium vapor cell filled with 60 kPa helium and 20 kPa ethane is used as laser medium. Using an output coupler with reflectivity of 48.79%, 1.26 W 894.57 nm CW laser is obtained at an incident pump power of 4.76 W, corresponding an optical-optical efficiency of 26.8% and a slope-efficiency of 28.8%, respectively. The threshold temperature is 67.5 °C. Stable pulsed cesium laser with a maximum average output power of 2.6 W is obtained at a repetition rate of 76 Hz, and the pulse repetition rate can be extend to 1 kHz with a pulse width of 18 μs.

Tandem Affinity Purification Approach Coupled to Mass Spectrometry to Identify Post-translational Modifications of Histones Associated with Chromatin-Binding Proteins.

Science.gov (United States)

Beyer, Sophie; Robin, Philippe; Ait-Si-Ali, Slimane

2017-01-01

Protein purification by tandem affinity purification (TAP)-tag coupled to mass spectrometry analysis is usually used to reveal protein complex composition. Here we describe a TAP-tag purification of chromatin-bound proteins along with associated nucleosomes, which allow exhaustive identification of protein partners. Moreover, this method allows exhaustive identification of the post-translational modifications (PTMs) of the associated histones. Thus, in addition to partner characterization, this approach reveals the associated epigenetic landscape that can shed light on the function and properties of the studied chromatin-bound protein.

Bound residues of 14C-carbofuran and 14C-malathion in stored soybean seeds and their toxicological effects on mice

International Nuclear Information System (INIS)

Mahdy, F.; Taha, H.

2005-01-01

Soybeans treated with 1 4C -carbofuran or 1 4C -malathion at a dose of 30 mg/kg and stored for 30 weeks showed that the organophosphorus insecticide malathion had a higher binding tendency to soybean seeds than the carbamate insecticide carbofuran. The amount of malathion bound 1 4C -residues were almost three times higher (as percentage of applied dose) than carbofuran bound residues. Sub-chronic feeding experiments on mice for 90 days with a diet containing the bound 1 4C -residues at a dose of 0.91 and 2.39 ppm/day/mouse for carbofuran and malathion, respectively, caused non-significant symptoms of toxicity during the period of the experiment. A slight inhibition in plasma and erythrocyte choline esterase activities was observed during the second and third months in both treated mice fed on carbofuran or malathion. Liver enzymes, total protein, albumin, cholesterol, triglycerides and kidney functions showed non-significant differences from control values in case of mice fed on 1 4C -bound carbofuran residues. However, these mentioned blood parameters showed only slight increase, as compared with controls, in treated mice fed on 1 4C -bound malathion residues

Uniform Bounds for Black--Scholes Implied Volatility

OpenAIRE

Tehranchi, Michael Rummine

2016-01-01

In this note, Black--Scholes implied volatility is expressed in terms of various optimization problems. From these representations, upper and lower bounds are derived which hold uniformly across moneyness and call price. Various symmetries of the Black--Scholes formula are exploited to derive new bounds from old. These bounds are used to reprove asymptotic formulas for implied volatility at extreme strikes and/or maturities. the Society for Industrial and Applied Mathematics 10.1137/14095248X

Post-mortem re-cloning of a transgenic red fluorescent protein dog.

Science.gov (United States)

Hong, So Gun; Koo, Ok Jae; Oh, Hyun Ju; Park, Jung Eun; Kim, Minjung; Kim, Geon-A; Park, Eun Jung; Jang, Goo; Lee, Byeong-Chun

2011-12-01

Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification.

MapA, an iron-regulated, cytoplasmic membrane protein in the cyanobacterium Synechococcus sp. strain PCC7942.

Science.gov (United States)

Webb, R; Troyan, T; Sherman, D; Sherman, L A

1994-08-01

Growth of Synechococcus sp. strain PCC 7942 in iron-deficient media leads to the accumulation of an approximately 34-kDa protein. The gene encoding this protein, mapA (membrane-associated protein A), has been cloned and sequenced (GenBank accession number, L01621). The mapA transcript is not detectable in normally grown cultures but is stably accumulated by cells grown in iron-deficient media. However, the promoter sequence for this gene does not resemble other bacterial iron-regulated promoters described to date. The carboxyl-terminal region of the derived amino acid sequence of MapA resembles bacterial proteins involved in iron acquisition, whereas the amino-terminal end of MapA has a high degree of amino acid identity with the abundant, chloroplast envelope protein E37. An approach employing improved cellular fractionation techniques as well as electron microscopy and immunocytochemistry was essential in localizing MapA protein to the cytoplasmic membrane of Synechococcus sp. strain PCC 7942. When these cells were grown under iron-deficient conditions, a significant fraction of MapA could also be localized to the thylakoid membranes.

[Biological characteristics of a chimeric rabies virus expressing canine parvovirus VP2 protein].

Science.gov (United States)

Niu, Xue-Feng; Liu, Xiao-Hui; Sun, Zhao-Jin; Shi, He-He; Chen, Jing; Jiang, Bido; Sun, Jing-Chen; Guo, Xiao-Feng

2009-09-01

To obtain a bivalence vaccine against canine rabies virus and canine parvovirus, a chimeric rabies virus expressing canine parvovirus VP2 protein was generated by the technique of reverse genetics. It was shown that the chimeric virus designated as HEP-Flury (VP2) grew well on BHK-21 cells and the VP2 gene could still be stably expressed after ten passages on BHK-21 cells. Experiments on the mice immunized with the chimeric virus HEP-Flury (VP2) demonstrated that specific antibodies against rabies virus and canine parvovirus were induced in immunized mice after vaccination with the live chimeric virus.

Proximity effect tunneling into virtual bound state alloys

International Nuclear Information System (INIS)

Tang, I.M.; Roongkkeadsakoon, S.

1984-01-01

The effects of a narrow virtual bound state formed by transition metal impurities dissolved in the normal layer of a superconducting proximity effect sandwich are studied. Using standard renormalization techniques, we obtain the changes in the transition temperatures and the jumps in the specific heat at T/sub c/ as a function of the thickness of the normal layer, of the widths of the virtual bound states, and of the impurity concentrations. It is seen that narrow virtual bound states lead to decrease in the transition temperatures, while broad virtual bound states do not. It if further seen that the narrow virtual bound state causes the reduced specific heat jump at T/sub c/ to deviate from the BCS behavior expected of the pure sandwich

A strongly quasiconvex PAC-Bayesian bound

DEFF Research Database (Denmark)

Thiemann, Niklas; Igel, Christian; Wintenberger, Olivier

2017-01-01

We propose a new PAC-Bayesian bound and a way of constructing a hypothesis space, so that the bound is convex in the posterior distribution and also convex in a trade-off parameter between empirical performance of the posterior distribution and its complexity. The complexity is measured by the Ku...

DEAD-box helicase DDX27 regulates 3′ end formation of ribosomal 47S RNA and stably associates with the PeBoW-complex

Energy Technology Data Exchange (ETDEWEB)

Kellner, Markus; Rohrmoser, Michaela [Department of Molecular Epigenetics, Helmholtz Center Munich, Center for Integrated Protein Science Munich (CIPSM), Marchioninistr. 25, Munich 81377 (Germany); Forné, Ignasi [Adolf Butenandt Institute, Ludwig Maximilians University of Munich, Center for Integrated Protein Science Munich (CIPSM), Schillerstr. 44, Munich 80336 (Germany); Voss, Kirsten; Burger, Kaspar; Mühl, Bastian; Gruber-Eber, Anita [Department of Molecular Epigenetics, Helmholtz Center Munich, Center for Integrated Protein Science Munich (CIPSM), Marchioninistr. 25, Munich 81377 (Germany); Kremmer, Elisabeth [Institute of Molecular Immunology, Helmholtz Center Munich, Marchioninistr. 25, Munich 81377 (Germany); Imhof, Axel [Adolf Butenandt Institute, Ludwig Maximilians University of Munich, Center for Integrated Protein Science Munich (CIPSM), Schillerstr. 44, Munich 80336 (Germany); Eick, Dirk, E-mail: eick@helmholtz-muenchen.de [Department of Molecular Epigenetics, Helmholtz Center Munich, Center for Integrated Protein Science Munich (CIPSM), Marchioninistr. 25, Munich 81377 (Germany)

2015-05-15

PeBoW, a trimeric complex consisting of pescadillo (Pes1), block of proliferation (Bop1), and the WD repeat protein 12 (WDR12), is essential for processing and maturation of mammalian 5.8S and 28S ribosomal RNAs. Applying a mass spectrometric analysis, we identified the DEAD-box helicase DDX27 as stably associated factor of the PeBoW-complex. DDX27 interacts with the PeBoW-complex via an evolutionary conserved F×F motif in the N-terminal domain and is recruited to the nucleolus via its basic C-terminal domain. This recruitment is RNA-dependent and occurs independently of the PeBoW-complex. Interestingly, knockdown of DDX27, but not of Pes1, induces the accumulation of an extended form of the primary 47S rRNA. We conclude that DDX27 can interact specifically with the Pes1 and Bop1 but fulfils critical function(s) for proper 3′ end formation of 47S rRNA independently of the PeBoW-complex. - Highlights: • DEAD-box helicase DDX27 is a new constituent of the PeBoW-complex. • The N-terminal F×F motif of DDX27 interacts with the PeBoW components Pes1 and Bop1. • Nucleolar anchoring of DDX27 via its basic C-terminal domain is RNA dependent. • Knockdown of DDX27 induces a specific defect in 3′ end formation of 47S rRNA.

Sharp bounds for periodic solutions of Lipschitzian differential equations

International Nuclear Information System (INIS)

Zevin, A A

2009-01-01

A general system of Lipschitzian differential equations, containing simultaneously terms without delay and with arbitrary constant and time-varying delays, is considered. For the autonomous case, a lower bound for the period of nonconstant periodic solutions, expressed in the respective supremum Lipschitz constants, is found. For nonautonomous periodic equations, explicit upper bounds for the amplitudes and maximum derivatives of periodic solutions are obtained. For all n ≥ 2, the bounds do not depend on n and, in general, are different from that for n = 1. All the bounds are sharp; they are attained in linear differential equations with piece-wise constant deviating arguments. A relation between the obtained bounds and the sharp bounds in other metrics is established

Bounds for nonlinear composites via iterated homogenization

Science.gov (United States)

Ponte Castañeda, P.

2012-09-01

Improved estimates of the Hashin-Shtrikman-Willis type are generated for the class of nonlinear composites consisting of two well-ordered, isotropic phases distributed randomly with prescribed two-point correlations, as determined by the H-measure of the microstructure. For this purpose, a novel strategy for generating bounds has been developed utilizing iterated homogenization. The general idea is to make use of bounds that may be available for composite materials in the limit when the concentration of one of the phases (say phase 1) is small. It then follows from the theory of iterated homogenization that it is possible, under certain conditions, to obtain bounds for more general values of the concentration, by gradually adding small amounts of phase 1 in incremental fashion, and sequentially using the available dilute-concentration estimate, up to the final (finite) value of the concentration (of phase 1). Such an approach can also be useful when available bounds are expected to be tighter for certain ranges of the phase volume fractions. This is the case, for example, for the "linear comparison" bounds for porous viscoplastic materials, which are known to be comparatively tighter for large values of the porosity. In this case, the new bounds obtained by the above-mentioned "iterated" procedure can be shown to be much improved relative to the earlier "linear comparison" bounds, especially at low values of the porosity and high triaxialities. Consistent with the way in which they have been derived, the new estimates are, strictly, bounds only for the class of multi-scale, nonlinear composites consisting of two well-ordered, isotropic phases that are distributed with prescribed H-measure at each stage in the incremental process. However, given the facts that the H-measure of the sequential microstructures is conserved (so that the final microstructures can be shown to have the same H-measure), and that H-measures are insensitive to length scales, it is conjectured

Mitochondrial Band-7 family proteins: scaffolds for respiratory chain assembly?

Directory of Open Access Journals (Sweden)

Bernadette eGehl

2014-04-01

Full Text Available The band-7 protein family comprises a diverse set of membrane-bound proteins characterised by the presence of a conserved domain. The exact function of this band-7 domain remains elusive, but examples from animal and bacterial stomatin-type proteins demonstrate binding to lipids and the ability to assemble into membrane-bound oligomers that form putative scaffolds. Some members, such as prohibitins and human stomatin-like protein 2 (HsSLP2, localise to the mitochondrial inner membrane where they function in cristae formation and hyperfusion. In Arabidopsis, the band-7 protein family has diversified and includes plant-specific members. Mitochondrial-localised members include prohibitins (AtPHBs and two stomatin-like proteins (AtSLP1 and -2. Studies into PHB function in plants have demonstrated an involvement in root meristem proliferation and putative scaffold formation for mAAA proteases, but it remains unknown how these roles are achieved at the molecular level. In this minireview we summarise the current status of band-7 protein functions in Arabidopsis, and speculate how the mitochondrial members might recruit specific lipids to form microdomains that could shape the organisation and functioning of the respiratory chain.

Four-quark bound states

International Nuclear Information System (INIS)

Zouzou, S.

1986-01-01

In the framework of simple non-relativistic potential models, we examine the system consisting of two quarks and two antiquarks with equal or unequal masses. We search for possible bound states below the threshold for the spontaneous dissociation into two mesons. We solve the four body problem by empirical or systematic variational methods and we include the virtual meson-meson components of the wave function. With standard two-body potentials, there is no proliferation of multiquarks. With unequal quark masses, we obtain however exotic (anti Qanti Qqq) bound states with a baryonic antidiquark-quark-quark structure very analogous to the heavy flavoured (Q'qq) baryons. (orig.)

Binding of dicamba to soluble and bound extracellular polymeric substances (EPS) from aerobic activated sludge: a fluorescence quenching study.

Science.gov (United States)

Pan, Xiangliang; Liu, Jing; Zhang, Daoyong; Chen, Xi; Song, Wenjuan; Wu, Fengchang

2010-05-15

Binding of dicamba to soluble EPS (SEPS) and bound EPS (BEPS) from aerobic activated sludge was investigated using fluorescence spectroscopy. Two protein-like fluorescence peaks (peak A with Ex/Em=225 nm/342-344 nm and peak B with Ex/Em=275/340-344 nm) were identified in SEPS and BEPS. Humic-like fluorescence peak C (Ex/Em=270-275 nm/450-460 nm) was only found in BEPS. Fluorescence of the peaks A and B for SEPS and peak A for BEPS were markedly quenched by dicamba at all temperatures whereas fluorescence of peaks B and C for BEPS was quenched only at 298 K. A dynamic process dominated the fluorescence quenching of peak A of both SEPS and BEPS. Fluorescence quenching of peak B and C was governed a static process. The effective quenching constants (logK(a)) were 4.725-5.293 for protein-like fluorophores of SEPS and 4.23-5.190 for protein-like fluorophores of BEPS, respectively. LogK(a) for humic-like substances was 3.85. Generally, SEPS had greater binding capacity for dicamba than BEPS, and protein-like substances bound dicamba more strongly than humic-like substances. Binding of dicamba to SEPS and BEPS was spontaneous and exothermic. Electrostatic force and hydrophobic interaction forces play a crucial role in binding of dicamba to EPS. Copyright © 2010 Elsevier Inc. All rights reserved.

Thermalization Time Bounds for Pauli Stabilizer Hamiltonians

Science.gov (United States)

Temme, Kristan

2017-03-01

We prove a general lower bound to the spectral gap of the Davies generator for Hamiltonians that can be written as the sum of commuting Pauli operators. These Hamiltonians, defined on the Hilbert space of N-qubits, serve as one of the most frequently considered candidates for a self-correcting quantum memory. A spectral gap bound on the Davies generator establishes an upper limit on the life time of such a quantum memory and can be used to estimate the time until the system relaxes to thermal equilibrium when brought into contact with a thermal heat bath. The bound can be shown to behave as {λ ≥ O(N^{-1} exp(-2β overline{ɛ}))}, where {overline{ɛ}} is a generalization of the well known energy barrier for logical operators. Particularly in the low temperature regime we expect this bound to provide the correct asymptotic scaling of the gap with the system size up to a factor of N -1. Furthermore, we discuss conditions and provide scenarios where this factor can be removed and a constant lower bound can be proven.

Information-Theoretic Bounded Rationality and ε-Optimality

Directory of Open Access Journals (Sweden)

Daniel A. Braun

2014-08-01

Full Text Available Bounded rationality concerns the study of decision makers with limited information processing resources. Previously, the free energy difference functional has been suggested to model bounded rational decision making, as it provides a natural trade-off between an energy or utility function that is to be optimized and information processing costs that are measured by entropic search costs. The main question of this article is how the information-theoretic free energy model relates to simple ε-optimality models of bounded rational decision making, where the decision maker is satisfied with any action in an ε-neighborhood of the optimal utility. We find that the stochastic policies that optimize the free energy trade-off comply with the notion of ε-optimality. Moreover, this optimality criterion even holds when the environment is adversarial. We conclude that the study of bounded rationality based on ε-optimality criteria that abstract away from the particulars of the information processing constraints is compatible with the information-theoretic free energy model of bounded rationality.

CDE-1 affects chromosome segregation through uridylation of CSR-1-bound siRNAs.

Science.gov (United States)

van Wolfswinkel, Josien C; Claycomb, Julie M; Batista, Pedro J; Mello, Craig C; Berezikov, Eugene; Ketting, René F

2009-10-02

We have studied the function of a conserved germline-specific nucleotidyltransferase protein, CDE-1, in RNAi and chromosome segregation in C. elegans. CDE-1 localizes specifically to mitotic chromosomes in embryos. This localization requires the RdRP EGO-1, which physically interacts with CDE-1, and the Argonaute protein CSR-1. We found that CDE-1 is required for the uridylation of CSR-1 bound siRNAs, and that in the absence of CDE-1 these siRNAs accumulate to inappropriate levels, accompanied by defects in both meiotic and mitotic chromosome segregation. Elevated siRNA levels are associated with erroneous gene silencing, most likely through the inappropriate loading of CSR-1 siRNAs into other Argonaute proteins. We propose a model in which CDE-1 restricts specific EGO-1-generated siRNAs to the CSR-1 mediated, chromosome associated RNAi pathway, thus separating it from other endogenous RNAi pathways. The conserved nature of CDE-1 suggests that similar sorting mechanisms may operate in other animals, including mammals.

Macro creatine kinases: results of isoenzyme electrophoresis and differentiation of the immunoglobulin-bound type by radioassay

International Nuclear Information System (INIS)

Bohner, J.; Stein, W.; Steinhart, R.; Wurzburg, U.; Eggstein, M.

1982-01-01

In 2.9% of sera from 1253 unselected patients we detected two different types of macromolecular creatine kinases (CK;EC 2.7.3.2). One macro type was represented by immunoglobulin-linked CK; in sera containing macro CK-BB isoenzyme, 125 I-labeled CK-BB was bound with high affinity to the immunoglobulin fraction. Furthermore, during electrophoresis, macro CK-BB mostly migrated between CK-MB and CK-MM, and was fixed to Protein A from Staphlococcus aureus. We therefore propose radioelectrophoresis as a specific, highly sensitive, and simple method for detecting this type of macro CK. This form occurs predominantly in elderly women, is not correlated to any specific disease, and persists in blood over a long period of time. In contrast, a second type (macro-CK type 2) never bound radiolabeled CK isoenzymes, and was not adsorbed to protein A. Electrophoretic migration of this macro-CK type 2 was generally cathodic to CK-MM. We observed this type in severely ill patients, frequently those suffering from malignant tumors. Clinical observations and biochemical data suggest that macro-CK type 2 is of mitochondrial origin

Structure, High Affinity, and Negative Cooperativity of the Escherichia coli Holo-(Acyl Carrier Protein):Holo-(Acyl Carrier Protein) Synthase Complex

Energy Technology Data Exchange (ETDEWEB)

Marcella, Aaron M.; Culbertson, Sannie J.; Shogren-Knaak, Michael A.; Barb, Adam W.

2017-11-01

The Escherichia coli holo-(acyl carrier protein) synthase (ACPS) catalyzes the coenzyme A-dependent activation of apo-ACPP to generate holo-(acyl carrier protein) (holo-ACPP) in an early step of fatty acid biosynthesis. E. coli ACPS is sufficiently different from the human fatty acid synthase to justify the development of novel ACPS-targeting antibiotics. Models of E. coli ACPS in unliganded and holo-ACPP-bound forms solved by X-ray crystallography to 2.05 and 4.10 Å, respectively, revealed that ACPS bound three product holo-ACPP molecules to form a 3:3 hexamer. Solution NMR spectroscopy experiments validated the ACPS binding interface on holo-ACPP using chemical shift perturbations and by determining the relative orientation of holo-ACPP to ACPS by fitting residual dipolar couplings. The binding interface is organized to arrange contacts between positively charged ACPS residues and the holo-ACPP phosphopantetheine moiety, indicating product contains more stabilizing interactions than expected in the enzyme:substrate complex. Indeed, holo-ACPP bound the enzyme with greater affinity than the substrate, apo-ACPP, and with negative cooperativity. The first equivalent of holo-ACPP bound with a KD = 62 ± 13 nM, followed by the binding of two more equivalents of holo-ACPP with KD = 1.2 ± 0.2 μM. Cooperativity was not observed for apo-ACPP which bound with KD = 2.4 ± 0.1 μM. Strong product binding and high levels of holo-ACPP in the cell identify a potential regulatory role of ACPS in fatty acid biosynthesis.

Copper carrier protein in copper toxic sheep liver

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