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.

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

Science.gov (United States)

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.

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

Recognition of higher order patterns in proteins: immunologic kernels.

Directory of Open Access Journals (Sweden)

Robert D Bremel

Full Text Available By applying analysis of the principal components of amino acid physical properties we predicted cathepsin cleavage sites, MHC binding affinity, and probability of B-cell epitope binding of peptides in tetanus toxin and in ten diverse additional proteins. Cross-correlation of these metrics, for peptides of all possible amino acid index positions, each evaluated in the context of a ±25 amino acid flanking region, indicated that there is a strongly repetitive pattern of short peptides of approximately thirty amino acids each bounded by cathepsin cleavage sites and each comprising B-cell linear epitopes, MHC-I and MHC-II binding peptides. Such "immunologic kernel" peptides comprise all signals necessary for adaptive immunologic cognition, response and recall. The patterns described indicate a higher order spatial integration that forms a symbolic logic coordinating the adaptive immune system.

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.

Protein patterning on polycrystalline silicon-germanium via standard UV lithography for bioMEMS applications

Energy Technology Data Exchange (ETDEWEB)

Lenci, S., E-mail: silvia.lenci@gmail.com [Dipartimento di Ingegneria dell' Informazione, University of Pisa, Via G. Caruso 16, I-56122 Pisa (Italy); imec, Kapeldreef 75, Leuven B-3001 (Belgium); Tedeschi, L.; Domenici, C.; Lande, C. [Istituto di Fisiologia Clinica, CNR, via G. Moruzzi 1, Pisa I-56124 (Italy); Nannini, A.; Pennelli, G.; Pieri, F. [Dipartimento di Ingegneria dell' Informazione, University of Pisa, Via G. Caruso 16, I-56122 Pisa (Italy); Severi, S. [imec, Kapeldreef 75, Leuven B-3001 (Belgium)

2010-10-12

Polycrystalline silicon-germanium (poly-SiGe) is a promising structural material for the post-processing of micro electro-mechanical systems (MEMS) on top of complementary metal-oxide-semiconductor (CMOS) substrates. Combining MEMS and CMOS allows for the development of high-performance devices. We present for the first time selective protein immobilization on top of poly-SiGe surfaces, an enabling technique for the development of novel poly-SiGe based MEMS biosensors. Active regions made of 3-aminopropyl-triethoxysilane (APTES) were defined using silane deposition onto photoresist patterns followed by lift-off in organic solvents. Subsequently, proteins were covalently bound on the created APTES patterns. Fluorescein-labeled human serum albumin (HSA) was used to verify the immobilization procedure while the binding capability of the protein layer was tested by an antigen-labeled antibody pair. Inspection by fluorescence microscopy showed protein immobilization inside the desired bioactive areas and low non-specific adsorption outside the APTES pattern. Furthermore, the quality of the silane patches was investigated by treatment with 30 nm-diameter gold nanoparticles and scanning electron microscope observation. The developed technique is therefore a promising first step towards the realization of poly-SiGe based biosensors.

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

Different protein-protein interface patterns predicted by different machine learning methods.

Science.gov (United States)

Wang, Wei; Yang, Yongxiao; Yin, Jianxin; Gong, Xinqi

2017-11-22

Different types of protein-protein interactions make different protein-protein interface patterns. Different machine learning methods are suitable to deal with different types of data. Then, is it the same situation that different interface patterns are preferred for prediction by different machine learning methods? Here, four different machine learning methods were employed to predict protein-protein interface residue pairs on different interface patterns. The performances of the methods for different types of proteins are different, which suggest that different machine learning methods tend to predict different protein-protein interface patterns. We made use of ANOVA and variable selection to prove our result. Our proposed methods taking advantages of different single methods also got a good prediction result compared to single methods. In addition to the prediction of protein-protein interactions, this idea can be extended to other research areas such as protein structure prediction and design.

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

Protein patterns of yeast during sporulation

International Nuclear Information System (INIS)

Litske Petersen, J.G.; Kielland-Brandt, M.C.; Nilsson-Tillgren, T.

1979-01-01

High resolution two-dimensional gel electrophoresis was used to study protein synthesis during synchronous meiosis and ascospore formation of Saccharomyces cerevisiae. The stained protein patterns of samples harvested at any stage between meiotic prophase and the four-spore stage in two sporulating strains showed the same approximately 250 polypeptides. Of these only a few seemed to increase or decrease in concentration during sporulation. The characteristic pattern of sporulating yeast was identical to the pattern of glucose-grown staitonary yeast cells adapted to respiration. The latter type of cells readily initiates meiosis when transferred to sporulation medium. This pattern differed from the protein patterns of exponentially growing cells in glucose or acetate presporulation medium. Five major proteins in stationary and sporulating yeast cells were not detected in either type of exponential culture. Two-dimensional autoradiograms of [ 35 S]methionine-labelled yeast proteins revealed that some proteins were preferentially labelled during sporulation, while other proteins were labelled at later stages. These patterns differed from the auroradiograms of exponentially growing yeast cells in glucose presporulation medium in a number of spots. No differences were observed when stained gels or autoradiograms of sporulating cultures and non-sporulating strains in sporulation medium were compared. (author)

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

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...

Chromosome driven spatial patterning of proteins in bacteria.

Directory of Open Access Journals (Sweden)

Saeed Saberi

Full Text Available The spatial patterning of proteins in bacteria plays an important role in many processes, from cell division to chemotaxis. In the asymmetrically dividing bacteria Caulobacter crescentus, a scaffolding protein, PopZ, localizes to both poles and aids the differential patterning of proteins between mother and daughter cells during division. Polar patterning of misfolded proteins in Escherichia coli has also been shown, and likely plays an important role in cellular ageing. Recent experiments on both of the above systems suggest that the presence of chromosome free regions along with protein multimerization may be a mechanism for driving the polar localization of proteins. We have developed a simple physical model for protein localization using only these two driving mechanisms. Our model reproduces all the observed patterns of PopZ and misfolded protein localization--from diffuse, unipolar, and bipolar patterns and can also account for the observed patterns in a variety of mutants. The model also suggests new experiments to further test the role of the chromosome in driving protein patterning, and whether such a mechanism is responsible for helping to drive the differentiation of the cell poles.

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

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)

Characterization and expression patterns of a membrane-bound trehalase from Spodoptera exigua

Directory of Open Access Journals (Sweden)

Xu Weihua

2008-05-01

Full Text Available Abstract Background The chitin biosynthesis pathway starts with trehalose in insects and the main functions of trehalases are hydrolysis of trehalose to glucose. Although insects possess two types, soluble trehalase (Tre-1 and membrane-bound trehalase (Tre-2, very little is known about Tre-2 and the difference in function between Tre-1 and Tre-2. Results To gain an insight into trehalase functions in insects, we investigated a putative membrane-bound trehalase from Spodoptera exigua (SeTre-2 cloned from the fat body. The deduced amino acid sequence of SeTre-2 contains 645 residues and has a predicted molecular weight of ~74 kDa and pI of 6.01. Alignment of SeTre-2 with other insect trehalases showed that it contains two trehalase signature motifs and a putative transmembrane domain, which is an important characteristic of Tre-2. Comparison of the genomic DNA and cDNA sequences demonstrated that SeTre-2 comprises 13 exons and 12 introns. Southern blot analysis revealed that S. exigua has two trehalase genes and that SeTre-2 is a single-copy gene. Northern blot analyses showed that the SeTre-2 transcript is expressed not only in the midgut, as previously reported for Bombyx mori, but also in the fat body and Malpighian tubules, although expression patterns differed between the midgut and fat body. SeTre-2 transcripts were detected in the midgut of feeding stage larvae, but not in pupae, whereas SeTre-2 mRNA was detected in the fat body of fifth instar larvae and pupae. Conclusion These findings provide new data on the tissue distribution, expression patterns and potential function of membrane-bound trehalase. The results suggest that the SeTre-2 gene may have different functions in the midgut and fat body.

An Interval Bound Algorithm of optimizing reactor core loading pattern by using reactivity interval schema

International Nuclear Information System (INIS)

Gong Zhaohu; Wang Kan; Yao Dong

2011-01-01

Highlights: → We present a new Loading Pattern Optimization method - Interval Bound Algorithm (IBA). → IBA directly uses the reactivity of fuel assemblies and burnable poison. → IBA can optimize fuel assembly orientation in a coupled way. → Numerical experiment shows that IBA outperforms genetic algorithm and engineers. → We devise DDWF technique to deal with multiple objectives and constraints. - Abstract: In order to optimize the core loading pattern in Nuclear Power Plants, the paper presents a new optimization method - Interval Bound Algorithm (IBA). Similar to the typical population based algorithms, e.g. genetic algorithm, IBA maintains a population of solutions and evolves them during the optimization process. IBA acquires the solution by statistical learning and sampling the control variable intervals of the population in each iteration. The control variables are the transforms of the reactivity of fuel assemblies or the worth of burnable poisons, which are the crucial heuristic information for loading pattern optimization problems. IBA can deal with the relationship between the dependent variables by defining the control variables. Based on the IBA algorithm, a parallel Loading Pattern Optimization code, named IBALPO, has been developed. To deal with multiple objectives and constraints, the Dynamic Discontinuous Weight Factors (DDWF) for the fitness function have been used in IBALPO. Finally, the code system has been used to solve a realistic reloading problem and a better pattern has been obtained compared with the ones searched by engineers and genetic algorithm, thus the performance of the code is proved.

Length-Bounded Hybrid CPU/GPU Pattern Matching Algorithm for Deep Packet Inspection

Directory of Open Access Journals (Sweden)

Yi-Shan Lin

2017-01-01

Full Text Available Since frequent communication between applications takes place in high speed networks, deep packet inspection (DPI plays an important role in the network application awareness. The signature-based network intrusion detection system (NIDS contains a DPI technique that examines the incoming packet payloads by employing a pattern matching algorithm that dominates the overall inspection performance. Existing studies focused on implementing efficient pattern matching algorithms by parallel programming on software platforms because of the advantages of lower cost and higher scalability. Either the central processing unit (CPU or the graphic processing unit (GPU were involved. Our studies focused on designing a pattern matching algorithm based on the cooperation between both CPU and GPU. In this paper, we present an enhanced design for our previous work, a length-bounded hybrid CPU/GPU pattern matching algorithm (LHPMA. In the preliminary experiment, the performance and comparison with the previous work are displayed, and the experimental results show that the LHPMA can achieve not only effective CPU/GPU cooperation but also higher throughput than the previous method.

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.

Network based approaches reveal clustering in protein point patterns

Science.gov (United States)

Parker, Joshua; Barr, Valarie; Aldridge, Joshua; Samelson, Lawrence E.; Losert, Wolfgang

2014-03-01

Recent advances in super-resolution imaging have allowed for the sub-diffraction measurement of the spatial location of proteins on the surfaces of T-cells. The challenge is to connect these complex point patterns to the internal processes and interactions, both protein-protein and protein-membrane. We begin analyzing these patterns by forming a geometric network amongst the proteins and looking at network measures, such the degree distribution. This allows us to compare experimentally observed patterns to models. Specifically, we find that the experimental patterns differ from heterogeneous Poisson processes, highlighting an internal clustering structure. Further work will be to compare our results to simulated protein-protein interactions to determine clustering mechanisms.

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.

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.

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

Science.gov (United States)

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.

Science.gov (United States)

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

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 ...

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

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...

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

Science.gov (United States)

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...

Upper bounds on the number of errors corrected by a convolutional code

DEFF Research Database (Denmark)

Justesen, Jørn

2004-01-01

We derive upper bounds on the weights of error patterns that can be corrected by a convolutional code with given parameters, or equivalently we give bounds on the code rate for a given set of error patterns. The bounds parallel the Hamming bound for block codes by relating the number of error...

Selective memory generalization by spatial patterning of protein synthesis.

Science.gov (United States)

O'Donnell, Cian; Sejnowski, Terrence J

2014-04-16

Protein synthesis is crucial for both persistent synaptic plasticity and long-term memory. De novo protein expression can be restricted to specific neurons within a population, and to specific dendrites within a single neuron. Despite its ubiquity, the functional benefits of spatial protein regulation for learning are unknown. We used computational modeling to study this problem. We found that spatially patterned protein synthesis can enable selective consolidation of some memories but forgetting of others, even for simultaneous events that are represented by the same neural population. Key factors regulating selectivity include the functional clustering of synapses on dendrites, and the sparsity and overlap of neural activity patterns at the circuit level. Based on these findings, we proposed a two-step model for selective memory generalization during REM and slow-wave sleep. The pattern-matching framework we propose may be broadly applicable to spatial protein signaling throughout cortex and hippocampus. Copyright © 2014 Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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.

A case study of Chinese bound feet: application of footprint analysis.

Science.gov (United States)

Reischl, Uwe; Nandikolla, Vidja; Colby, Conrad; Mijović, Budimir; Wei, Hsian-Chuen

2008-06-01

Foot print patterns of the bound feet of a 90-year-old Chinese female were made to obtain insight into the ergonomic consequences of a Chinese custom that caused significant disabilities for many women throughout history. Pressure patterns were evaluated using the techniques applied to standard thumb print analsyis. A digital summary of the pressure patterns were compared to the patterns obtained from a normal subject. The outcomes indicated that the bound foot produced greater plantar tissue pressures than the non-bound foot. These observations help explain the discomfort, gait abnormalities, and disabilities exhibited by many older women with bound feet living in China today. Although foot-binding is no longer practiced, this study offers an ergonomic perspective on a custom practiced in China for centuries.

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.

Directory of Open Access Journals (Sweden)

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.

Rapid photochemical surface patterning of proteins in thiol-ene based microfluidic devices

DEFF Research Database (Denmark)

Lafleur, Josiane P.; Kwapiszewski, Radoslaw; Jensen, Thomas Glasdam

2013-01-01

! 17 SH nm"2. Biotin alkyne was patterned directly inside thiol–ene microchannels prior to conjugation with fluorescently labelled streptavidin. The surface bound conjugates were detected by evanescent waveinduced fluorescence (EWIF), demonstrating the success of the grafting procedure and its...

Covalent microcontact printing of proteins fro cell patterning

NARCIS (Netherlands)

Rozkiewicz, D.I.; Kraan, Yvonne M.; Werten, Marc W.T.; de Wolf, Frits A.; Subramaniam, Vinod; Ravoo, B.J.; Reinhoudt, David

2006-01-01

We describe a straightforward approach to the covalent immobilization of cytophilic proteins by microcontact printing, which can be used to pattern cells on substrates. Cytophilic proteins are printed in micropatterns on reactive self-assembled monolayers by using imine chemistry. An

An efficient, versatile and scalable pattern growth approach to mine frequent patterns in unaligned protein sequences.

Science.gov (United States)

Ye, Kai; Kosters, Walter A; Ijzerman, Adriaan P

2007-03-15

Pattern discovery in protein sequences is often based on multiple sequence alignments (MSA). The procedure can be computationally intensive and often requires manual adjustment, which may be particularly difficult for a set of deviating sequences. In contrast, two algorithms, PRATT2 (http//www.ebi.ac.uk/pratt/) and TEIRESIAS (http://cbcsrv.watson.ibm.com/) are used to directly identify frequent patterns from unaligned biological sequences without an attempt to align them. Here we propose a new algorithm with more efficiency and more functionality than both PRATT2 and TEIRESIAS, and discuss some of its applications to G protein-coupled receptors, a protein family of important drug targets. In this study, we designed and implemented six algorithms to mine three different pattern types from either one or two datasets using a pattern growth approach. We compared our approach to PRATT2 and TEIRESIAS in efficiency, completeness and the diversity of pattern types. Compared to PRATT2, our approach is faster, capable of processing large datasets and able to identify the so-called type III patterns. Our approach is comparable to TEIRESIAS in the discovery of the so-called type I patterns but has additional functionality such as mining the so-called type II and type III patterns and finding discriminating patterns between two datasets. The source code for pattern growth algorithms and their pseudo-code are available at http://www.liacs.nl/home/kosters/pg/.

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.

Deleted in malignant brain tumors-1 protein (DMBT1): a pattern recognition receptor with multiple binding sites.

Science.gov (United States)

Ligtenberg, Antoon J M; Karlsson, Niclas G; Veerman, Enno C I

2010-01-01

Deleted in Malignant Brain Tumors-1 protein (DMBT1), salivary agglutinin (DMBT1(SAG)), and lung glycoprotein-340 (DMBT1(GP340)) are three names for glycoproteins encoded by the same DMBT1 gene. All these proteins belong to the scavenger receptor cysteine-rich (SRCR) superfamily of proteins: a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. In addition to SRCR domains, all DMBT1s contain two CUB domains and one zona pellucida domain. The SRCR domains play a role in the function of DMBT1s, which is the binding of a broad range of pathogens including cariogenic streptococci, Helicobacter pylori and HIV. Mucosal defense proteins like IgA, surfactant proteins and lactoferrin also bind to DMBT1s through their SRCR domains. The binding motif on the SRCR domains comprises an 11-mer peptide in which a few amino acids are essential for binding (GRVEVLYRGSW). Adjacent to each individual SRCR domain are glycosylation domains, where the attached carbohydrate chains play a role in the binding of influenza A virus and Helicobacter pylori. The composition of the carbohydrate chains is not only donor specific, but also varies between different organs. These data demonstrate a role for DMBT1s as pattern recognition molecules containing various peptide and carbohydrate binding motifs.

Deleted in Malignant Brain Tumors-1 Protein (DMBT1: A Pattern Recognition Receptor with Multiple Binding Sites

Directory of Open Access Journals (Sweden)

Enno C. I. Veerman

2010-12-01

Full Text Available Deleted in Malignant Brain Tumors-1 protein (DMBT1, salivary agglutinin (DMBT1SAG, and lung glycoprotein-340 (DMBT1GP340 are three names for glycoproteins encoded by the same DMBT1 gene. All these proteins belong to the scavenger receptor cysteine-rich (SRCR superfamily of proteins: a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. In addition to SRCR domains, all DMBT1s contain two CUB domains and one zona pellucida domain. The SRCR domains play a role in the function of DMBT1s, which is the binding of a broad range of pathogens including cariogenic streptococci, Helicobacter pylori and HIV. Mucosal defense proteins like IgA, surfactant proteins and lactoferrin also bind to DMBT1s through their SRCR domains. The binding motif on the SRCR domains comprises an 11-mer peptide in which a few amino acids are essential for binding (GRVEVLYRGSW. Adjacent to each individual SRCR domain are glycosylation domains, where the attached carbohydrate chains play a role in the binding of influenza A virus and Helicobacter pylori. The composition of the carbohydrate chains is not only donor specific, but also varies between different organs. These data demonstrate a role for DMBT1s as pattern recognition molecules containing various peptide and carbohydrate binding motifs.

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.

Ficolins and FIBCD1: Soluble and membrane bound pattern recognition molecules with acetyl group selectivity

DEFF Research Database (Denmark)

Thomsen, Theresa; Schlosser, Anders; Holmskov, Uffe

2011-01-01

as pattern recognition molecules. Ficolins are soluble oligomeric proteins composed of trimeric collagen-like regions linked to fibrinogen-related domains (FReDs) that have the ability to sense molecular patterns on both pathogens and apoptotic cell surfaces and activate the complement system. The ficolins......D-containing molecules, and discusses structural resemblance but also diversity in recognition of acetylated ligands....

Patterning protein complexes on DNA nanostructures using a GFP nanobody.

Science.gov (United States)

Sommese, R F; Hariadi, R F; Kim, K; Liu, M; Tyska, M J; Sivaramakrishnan, S

2016-11-01

DNA nanostructures have become an important and powerful tool for studying protein function over the last 5 years. One of the challenges, though, has been the development of universal methods for patterning protein complexes on DNA nanostructures. Herein, we present a new approach for labeling DNA nanostructures by functionalizing them with a GFP nanobody. We demonstrate the ability to precisely control protein attachment via our nanobody linker using two enzymatic model systems, namely adenylyl cyclase activity and myosin motility. Finally, we test the power of this attachment method by patterning unpurified, endogenously expressed Arp2/3 protein complex from cell lysate. By bridging DNA nanostructures with a fluorescent protein ubiquitous throughout cell and developmental biology and protein biochemistry, this approach significantly streamlines the application of DNA nanostructures as a programmable scaffold in biological studies. © 2016 The Protein Society.

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.)

Urinary excretion and patterns of protein binding of iodipamide (Biligrafin forte). A comparison of the infusion technique with the single bolus injection in intravenous cholangiography

Energy Technology Data Exchange (ETDEWEB)

Husband, J; Saxton, H M [Guy' s Hospital, London (UK)

1978-01-01

It has been suggested that the bile ducts are seen better during intravenous cholangiography when the contrast medium is given by infusion rather than by injection in a single bolus. As an explanation, it has been proposed that a greater amount of contrast medium is bound to plasma proteins after infusion, resulting in a smaller quantity of contrast medium being excreted in the urine, so leaving a larger total for excretion by the liver. In this study, the urinary excretion of /sup 125/I labelled iodipamide methylglucamine (50% w/v Biligrafin forte) has been measured in 42 patients. The patients were divided into two groups. One group received a slow infusion of radioactive iodipamide over 45 minutes and the other an intravenous injection over five minutes. When a relatively high dose (0.6 mg/kg body weight) was used, no difference in urinary excretion was noted between these two groups; but with a lower dose (0.2 mg/kg body weight), slow infusion resulted in a reduced urinary excretion; however the total difference in contrast lost in the urine was too small to affect biliary concentration. The patterns of protein binding of iodipamide have been examined in 12 of these patients. The results showed that at the low dose a higher percentage of radioactive iodipamide was bound to protein in patients given contrast by infusion. There was clear evidence that the contrast binding capacity of plasma was limited so that with higher doses, much contrast remained unbound. At any given dose level, there was inverse correlation between the proportion of contrast bound to protein and the urinary excretion. The factors affecting contrast binding in individual subjects were not clear.

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.

Von Willebrand protein binds to extracellular matrices independently of collagen.

OpenAIRE

Wagner, D D; Urban-Pickering, M; Marder, V J

1984-01-01

Von Willebrand protein is present in the extracellular matrix of endothelial cells where it codistributes with fibronectin and types IV and V collagen. Bacterial collagenase digestion of endothelial cells removed fibrillar collagen, but the pattern of fibronectin and of von Willebrand protein remained undisturbed. Exogenous von Willebrand protein bound to matrices of different cells, whether rich or poor in collagen. von Willebrand protein also decorated the matrix of cells grown in the prese...

Peptide Pattern Recognition for high-throughput protein sequence analysis and clustering

DEFF Research Database (Denmark)

Busk, Peter Kamp

2017-01-01

Large collections of protein sequences with divergent sequences are tedious to analyze for understanding their phylogenetic or structure-function relation. Peptide Pattern Recognition is an algorithm that was developed to facilitate this task but the previous version does only allow a limited...... number of sequences as input. I implemented Peptide Pattern Recognition as a multithread software designed to handle large numbers of sequences and perform analysis in a reasonable time frame. Benchmarking showed that the new implementation of Peptide Pattern Recognition is twenty times faster than...... the previous implementation on a small protein collection with 673 MAP kinase sequences. In addition, the new implementation could analyze a large protein collection with 48,570 Glycosyl Transferase family 20 sequences without reaching its upper limit on a desktop computer. Peptide Pattern Recognition...

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.

Protein patterns of black fungi under simulated Mars-like conditions.

Science.gov (United States)

Zakharova, Kristina; Marzban, Gorji; de Vera, Jean-Pierre; Lorek, Andreas; Sterflinger, Katja

2014-05-29

Two species of microcolonial fungi - Cryomyces antarcticus and Knufia perforans - and a species of black yeasts-Exophiala jeanselmei - were exposed to thermo-physical Mars-like conditions in the simulation chamber of the German Aerospace Center. In this study the alterations at the protein expression level from various fungi species under Mars-like conditions were analyzed for the first time using 2D gel electrophoresis. Despite of the expectations, the fungi did not express any additional proteins under Mars simulation that could be interpreted as stress induced HSPs. However, up-regulation of some proteins and significant decreasing of protein number were detected within the first 24 hours of the treatment. After 4 and 7 days of the experiment protein spot number was increased again and the protein patterns resemble the protein patterns of biomass from normal conditions. It indicates the recovery of the metabolic activity under Martian environmental conditions after one week of exposure.

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

Directory of Open Access Journals (Sweden)

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

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

Geomfinder: a multi-feature identifier of similar three-dimensional protein patterns: a ligand-independent approach.

Science.gov (United States)

Núñez-Vivanco, Gabriel; Valdés-Jiménez, Alejandro; Besoaín, Felipe; Reyes-Parada, Miguel

2016-01-01

Since the structure of proteins is more conserved than the sequence, the identification of conserved three-dimensional (3D) patterns among a set of proteins, can be important for protein function prediction, protein clustering, drug discovery and the establishment of evolutionary relationships. Thus, several computational applications to identify, describe and compare 3D patterns (or motifs) have been developed. Often, these tools consider a 3D pattern as that described by the residues surrounding co-crystallized/docked ligands available from X-ray crystal structures or homology models. Nevertheless, many of the protein structures stored in public databases do not provide information about the location and characteristics of ligand binding sites and/or other important 3D patterns such as allosteric sites, enzyme-cofactor interaction motifs, etc. This makes necessary the development of new ligand-independent methods to search and compare 3D patterns in all available protein structures. Here we introduce Geomfinder, an intuitive, flexible, alignment-free and ligand-independent web server for detailed estimation of similarities between all pairs of 3D patterns detected in any two given protein structures. We used around 1100 protein structures to form pairs of proteins which were assessed with Geomfinder. In these analyses each protein was considered in only one pair (e.g. in a subset of 100 different proteins, 50 pairs of proteins can be defined). Thus: (a) Geomfinder detected identical pairs of 3D patterns in a series of monoamine oxidase-B structures, which corresponded to the effectively similar ligand binding sites at these proteins; (b) we identified structural similarities among pairs of protein structures which are targets of compounds such as acarbose, benzamidine, adenosine triphosphate and pyridoxal phosphate; these similar 3D patterns are not detected using sequence-based methods; (c) the detailed evaluation of three specific cases showed the versatility

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

Directory of Open Access Journals (Sweden)

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.

Canine serum protein patterns using high-resolution electrophoresis (HRE).

Science.gov (United States)

Abate, O; Zanatta, R; Malisano, T; Dotta, U

2000-03-01

Serum protein values were determined in 26 healthy dogs using agarose gel electrophoresis (SPE), splitting the electrophoretic separation into six regions: albumin, alpha(1), alpha(2), beta(1), beta(2)and gamma globulins. High-resolution electrophoresis (HRE) was used to separate single proteins. Serum proteins from dogs (26 healthy and 20 affected by various diseases) were then characterized by electrophoretic immunofixation (IFE) and Sudan black staining on HRE film. Haemoglobin and normal canine plasma and serum were used to identify haptoglobin and fibrinogen, respectively. In the standard pattern, determined by HRE, the following proteins were identified: albumin, alpha(1)-lipoprotein (alpha(1)-region), haptoglobin and alpha(2)-macroglobulin (alpha(2)-region), beta -lipoprotein and C3 (beta(1)-region), transferrin and IgM (beta(2)-region), IgG (mostly in gamma -region and partly in beta(2)-region). The HRE pattern shown by healthy dogs could be compared with those of dogs affected by various diseases to obtain clinical information. Copyright 2000 Harcourt Publishers Ltd.

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.

Directory of Open Access Journals (Sweden)

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.

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.

Comparison of serum protein electrophoretic pattern in cows and small ruminants

Directory of Open Access Journals (Sweden)

Oskar Nagy

2015-01-01

Full Text Available Determination of the physiological electrophoretic patterns in animals is very useful for clinicians in diagnosing healthy and sick animals. The objective of this study was to investigate the serum protein electrophoretic pattern in cows, sheep, and goats in order to evaluate the differences in the size and number of protein fractions between the evaluated ruminant species. Ten adult multiparous high-yielding dairy cows, 10 adult female sheep and 10 adult female goats were included in this study. All the evaluated animals were clinically healthy. Serum was analyzed for total serum protein concentrations, and for the relative and absolute values of protein fractions with calculation of albumin/globulin ratios. Serum protein fractions were separated by zone electrophoresis on buffered agarose gel. Serum protein electrophoresis identified 6 distinct bands, comprising albumin, alpha1- (α1, alpha2- (α2, beta1- (β1, beta2- (β2, and gamma- (γ globulins in cows. In sheep, serum proteins exhibited 6 fractions: albumin, α1-, α2-, β-, γ1- and γ2-globulins. In goats, serum proteins were separated into 5 fractions: albumin, α1-, α2-, β- and γ-globulins. Significant differences in the relative as well as absolute means were found for the albumin/globulin ratio and most of the protein fractions, except γ-globulins. No significant differences were found in the concentration of total proteins. These results describe the marked species differences in most of serum protein fractions between the evaluated groups of animals, and contribute to the current knowledge about the physiological electrophoretic pattern of serum proteins in ruminants, which can be used for diagnostic purposes.

The use of protein patterns in genetic diversity analysis in some Brassica napus cultivars

Directory of Open Access Journals (Sweden)

Roya Razavizadeh

2013-11-01

Full Text Available In this study, protein variations of seeds and five-day old cotyledonal leaves of four selected Brassica napus cultivars including Elite, Ocapy, Tasilo and Zarfam were analyzed by SDS-PAGE to identify protein markers. The amount of total soluble protein of seed storage proteins did not show significant differences in all cultivars whereas it was different in cotyledonal leaves. Protein patterns of seeds and cotyledonal leaves showed significant differences using SDS-PAGE and consequence analysis of bands by ImageJ program. Relative expression of six protein bands in seeds and five-day old cotyledonal leaves were significantly different. Three protein markers were identified by protein patterns of seed and cotyledonal leaves. The results of relationship analysis based on presence and absence of the specific protein bands in protein pattern of seed storage proteins showed that Tasilo and Elite cultivars had the highest similarities.

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.

Pattern of occurrence and occupancy of carbonylation sites in proteins

DEFF Research Database (Denmark)

Rao, R Shyama Prasad; Møller, Ian Max

2011-01-01

sites. Comparison of metal-catalyzed oxidation of two closely related proteins indicates that this type of carbonylation might not be very specific in proteins. Interestingly, carbonylated sites show a very strong tendency to cluster together in the protein primary sequence hinting at some sort......Proteins are targets for modification by reactive oxygen species, and carbonylation is an important irreversible modification that increases during oxidative stress. While information on protein carbonylation is accumulating, its pattern is not yet understood. We have made a meta...

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.

Computational mining for hypothetical patterns of amino acid side chains in protein data bank (PDB)

Science.gov (United States)

Ghani, Nur Syatila Ab; Firdaus-Raih, Mohd

2018-04-01

The three-dimensional structure of a protein can provide insights regarding its function. Functional relationship between proteins can be inferred from fold and sequence similarities. In certain cases, sequence or fold comparison fails to conclude homology between proteins with similar mechanism. Since the structure is more conserved than the sequence, a constellation of functional residues can be similarly arranged among proteins of similar mechanism. Local structural similarity searches are able to detect such constellation of amino acids among distinct proteins, which can be useful to annotate proteins of unknown function. Detection of such patterns of amino acids on a large scale can increase the repertoire of important 3D motifs since available known 3D motifs currently, could not compensate the ever-increasing numbers of uncharacterized proteins to be annotated. Here, a computational platform for an automated detection of 3D motifs is described. A fuzzy-pattern searching algorithm derived from IMagine an Amino Acid 3D Arrangement search EnGINE (IMAAAGINE) was implemented to develop an automated method for searching of hypothetical patterns of amino acid side chains in Protein Data Bank (PDB), without the need for prior knowledge on related sequence or structure of pattern of interest. We present an example of the searches, which is the detection of a hypothetical pattern derived from known structural motif of C2H2 structural pattern from zinc fingers. The conservation of particular patterns of amino acid side chains in unrelated proteins is highlighted. This approach can act as a complementary method for available structure- and sequence-based platforms and may contribute in improving functional association between proteins.

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

Protein functional features are reflected in the patterns of mRNA translation speed.

Science.gov (United States)

López, Daniel; Pazos, Florencio

2015-07-09

The degeneracy of the genetic code makes it possible for the same amino acid string to be coded by different messenger RNA (mRNA) sequences. These "synonymous mRNAs" may differ largely in a number of aspects related to their overall translational efficiency, such as secondary structure content and availability of the encoded transfer RNAs (tRNAs). Consequently, they may render different yields of the translated polypeptides. These mRNA features related to translation efficiency are also playing a role locally, resulting in a non-uniform translation speed along the mRNA, which has been previously related to some protein structural features and also used to explain some dramatic effects of "silent" single-nucleotide-polymorphisms (SNPs). In this work we perform the first large scale analysis of the relationship between three experimental proxies of mRNA local translation efficiency and the local features of the corresponding encoded proteins. We found that a number of protein functional and structural features are reflected in the patterns of ribosome occupancy, secondary structure and tRNA availability along the mRNA. One or more of these proxies of translation speed have distinctive patterns around the mRNA regions coding for certain protein local features. In some cases the three patterns follow a similar trend. We also show specific examples where these patterns of translation speed point to the protein's important structural and functional features. This support the idea that the genome not only codes the protein functional features as sequences of amino acids, but also as subtle patterns of mRNA properties which, probably through local effects on the translation speed, have some consequence on the final polypeptide. These results open the possibility of predicting a protein's functional regions based on a single genomic sequence, and have implications for heterologous protein expression and fine-tuning protein function.

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

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.

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...

Protein Tyrosine Phosphatase-PEST and β8 Integrin Regulate Spatiotemporal Patterns of RhoGDI1 Activation in Migrating Cells

Science.gov (United States)

Lee, Hye Shin; Cheerathodi, Mujeeburahiman; Chaki, Sankar P.; Reyes, Steve B.; Zheng, Yanhua; Lu, Zhimin; Paidassi, Helena; DerMardirossian, Celine; Lacy-Hulbert, Adam; Rivera, Gonzalo M.

2015-01-01

Directional cell motility is essential for normal development and physiology, although how motile cells spatiotemporally activate signaling events remains largely unknown. Here, we have characterized an adhesion and signaling unit comprised of protein tyrosine phosphatase (PTP)-PEST and the extracellular matrix (ECM) adhesion receptor β8 integrin that plays essential roles in directional cell motility. β8 integrin and PTP-PEST form protein complexes at the leading edge of migrating cells and balance patterns of Rac1 and Cdc42 signaling by controlling the subcellular localization and phosphorylation status of Rho GDP dissociation inhibitor 1 (RhoGDI1). Translocation of Src-phosphorylated RhoGDI1 to the cell's leading edge promotes local activation of Rac1 and Cdc42, whereas dephosphorylation of RhoGDI1 by integrin-bound PTP-PEST promotes RhoGDI1 release from the membrane and sequestration of inactive Rac1/Cdc42 in the cytoplasm. Collectively, these data reveal a finely tuned regulatory mechanism for controlling signaling events at the leading edge of directionally migrating cells. PMID:25666508

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

A Versatile Method of Patterning Proteins and Cells.

Science.gov (United States)

Shrirao, Anil B; Kung, Frank H; Yip, Derek; Firestein, Bonnie L; Cho, Cheul H; Townes-Anderson, Ellen

2017-02-26

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

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.

Cytometry of chromatin bound Mcm6 and PCNA identifies two states in G1 that are separated functionally by the G1 restriction point1

Directory of Open Access Journals (Sweden)

Jacobberger James W

2010-04-01

Full Text Available Abstract Background Cytometric measurements of DNA content and chromatin-bound Mcm2 have demonstrated bimodal patterns of expression in G1. These patterns, the replication licensing function of Mcm proteins, and a correlation between Mcm loading and cell cycle commitment for cells re-entering the cell cycle, led us to test the idea that cells expressing a defined high level of chromatin-bound Mcm6 in G1 are committed - i.e., past the G1 restriction point. We developed a cell-based assay for tightly-bound PCNA (PCNA* and Mcm6 (Mcm6*, DNA content, and a mitotic marker to clearly define G1, S, G2, and M phases of the cell cycle. hTERT-BJ1, hTERT-RPE-1, and Molt4 cells were extracted with Triton X-100 followed by methanol fixation, stained with antibodies and DAPI, then measured by cytometry. Results Bivariate analysis of cytometric data demonstrated complex patterns with distinct clustering for all combinations of the 4 variables. In G1, cells clustered in two groups characterized by low and high Mcm6* expression. Serum starvation and release experiments showed that residence in the high group was in late G1, just prior to S phase. Kinetic experiments, employing serum withdrawal, and stathmokinetic analysis with aphidicolin, mimosine or nocodazole demonstrated that cells with high levels of Mcm6* cycled with the committed phases of the cell cycle (S, G2, and M. Conclusions A multivariate assay for Mcm6*, PCNA*, DNA content, and a mitotic marker provides analysis capable of estimating the fraction of pre and post-restriction point G1 cells and supports the idea that there are at least two states in G1 defined by levels of chromatin bound Mcm proteins.

Regular Nanoscale Protein Patterns via Directed Adsorption through Self-Assembled DNA Origami Masks.

Science.gov (United States)

Ramakrishnan, Saminathan; Subramaniam, Sivaraman; Stewart, A Francis; Grundmeier, Guido; Keller, Adrian

2016-11-16

DNA origami has become a widely used method for synthesizing well-defined nanostructures with promising applications in various areas of nanotechnology, biophysics, and medicine. Recently, the possibility to transfer the shape of single DNA origami nanostructures into different materials via molecular lithography approaches has received growing interest due to the great structural control provided by the DNA origami technique. Here, we use ordered monolayers of DNA origami nanostructures with internal cavities on mica surfaces as molecular lithography masks for the fabrication of regular protein patterns over large surface areas. Exposure of the masked sample surface to negatively charged proteins results in the directed adsorption of the proteins onto the exposed surface areas in the holes of the mask. By controlling the buffer and adsorption conditions, the protein coverage of the exposed areas can be varied from single proteins to densely packed monolayers. To demonstrate the versatility of this approach, regular nanopatterns of four different proteins are fabricated: the single-strand annealing proteins Redβ and Sak, the iron-storage protein ferritin, and the blood protein bovine serum albumin (BSA). We furthermore demonstrate the desorption of the DNA origami mask after directed protein adsorption, which may enable the fabrication of hierarchical patterns composed of different protein species. Because selectivity in adsorption is achieved by electrostatic interactions between the proteins and the exposed surface areas, this approach may enable also the large-scale patterning of other charged molecular species or even nanoparticles.

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 ...

PatternQuery: web application for fast detection of biomacromolecular structural patterns in the entire Protein Data Bank.

Science.gov (United States)

Sehnal, David; Pravda, Lukáš; Svobodová Vařeková, Radka; Ionescu, Crina-Maria; Koča, Jaroslav

2015-07-01

Well defined biomacromolecular patterns such as binding sites, catalytic sites, specific protein or nucleic acid sequences, etc. precisely modulate many important biological phenomena. We introduce PatternQuery, a web-based application designed for detection and fast extraction of such patterns. The application uses a unique query language with Python-like syntax to define the patterns that will be extracted from datasets provided by the user, or from the entire Protein Data Bank (PDB). Moreover, the database-wide search can be restricted using a variety of criteria, such as PDB ID, resolution, and organism of origin, to provide only relevant data. The extraction generally takes a few seconds for several hundreds of entries, up to approximately one hour for the whole PDB. The detected patterns are made available for download to enable further processing, as well as presented in a clear tabular and graphical form directly in the browser. The unique design of the language and the provided service could pave the way towards novel PDB-wide analyses, which were either difficult or unfeasible in the past. The application is available free of charge at http://ncbr.muni.cz/PatternQuery. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

HIV and serum protein electrophoresis patterns in KwaZulu-Natal: a ...

African Journals Online (AJOL)

Objective. To describe the effect of HIV serostatus on serum proteins, serum protein electrophoresis (SPEP) patterns and monoclonal bands. Setting. Inkosi Albert Luthuli Central Hospital, Durban. Design. Retrospective, anonymous analysis of routine laboratory results. Results. Monoclonal bands were not increased in ...

The Pattern of Tyrosine Phosphorylation in Human Sperm in Response to Binding to Zona Pellucida or Hyaluronic Acid

Science.gov (United States)

Sati, Leyla; Cayli, Sevil; Delpiano, Elena; Sakkas, Denny

2014-01-01

In mammalian species, acquisition of sperm fertilization competence is dependent on the phenomenon of sperm capacitation. One of the key elements of capacitation is protein tyrosine phosphorylation (TP) in various sperm membrane regions. In previous studies performed, the pattern of TP was examined in human sperm bound to zona pellucida of oocytes. In the present comparative study, TP patterns upon sperm binding to the zona pellucida or hyaluronic acid (HA) were investigated in spermatozoa arising from the same semen samples. Tyrosine phosphorylation, visualized by immunofluorescence, was localized within the acrosomal cap, equatorial head region, neck, and the principal piece. Tyrosine phosphorylation has increased in a time-related manner as capacitation progressed, and the phosphorylation pattern was identical within the principal piece and neck, regardless of the sperm bound to the zona pellucida or HA. Thus, the data demonstrated that the patterns of sperm activation-related TP were similar regardless of the spermatozoa bound to zona pellucida or HA. Further, sperm with incomplete development, as detected by excess cytoplasmic retention, failed to exhibit TP. PMID:24077441

Patterns of Protein Food Intake Are Associated with Nutrient Adequacy in the General French Adult Population.

Science.gov (United States)

Gavelle, Erwan de; Huneau, Jean-François; Mariotti, François

2018-02-17

Protein food intake appears to partially structure dietary patterns, as most current emergent diets (e.g., vegetarian and flexitarian) can be described according to their levels of specific protein sources. However, few data are available on dietary protein patterns in the general population and their association with nutrient adequacy. Based on protein food intake data concerning 1678 adults from a representative French national dietary survey, and non-negative-matrix factorization followed by cluster analysis, we were able to identify distinctive dietary protein patterns and compare their nutrient adequacy (using PANDiet probabilistic scoring). The findings revealed eight patterns that clearly discriminate protein intakes and were characterized by the intakes of one or more specific protein foods: 'Processed meat', 'Poultry', 'Pork', 'Traditional', 'Milk', 'Take-away', 'Beef' and 'Fish'. 'Fish eaters' and 'Milk drinkers' had the highest overall nutrient adequacy, whereas that of 'Pork' and 'Take-away eaters' was the lowest. Nutrient adequacy could often be accounted for by the characteristics of the food contributing to protein intake: 'Meat eaters' had high probability of adequacy for iron and zinc, for example. We concluded that protein patterns constitute strong elements in the background structure of the dietary intake and are associated with the nutrient profile that they convey.

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.

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.

Changes in protein patterns and in vivo protein synthesis during senescence of hibiscus petals

International Nuclear Information System (INIS)

Woodson, W.R.; Handa, A.K.

1986-01-01

Changes in proteins associated with senescence of the flowers of Hibiscus rosa-sinensis was studied using SDS-PAGE. Total extractable protein from petals decreased with senescence. Changes were noted in patterns of proteins from aging petals. Flower opening and senescence was associated with appearance and disappearance of several polypeptides. One new polypeptide with an apparent mw of 41 kd was first seen the day of flower opening and increased to over 9% of the total protein content of senescent petal tissue. Protein synthesis during aging was investigated by following uptake and incorporation of 3 H-leucine into TCA-insoluble fraction of petal discs. Protein synthesis, as evidenced by the percent of label incorporated into the TCA-insoluble fraction, was greatest (32%) the day before flower opening. Senescent petal tissue incorporated 4% of label taken up into protein. Proteins were separated by SDS-PAGE and labelled polypeptides identified by fluorography. In presenescent petal tissue, radioactivity was distributed among several major polypeptides. In senescent tissue, much of the radioactivity was concentrated in the 41 kd polypeptide

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

DETECTION OF TOPOLOGICAL PATTERNS IN PROTEIN NETWORKS.

Energy Technology Data Exchange (ETDEWEB)

MASLOV,S.SNEPPEN,K.

2003-11-17

interesting property of many biological networks that was recently brought to attention of the scientific community [3, 4, 5] is an extremely broad distribution of node connectivities defined as the number of immediate neighbors of a given node in the network. While the majority of nodes have just a few edges connecting them to other nodes in the network, there exist some nodes, that we will refer to as ''hubs'', with an unusually large number of neighbors. The connectivity of the most connected hub in such a network is typically several orders of magnitude larger than the average connectivity in the network. Often the distribution of connectivities of individual nodes can be approximated by a scale-free power law form [3] in which case the network is referred to as scale-free. Among biological networks distributions of node connectivities in metabolic [4], protein interaction [5], and brain functional [6] networks can be reasonably approximated by a power law extending for several orders of magnitude. The set of connectivities of individual nodes is an example of a low-level (single-node) topological property of a network. While it answers the question about how many neighbors a given node has, it gives no information about the identity of those neighbors. It is clear that most functional properties of networks are defined at a higher topological level in the exact pattern of connections of nodes to each other. However, such multi-node connectivity patterns are rather difficult to quantify and compare between networks. In this work we concentrate on multi-node topological properties of protein networks. These networks (as any other biological networks) lack the top-down design. Instead, selective forces of biological evolution shape them from raw material provided by random events such as mutations within individual genes, and gene duplications. As a result their connections are characterized by a large degree of randomness. One may wonder which

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.

Compare local pocket and global protein structure models by small structure patterns

KAUST Repository

Cui, Xuefeng

2015-09-09

Researchers proposed several criteria to assess the quality of predicted protein structures because it is one of the essential tasks in the Critical Assessment of Techniques for Protein Structure Prediction (CASP) competitions. Popular criteria include root mean squared deviation (RMSD), MaxSub score, TM-score, GDT-TS and GDT-HA scores. All these criteria require calculation of rigid transformations to superimpose the the predicted protein structure to the native protein structure. Yet, how to obtain the rigid transformations is unknown or with high time complexity, and, hence, heuristic algorithms were proposed. In this work, we carefully design various small structure patterns, including the ones specifically tuned for local pockets. Such structure patterns are biologically meaningful, and address the issue of relying on a sufficient number of backbone residue fragments for existing methods. We sample the rigid transformations from these small structure patterns; and the optimal superpositions yield by these small structures are refined and reported. As a result, among 11; 669 pairs of predicted and native local protein pocket models from the CASP10 dataset, the GDT-TS scores calculated by our method are significantly higher than those calculated by LGA. Moreover, our program is computationally much more efficient. Source codes and executables are publicly available at http://www.cbrc.kaust.edu.sa/prosta/

Characterization of white grub (Melolonthidae; Coleoptera in salak plantation based on morphology and protein banding pattern

Directory of Open Access Journals (Sweden)

SUGIYARTO

2009-07-01

Full Text Available Maryati KT, Sugiyarto. 2010. Characterization of white grub (Melolonthidae; Coleoptera in salak plantation based on morphology and protein banding pattern. Nusantara Bioscience 1: 72-77. This research aims to find out the white grub (Melolonthidae; Coleoptera variability based on the morphological characteristic and protein banding pattern found in ”salak pondoh” farm in Regencies of Sleman, Yogyakarta and Magelang, Central Java. Each area has five sampling points. Morphological analysis on white grub was conducted using descriptive method and analysis on protein banding pattern was conducted using qualitative analysis based on the presence or absent of band pattern on the gel, and qualitatively based on the relative mobility value (Rf of protein. The result indicated that the white grub in Sleman and Magelang, based on morphology characteristic is only one species, namely Holothricia sp. Based on the protein banding pattern, the white grub sample have differences of protein band number and protein molecular weight. Key words: Salacca zalacca, white grub, morphology, protein banding pattern.Abstrak. Maryati KT, Sugiyarto. 2010. Karakterisasi lundi putih (Melolonthidae: Coleoptera pada pertanaman salak berdasarkan ciri morfologi dan pola pita protein. Nusantara Bioscience 1: 72-77. Penelitian ini bertujuan untuk mengetahui keanekaragaman lundi putih (Melolonthidae; Coleoptera berdasarkan ciri morfologi dan pola pita protein yang ditemukan di lahan pertanaman salak pondoh di Kabupaten Sleman, Yogyakarta dan Kabupaten Magelang, Jawa Tengah. Pada masing-masing wilayah diambil lima titik sampling. Analisis morfologi lundi putih digunakan metode deskriptif, dan analisis pola pita protein digunakan analisis kualitatif berdasarkan muncul tidaknya pola pita pada gel, dan secara kuantitatif berdasarkan nilai mobilitas relatif protein (RF. Hasil penelitian menunjukkan bahwa sampel lundi putih di Kabupaten Sleman dan Magelang, berdasar karakter

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.

Protein Attachment on Nanodiamonds.

Science.gov (United States)

Lin, Chung-Lun; Lin, Cheng-Huang; Chang, Huan-Cheng; Su, Meng-Chih

2015-07-16

A recent advance in nanotechnology is the scale-up production of small and nonaggregated diamond nanoparticles suitable for biological applications. Using detonation nanodiamonds (NDs) with an average diameter of ∼4 nm as the adsorbents, we have studied the static attachment of three proteins (myoglobin, bovine serum albumin, and insulin) onto the nanoparticles by optical spectroscopy, mass spectrometry, and dynamic light scattering, and electrophoretic zeta potential measurements. Results show that the protein surface coverage is predominantly determined by the competition between protein-protein and protein-ND interactions, giving each protein a unique and characteristic structural configuration in its own complex. Specifically, both myoglobin and bovine serum albumin show a Langmuir-type adsorption behavior, forming 1:1 complexes at saturation, whereas insulin folds into a tightly bound multimer before adsorption. The markedly different adsorption patterns appear to be independent of the protein concentration and are closely related to the affinity of the individual proteins for the NDs. The present study provides a fundamental understanding for the use of NDs as a platform for nanomedical drug delivery.

Using Protein Dimers to Maximize the Protein Hybridization Efficiency with Multisite DNA Origami Scaffolds

Science.gov (United States)

Verma, Vikash; Mallik, Leena; Hariadi, Rizal F.; Sivaramakrishnan, Sivaraj; Skiniotis, Georgios; Joglekar, Ajit P.

2015-01-01

DNA origami provides a versatile platform for conducting ‘architecture-function’ analysis to determine how the nanoscale organization of multiple copies of a protein component within a multi-protein machine affects its overall function. Such analysis requires that the copy number of protein molecules bound to the origami scaffold exactly matches the desired number, and that it is uniform over an entire scaffold population. This requirement is challenging to satisfy for origami scaffolds with many protein hybridization sites, because it requires the successful completion of multiple, independent hybridization reactions. Here, we show that a cleavable dimerization domain on the hybridizing protein can be used to multiplex hybridization reactions on an origami scaffold. This strategy yields nearly 100% hybridization efficiency on a 6-site scaffold even when using low protein concentration and short incubation time. It can also be developed further to enable reliable patterning of a large number of molecules on DNA origami for architecture-function analysis. PMID:26348722

Using Protein Dimers to Maximize the Protein Hybridization Efficiency with Multisite DNA Origami Scaffolds.

Directory of Open Access Journals (Sweden)

Vikash Verma

Full Text Available DNA origami provides a versatile platform for conducting 'architecture-function' analysis to determine how the nanoscale organization of multiple copies of a protein component within a multi-protein machine affects its overall function. Such analysis requires that the copy number of protein molecules bound to the origami scaffold exactly matches the desired number, and that it is uniform over an entire scaffold population. This requirement is challenging to satisfy for origami scaffolds with many protein hybridization sites, because it requires the successful completion of multiple, independent hybridization reactions. Here, we show that a cleavable dimerization domain on the hybridizing protein can be used to multiplex hybridization reactions on an origami scaffold. This strategy yields nearly 100% hybridization efficiency on a 6-site scaffold even when using low protein concentration and short incubation time. It can also be developed further to enable reliable patterning of a large number of molecules on DNA origami for architecture-function analysis.

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.

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

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...

Variation of morphology, karyotype and protein band pattern of adenium (Adenium obesum varieties

Directory of Open Access Journals (Sweden)

PRABANG SETYONO

2009-07-01

Full Text Available Hastuti D, Suranto, Setyono P. 2009. Variation of morphology, karyotype and protein band pattern of adenium (Adenium obesum varieties. Nusantara Bioscience 1: 78-83. The aim of this research to find out the Adenium obesum variation from six varieties, namely: obesum, cery, red lucas, red fanta , white bigben and harry potter based on morphology, karyotype, as well as protein banding pattern. The chromosome preparation was made using semi-permanent squash method from the tip of root plant; while protein banding pattern was made using SDS-PAGE method. Qualitative data included shape and color of the leave and flower described from each variety. Data were presented in morphometry and analyzed using ANOVA and then followed by DMRT with 5% of confidence levels, indicated significance difference. Protein banding pattern, the root, stem, leave and all organs were analyzed using Hierarchical Cluster Analysis method with Average Linkage (between Groups using SPSS 10.0. The result of research shows that the six A. obesum varieties have morphological character with no variation of light green to dark green leave, not hairy, smooth leave bone, meanwhile for light red to dark red flower crown color although some of them are white and the same funnel color, yellow. All varieties of A. obesum have same number of chromosome, 2n = 22 and shows the difference ranging from 2.56 to 5.13 um. In the banding pattern formed qualitatively, there is variation among the six varieties.

Protein recognition by a pattern-generating fluorescent molecular probe

Science.gov (United States)

Pode, Zohar; Peri-Naor, Ronny; Georgeson, Joseph M.; Ilani, Tal; Kiss, Vladimir; Unger, Tamar; Markus, Barak; Barr, Haim M.; Motiei, Leila; Margulies, David

2017-12-01

Fluorescent molecular probes have become valuable tools in protein research; however, the current methods for using these probes are less suitable for analysing specific populations of proteins in their native environment. In this study, we address this gap by developing a unimolecular fluorescent probe that combines the properties of small-molecule-based probes and cross-reactive sensor arrays (the so-called chemical 'noses/tongues'). On the one hand, the probe can detect different proteins by generating unique identification (ID) patterns, akin to cross-reactive arrays. On the other hand, its unimolecular scaffold and selective binding enable this ID-generating probe to identify combinations of specific protein families within complex mixtures and to discriminate among isoforms in living cells, where macroscopic arrays cannot access. The ability to recycle the molecular device and use it to track several binding interactions simultaneously further demonstrates how this approach could expand the fluorescent toolbox currently used to detect and image proteins.

Inheritance patterns of enzymes and serum proteins of mallard-black duck hybrids

Science.gov (United States)

Morgan, R.P.; Meritt, D.W.; Block, S.B.; Cole, M.A.; Sulkin, S.T.; Lee, F.B.; Henny, C.J.

1984-01-01

From 1974 to 1976, a breeding program was used to produce hybrids of black ducks and mallards for the evaluation of inheritance patterns of serum proteins and serum, liver and muscle enzymes. In addition to the crosses designed to produce hybrids, a series of matings in 1975 and 1976 were designed to evaluate inheritance patterns of a hybrid with either a black duck or mallard. At the F1 level, hybrids were easily distinguished using serum proteins. However, once a hybrid was crossed back to either a mallard or black duck, only 12?23% of the progeny were distinguishable from black ducks or mallards using serum proteins and 23?39% using esterases. Muscle, serum and liver enzymes were similar between the two species.

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

Identification of genes encoding granule-bound starch synthase involved in amylose metabolism in banana fruit.

Directory of Open Access Journals (Sweden)

Hongxia Miao

Full Text Available Granule-bound starch synthase (GBSS is responsible for amylose synthesis, but the role of GBSS genes and their encoded proteins remains poorly understood in banana. In this study, amylose content and GBSS activity gradually increased during development of the banana fruit, and decreased during storage of the mature fruit. GBSS protein in banana starch granules was approximately 55.0 kDa. The protein was up-regulated expression during development while it was down-regulated expression during storage. Six genes, designated as MaGBSSI-1, MaGBSSI-2, MaGBSSI-3, MaGBSSI-4, MaGBSSII-1, and MaGBSSII-2, were cloned and characterized from banana fruit. Among the six genes, the expression pattern of MaGBSSI-3 was the most consistent with the changes in amylose content, GBSS enzyme activity, GBSS protein levels, and the quantity or size of starch granules in banana fruit. These results suggest that MaGBSSI-3 might regulate amylose metabolism by affecting the variation of GBSS levels and the quantity or size of starch granules in banana fruit during development or storage.

Identification of Genes Encoding Granule-Bound Starch Synthase Involved in Amylose Metabolism in Banana Fruit

Science.gov (United States)

Liu, Weixin; Xu, Biyu; Jin, Zhiqiang

2014-01-01

Granule-bound starch synthase (GBSS) is responsible for amylose synthesis, but the role of GBSS genes and their encoded proteins remains poorly understood in banana. In this study, amylose content and GBSS activity gradually increased during development of the banana fruit, and decreased during storage of the mature fruit. GBSS protein in banana starch granules was approximately 55.0 kDa. The protein was up-regulated expression during development while it was down-regulated expression during storage. Six genes, designated as MaGBSSI-1, MaGBSSI-2, MaGBSSI-3, MaGBSSI-4, MaGBSSII-1, and MaGBSSII-2, were cloned and characterized from banana fruit. Among the six genes, the expression pattern of MaGBSSI-3 was the most consistent with the changes in amylose content, GBSS enzyme activity, GBSS protein levels, and the quantity or size of starch granules in banana fruit. These results suggest that MaGBSSI-3 might regulate amylose metabolism by affecting the variation of GBSS levels and the quantity or size of starch granules in banana fruit during development or storage. PMID:24505384

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.

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.

Expression Pattern of Fatty Acid Binding Proteins in Celiac Disease Enteropathy

Directory of Open Access Journals (Sweden)

Natalia M. Bottasso Arias

2015-01-01

Full Text Available Celiac disease (CD is an immune-mediated enteropathy that develops in genetically susceptible individuals following exposure to dietary gluten. Severe changes at the intestinal mucosa observed in untreated CD patients are linked to changes in the level and in the pattern of expression of different genes. Fully differentiated epithelial cells express two isoforms of fatty acid binding proteins (FABPs: intestinal and liver, IFABP and LFABP, respectively. These proteins bind and transport long chain fatty acids and also have other important biological roles in signaling pathways, particularly those related to PPARγ and inflammatory processes. Herein, we analyze the serum levels of IFABP and characterize the expression of both FABPs at protein and mRNA level in small intestinal mucosa in severe enteropathy and normal tissue. As a result, we observed higher levels of circulating IFABP in untreated CD patients compared with controls and patients on gluten-free diet. In duodenal mucosa a differential FABPs expression pattern was observed with a reduction in mRNA levels compared to controls explained by the epithelium loss in severe enteropathy. In conclusion, we report changes in FABPs’ expression pattern in severe enteropathy. Consequently, there might be alterations in lipid metabolism and the inflammatory process in the small intestinal mucosa.

Multivariate analysis of 2-DE protein patterns - Practical approaches

DEFF Research Database (Denmark)

Jacobsen, Charlotte; Jacobsen, Susanne; Grove, H.

2007-01-01

Practical approaches to the use of multivariate data analysis of 2-DE protein patterns are demonstrated by three independent strategies for the image analysis and the multivariate analysis on the same set of 2-DE data. Four wheat varieties were selected on the basis of their baking quality. Two...... of the varieties were of strong baking quality and hard wheat kernel and two were of weak baking quality and soft kernel. Gliadins at different stages of grain development were analyzed by the application of multivariate data analysis on images of 2-DEs. Patterns related to the wheat varieties, harvest times...

Chromosome-wise Protein Interaction Patterns and Their Impact on Functional Implications of Large-Scale Genomic Aberrations

DEFF Research Database (Denmark)

Kirk, Isa Kristina; Weinhold, Nils; Belling, Kirstine González-Izarzugaza

2017-01-01

Gene copy-number changes influence phenotypes through gene-dosage alteration and subsequent changes of protein complex stoichiometry. Human trisomies where gene copy numbers are increased uniformly over entire chromosomes provide generic cases for studying these relationships. In most trisomies......, gene and protein level alterations have fatal consequences. We used genome-wide protein-protein interaction data to identify chromosome-specific patterns of protein interactions. We found that some chromosomes encode proteins that interact infrequently with each other, chromosome 21 in particular. We...... combined the protein interaction data with transcriptome data from human brain tissue to investigate how this pattern of global interactions may affect cellular function. We identified highly connected proteins that also had coordinated gene expression. These proteins were associated with important...

Evolutionary Pattern of N-Glycosylation Sequon Numbers  in Eukaryotic ABC Protein Superfamilies

Directory of Open Access Journals (Sweden)

R. Shyama Prasad Rao

2010-02-01

Full Text Available Many proteins contain a large number of NXS/T sequences (where X is any amino acid except proline which are the potential sites of asparagine (N linked glycosylation. However, the patterns of occurrence of these N-glycosylation sequons in related proteins or groups of proteins and their underlying causes have largely been unexplored. We computed the actual and probabilistic occurrence of NXS/T sequons in ABC protein superfamilies from eight diverse eukaryotic organisms. The ABC proteins contained significantly higher NXS/T sequon numbers compared to respective genome-wide average, but the sequon density was significantly lower owing to the increase in protein size and decrease in sequon specific amino acids. However, mammalian ABC proteins have significantly higher sequon density, and both serine and threonine containing sequons (NXS and NXT have been positively selected—against the recent findings of only threonine specific Darwinian selection of sequons in proteins. The occurrence of sequons was positively correlated with the frequency of sequon specific amino acids and negatively correlated with proline and the NPS/T sequences. Further, the NPS/T sequences were significantly higher than expected in plant ABC proteins which have the lowest number of NXS/T sequons. Accord- ingly, compared to overall proteins, N-glycosylation sequons in ABC protein superfamilies have a distinct pattern of occurrence, and the results are discussed in an evolutionary perspective.

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

Electrophoretic pattern of blood serum proteins of some of the vertebrates of Pakistan

International Nuclear Information System (INIS)

Shakoori, Abdul Rauf; Zaheer, Saleem Akhtar; Ahmad, Muhammad Salih.

1976-01-01

The electrophoretic pattern of blood serum proteins of some of the common fishes e.g. Catla catla, Cirrhina mrigala, Channa punctatus, Channa marulius, Wallago attu, Heterop-neustes fossilis; amphibia e.g., Rana tigrina, Rana cyanophlyctis, Bufo melanostictus; reptiles e.g. Varanus bengalensis, Uromastix hardwickii; birds e.g. Columba livia, Gallus domesticus, Passer domestica, Anas platyrhynchos; and mammals e.g. Homo sapiens, Mus musculus, Lepus cuniculus have been described. The mobility of proteins of blood sera has been studied over cellulose acetate paper and then a comparative pattern analysed

Influence of protein source on amino acid uptake patterns and protein utilization in rainbow trout Oncorhynchus mykiss

DEFF Research Database (Denmark)

Rolland, Marine; Holm, Jørgen; Dalsgaard, Anne Johanne Tang

induces reduced growth performances that remain partly unexplained. The aim of the current study was to investigate the effect of exchanging the protein source on protein utilization. Marine (fish meal) and vegetable (pea protein) sources were used with or without supplementation of crystalline amino......Matrixes of different protein sources (fish and plant products) combined with the use of crystalline amino acids allow for formulation of diets that meet fish requirements with little or no effect on protein digestibility and/or feed intake. Despite this, a total or partial replacement of fish meal...... acids to the fishmeal diet level (see Table 1). Amino acid uptake patterns were assessed by the appearance of amino acids in the blood stream following the ingestion of a meal, while dietary protein utilization was evaluated by examining the metabolic response to digestion and ammonium and urea...

Characterization of seed storage protein patterns of Heliotropium digynum.

Science.gov (United States)

Alwhibi, Mona Soliman

2017-09-01

Heliotropium digynum , is a shrub that has ecological importance. The height of the plant differs from one population to another and the difference in length of the inflorescence can be attributed to environmental factors, such as rainfall or type of soil and temperature. To date, no study has shed light on estimation in seed samples of H. digynum in Saudi Arabia. So, the aim is to evaluate and characterize the protein patterns of seed storage proteins of H. digynum to be used as fingerprint of this plant in Saudi Arabia. It is collected from different locations in the central region of Saudi Arabia and total protein extraction from plant was compared in SDS-PAGE. The genetic relationships among all cultivars were analyzed using UPGMA and NJ using Total Lab TL and in the same way using Jaccard Similarity Coefficient dendrogram using STATISTICA (ver.8) software. Results, our data show that amounts of protein are different, although they are of the same type or from the same geographical region. Amounts ranged between 22 and 1.5 mg/g of dry weight. Less amount of protein was obtained from the group of samples collected from Dir'iyah area, and the highest amount of protein was from the group of samples collected from Dyrab area in general.

Automated classification of immunostaining patterns in breast tissue from the human protein atlas.

Science.gov (United States)

Swamidoss, Issac Niwas; Kårsnäs, Andreas; Uhlmann, Virginie; Ponnusamy, Palanisamy; Kampf, Caroline; Simonsson, Martin; Wählby, Carolina; Strand, Robin

2013-01-01

The Human Protein Atlas (HPA) is an effort to map the location of all human proteins (http://www.proteinatlas.org/). It contains a large number of histological images of sections from human tissue. Tissue micro arrays (TMA) are imaged by a slide scanning microscope, and each image represents a thin slice of a tissue core with a dark brown antibody specific stain and a blue counter stain. When generating antibodies for protein profiling of the human proteome, an important step in the quality control is to compare staining patterns of different antibodies directed towards the same protein. This comparison is an ultimate control that the antibody recognizes the right protein. In this paper, we propose and evaluate different approaches for classifying sub-cellular antibody staining patterns in breast tissue samples. The proposed methods include the computation of various features including gray level co-occurrence matrix (GLCM) features, complex wavelet co-occurrence matrix (CWCM) features, and weighted neighbor distance using compound hierarchy of algorithms representing morphology (WND-CHARM)-inspired features. The extracted features are used into two different multivariate classifiers (support vector machine (SVM) and linear discriminant analysis (LDA) classifier). Before extracting features, we use color deconvolution to separate different tissue components, such as the brownly stained positive regions and the blue cellular regions, in the immuno-stained TMA images of breast tissue. We present classification results based on combinations of feature measurements. The proposed complex wavelet features and the WND-CHARM features have accuracy similar to that of a human expert. Both human experts and the proposed automated methods have difficulties discriminating between nuclear and cytoplasmic staining patterns. This is to a large extent due to mixed staining of nucleus and cytoplasm. Methods for quantification of staining patterns in histopathology have many

Automated classification of immunostaining patterns in breast tissue from the human protein Atlas

Directory of Open Access Journals (Sweden)

Issac Niwas Swamidoss

2013-01-01

Full Text Available Background: The Human Protein Atlas (HPA is an effort to map the location of all human proteins (http://www.proteinatlas.org/. It contains a large number of histological images of sections from human tissue. Tissue micro arrays (TMA are imaged by a slide scanning microscope, and each image represents a thin slice of a tissue core with a dark brown antibody specific stain and a blue counter stain. When generating antibodies for protein profiling of the human proteome, an important step in the quality control is to compare staining patterns of different antibodies directed towards the same protein. This comparison is an ultimate control that the antibody recognizes the right protein. In this paper, we propose and evaluate different approaches for classifying sub-cellular antibody staining patterns in breast tissue samples. Materials and Methods: The proposed methods include the computation of various features including gray level co-occurrence matrix (GLCM features, complex wavelet co-occurrence matrix (CWCM features, and weighted neighbor distance using compound hierarchy of algorithms representing morphology (WND-CHARM-inspired features. The extracted features are used into two different multivariate classifiers (support vector machine (SVM and linear discriminant analysis (LDA classifier. Before extracting features, we use color deconvolution to separate different tissue components, such as the brownly stained positive regions and the blue cellular regions, in the immuno-stained TMA images of breast tissue. Results: We present classification results based on combinations of feature measurements. The proposed complex wavelet features and the WND-CHARM features have accuracy similar to that of a human expert. Conclusions: Both human experts and the proposed automated methods have difficulties discriminating between nuclear and cytoplasmic staining patterns. This is to a large extent due to mixed staining of nucleus and cytoplasm. Methods for

Multiplex protein pattern unmixing using a non-linear variable-weighted support vector machine as optimized by a particle swarm optimization algorithm.

Science.gov (United States)

Yang, Qin; Zou, Hong-Yan; Zhang, Yan; Tang, Li-Juan; Shen, Guo-Li; Jiang, Jian-Hui; Yu, Ru-Qin

2016-01-15

Most of the proteins locate more than one organelle in a cell. Unmixing the localization patterns of proteins is critical for understanding the protein functions and other vital cellular processes. Herein, non-linear machine learning technique is proposed for the first time upon protein pattern unmixing. Variable-weighted support vector machine (VW-SVM) is a demonstrated robust modeling technique with flexible and rational variable selection. As optimized by a global stochastic optimization technique, particle swarm optimization (PSO) algorithm, it makes VW-SVM to be an adaptive parameter-free method for automated unmixing of protein subcellular patterns. Results obtained by pattern unmixing of a set of fluorescence microscope images of cells indicate VW-SVM as optimized by PSO is able to extract useful pattern features by optimally rescaling each variable for non-linear SVM modeling, consequently leading to improved performances in multiplex protein pattern unmixing compared with conventional SVM and other exiting pattern unmixing methods. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Protein immobilization on Ni(II) ion patterns prepared by microcontact printing and dip-pen nanolithography

NARCIS (Netherlands)

Wu, Chien-Ching; Reinhoudt, David N; Otto, Cees; Velders, Aldrik H; Subramaniam, Vinod

2010-01-01

An indirect method of protein patterning by using Ni(II) ion templates for immobilization via a specific metal-protein interaction is described. A nitrilotriacetic acid (NTA)-terminated self-assembled monolayer (SAM) allows oriented binding of histidine-tagged proteins via complexation with late

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

Patterned layers of adsorbed extracellular matrix proteins: influence on mammalian cell adhesion.

Science.gov (United States)

Dupont-Gillain, C C; Alaerts, J A; Dewez, J L; Rouxhet, P G

2004-01-01

Three patterned systems aiming at the control of mammalian cell behavior are presented. The determinant feature common to these systems is the spatial distribution of extracellular matrix (ECM) proteins (mainly collagen) on polymer substrates. This distribution differs from one system to another with respect to the scale at which it is affected, from the supracellular to the supramolecular scale, and with respect to the way it is produced. In the first system, the surface of polystyrene was oxidized selectively to form micrometer-scale patterns, using photolithography. Adsorption of ECM proteins in presence of a competitor was enhanced on the oxidized domains, allowing selective cell adhesion to be achieved. In the second system, electron beam lithography was used to engrave grooves (depth and width approximately 1 microm) on a poly(methyl methacrylate) (PMMA) substratum. No modification of the surface chemistry associated to the created topography could be detected. Cell orientation along the grooves was only observed when collagen was preadsorbed on the substratum. In the third system, collagen adsorbed on PMMA was dried in conditions ensuring the formation of a nanometer-scale pattern. Cell adhesion was enhanced on such patterned collagen layers compared to smooth collagen layers.

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

Characterization of seed storage protein patterns of Heliotropium digynum

Directory of Open Access Journals (Sweden)

Mona Soliman Alwhibi

2017-09-01

Full Text Available Heliotropium digynum, is a shrub that has ecological importance. The height of the plant differs from one population to another and the difference in length of the inflorescence can be attributed to environmental factors, such as rainfall or type of soil and temperature. To date, no study has shed light on estimation in seed samples of H. digynum in Saudi Arabia. So, the aim is to evaluate and characterize the protein patterns of seed storage proteins of H. digynum to be used as fingerprint of this plant in Saudi Arabia. It is collected from different locations in the central region of Saudi Arabia and total protein extraction from plant was compared in SDS-PAGE. The genetic relationships among all cultivars were analyzed using UPGMA and NJ using Total Lab TL and in the same way using Jaccard Similarity Coefficient dendrogram using STATISTICA (ver.8 software. Results, our data show that amounts of protein are different, although they are of the same type or from the same geographical region. Amounts ranged between 22 and 1.5 mg/g of dry weight. Less amount of protein was obtained from the group of samples collected from Dir’iyah area, and the highest amount of protein was from the group of samples collected from Dyrab area in general.

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...

Mycelium-Bound Lipase from a Locally Isolated Strain of Geotrichum candidum

Directory of Open Access Journals (Sweden)

Joo Ling Loo

2014-06-01

Full Text Available Mycelium-bound lipase (MBL, from a locally isolated Geotrichum candidum strain, was produced and characterized as a natural immobilized lipase. A time course study of its lipolytic activity in 1 L liquid broth revealed the maximum MBL activity at 4 h for mycelium cells harvested after 54 h. The yield and specific activity of MBL were 3.87 g/L dry weight and 508.33 U/g protein, respectively, while less than 0.2 U/mL lipase activity was detected in the culture supernatant. Prolonged incubation caused release of the bound lipase into the growth medium. The growth pattern of G. candidum, and production and properties of MBL were not affected by the scale. The stability of mycelia harboring lipase (MBL, harvested and lyophilized after 54 h, studied at 4 °C depicted a loss of 4.3% and 30% in MBL activity after 1 and 8 months, while the activity of free lipase was totally lost after 14 days of storage. The MBL from G. candidum displayed high substrate selectivity for unsaturated fatty acids containing a cis-9 double bond, even in crude form. This unique specificity of MBL could be a direct, simple and inexpensive way in the fats and oil industry for the selective hydrolysis or transesterification of cis-9 fatty acid residues in natural triacylglycerols.

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.

Automated Learning of Subcellular Variation among Punctate Protein Patterns and a Generative Model of Their Relation to Microtubules.

Directory of Open Access Journals (Sweden)

Gregory R Johnson

2015-12-01

Full Text Available Characterizing the spatial distribution of proteins directly from microscopy images is a difficult problem with numerous applications in cell biology (e.g. identifying motor-related proteins and clinical research (e.g. identification of cancer biomarkers. Here we describe the design of a system that provides automated analysis of punctate protein patterns in microscope images, including quantification of their relationships to microtubules. We constructed the system using confocal immunofluorescence microscopy images from the Human Protein Atlas project for 11 punctate proteins in three cultured cell lines. These proteins have previously been characterized as being primarily located in punctate structures, but their images had all been annotated by visual examination as being simply "vesicular". We were able to show that these patterns could be distinguished from each other with high accuracy, and we were able to assign to one of these subclasses hundreds of proteins whose subcellular localization had not previously been well defined. In addition to providing these novel annotations, we built a generative approach to modeling of punctate distributions that captures the essential characteristics of the distinct patterns. Such models are expected to be valuable for representing and summarizing each pattern and for constructing systems biology simulations of cell behaviors.

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.

Protein Adsorption Patterns and Analysis on IV Nanoemulsions-The Key Factor Determining the Organ Distribution.

Science.gov (United States)

Keck, Cornelia M; Jansch, Mirko; Müller, Rainer H

2012-12-21

Intravenous nanoemulsions have been on the market for parenteral nutrition since the 1950s; meanwhile, they have also been used successfully for IV drug delivery. To be well tolerable, the emulsions should avoid uptake by the MPS cells of the body; for drug delivery, they should be target-specific. The organ distribution is determined by the proteins adsorbing them after injection from the blood (protein adsorption pattern), typically analyzed by two-dimensional polyacrylamide gel electrophoresis, 2-D PAGE. The article reviews the 2-D PAGE method, the analytical problems to be faced and the knowledge available on how the composition of emulsions affects the protein adsorption patterns, e.g., the composition of the oil phase, stabilizer layer and drug incorporation into the interface or oil core. Data were re-evaluated and compared, and the implications for the in vivo distribution are discussed. Major results are that the interfacial composition of the stabilizer layer is the main determining factor and that this composition can be modulated by simple processes. Drug incorporation affects the pattern depending on the localization of the drug (oil core versus interface). The data situation regarding in vivo effects is very limited; mainly, it has to be referred to in the in vivo data of polymeric nanoparticles. As a conclusion, determination of the protein adsorption patterns can accelerate IV nanoemulsion formulation development regarding optimized organ distribution and related pharmacokinetics.

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.

Dietary protein is associated with musculoskeletal health independently of dietary pattern: the Framingham Third Generation Study.

Science.gov (United States)

Mangano, Kelsey M; Sahni, Shivani; Kiel, Douglas P; Tucker, Katherine L; Dufour, Alyssa B; Hannan, Marian T

2017-03-01

Background: Above-average dietary protein, as a single nutrient, improves musculoskeletal health. Evaluating the link between dietary protein and musculoskeletal health from a whole-diet perspective is important, as dietary guidelines focus on dietary patterns. Objective: We examined the prospective association of novel dietary protein food clusters (derived from established dietary pattern techniques) with appendicular lean mass (ALM), quadriceps strength (QS), and bone mineral density (BMD) in 2986 men and women, aged 19-72 y, from the Framingham Third Generation Study. Design: Total protein intake was estimated by food-frequency questionnaire in 2002-2005. A cluster analysis was used to classify participants into mutually exclusive groups, which were determined by using the percentage of contribution of food intake to overall protein intake. General linear modeling was used to 1 ) estimate the association between protein intake (grams per day) and BMD, ALM, appendicular lean mass normalized for height (ALM/ht 2 ), and QS (2008-2011) and to 2 ) calculate adjusted least-squares mean outcomes across quartiles of protein (grams per day) and protein food clusters. Results: The mean ± SD age of subjects was 40 ± 9 y; 82% of participants met the Recommended Daily Allowance (0.8 g · kg body weight -1 · d -1 ). The following 6 dietary protein food clusters were identified: fast food and full-fat dairy, fish, red meat, chicken, low-fat milk, and legumes. BMD was not different across quartiles of protein intake ( P -trend range = 0.32-0.82); but significant positive trends were observed for ALM, ALM/ht 2 ( P dietary protein is associated with ALM and QS but not with BMD. In this study, dietary protein food patterns do not provide further insight into beneficial protein effects on muscle outcomes. © 2017 American Society for Nutrition.

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

Antigenic properties of the human immunodeficiency virus envelope glycoprotein gp120 on virions bound to target cells.

Directory of Open Access Journals (Sweden)

Meron Mengistu

2015-03-01

Full Text Available The HIV-1 envelope glycoprotein, gp120, undergoes multiple molecular interactions and structural rearrangements during the course of host cell attachment and viral entry, which are being increasingly defined at the atomic level using isolated proteins. In comparison, antigenic markers of these dynamic changes are essentially unknown for single HIV-1 particles bound to target cells. Such markers should indicate how neutralizing and/or non-neutralizing antibodies might interdict infection by either blocking infection or sensitizing host cells for elimination by Fc-mediated effector function. Here we address this deficit by imaging fluorescently labeled CCR5-tropic HIV-1 pseudoviruses using confocal and superresolution microscopy to track the exposure of neutralizing and non-neutralizing epitopes as they appear on single HIV-1 particles bound to target cells. Epitope exposure was followed under conditions permissive or non-permissive for viral entry to delimit changes associated with virion binding from those associated with post-attachment events. We find that a previously unexpected array of gp120 epitopes is exposed rapidly upon target cell binding. This array comprises both neutralizing and non-neutralizing epitopes, the latter being hidden on free virions yet capable of serving as potent targets for Fc-mediated effector function. Under non-permissive conditions for viral entry, both neutralizing and non-neutralizing epitope exposures were relatively static over time for the majority of bound virions. Under entry-permissive conditions, epitope exposure patterns changed over time on subsets of virions that exhibited concurrent variations in virion contents. These studies reveal that bound virions are distinguished by a broad array of both neutralizing and non-neutralizing gp120 epitopes that potentially sensitize a freshly engaged target cell for destruction by Fc-mediated effector function and/or for direct neutralization at a post-binding step

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.

Effect of resistance training and protein intake pattern on myofibrillar protein synthesis and proteome kinetics in older men in energy restriction.

Science.gov (United States)

Murphy, Caoileann H; Shankaran, Mahalakshmi; Churchward-Venne, Tyler A; Mitchell, Cameron J; Kolar, Nathan M; Burke, Louise M; Hawley, John A; Kassis, Amira; Karagounis, Leonidas G; Li, Kelvin; King, Chelsea; Hellerstein, Marc; Phillips, Stuart M

2018-06-01

Strategies to enhance the loss of fat while preserving muscle mass during energy restriction are of great importance to prevent sarcopenia in overweight older adults. We show for the first time that the integrated rate of synthesis of numerous individual contractile, cytosolic and mitochondrial skeletal muscle proteins was increased by resistance training (RT) and unaffected by dietary protein intake pattern during energy restriction in free-living, obese older men. We observed a correlation between the synthetic rates of skeletal muscle-derived proteins obtained in serum (creatine kinase M-type, carbonic anhydrase 3) and the synthetic rates of proteins obtained via muscle sampling; and that the synthesis rates of these proteins in serum revealed the stimulatory effects of RT. These results have ramifications for understanding the influence of RT on skeletal muscle and are consistent with the role of RT in maintaining muscle protein synthesis and potentially supporting muscle mass preservation during weight loss. We determined how the pattern of protein intake and resistance training (RT) influenced longer-term (2 weeks) integrated myofibrillar protein synthesis (MyoPS) during energy restriction (ER). MyoPS and proteome kinetics were measured during 2 weeks of ER alone and 2 weeks of ER plus RT (ER + RT) in overweight/obese older men. Participants were randomized to consume dietary protein in a balanced (BAL: 25% daily protein per meal × 4 meals) or skewed (SKEW: 7:17:72:4% daily protein per meal) pattern (n = 10 per group). Participants ingested deuterated water during the consecutive 2-week periods, and skeletal muscle biopsies and serum were obtained at the beginning and conclusion of ER and ER + RT. Bulk MyoPS (i.e. synthesis of the myofibrillar protein sub-fraction) and the synthetic rates of numerous individual skeletal muscle proteins were quantified. Bulk MyoPS was not affected by protein distribution during ER or ER + RT (ER: BAL = 1.24�

A Pilot Study on Gait Kinematics of Old Women with Bound Feet

Directory of Open Access Journals (Sweden)

Yan Zhang

2015-01-01

Full Text Available Foot binding has a long and influential history in China. Little is known about biomechanical changes in gait caused by bound foot. The purpose of this study was to investigate the differences in lower limb kinematics between old women with bound feet and normal feet during walking. Six old women subjects (three with bound feet and three controls with normal feet volunteered to participate in this study. Video data were recorded with a high speed video camera and analysed in the SIMI motion analysis software. Compared to normal controls, bound feet subjects had faster gait cadence with shorter stride length as well as smaller ankle and knee range of motion (ROM. During preswing phase, ankle remained to be dorsiflexion for bound foot subjects. The data from bound foot group also demonstrated that toe vertical displacement increased continuously during whole swing phase without a minimum toe clearance (MTC. The findings indicate that older women with bound feet exhibit significant differences in gait pattern compared to those with normal feet, which is characterised by disappeared propulsion/push-off and reduced mobility of lower limb segments.

Effect of Gamma Radiation and Electron Beam on Microbiological Quality and Protein Patterns of 4 Selected Beans

International Nuclear Information System (INIS)

Chookaew, S.; Eamsir, J.; Pewlong, W.; Sajjabut, S.

2014-01-01

The aim of the present study was to evaluate the effect of gamma ray and electron beam on microbiological quality and protein pattern of four selected beans: mung beans, soy beans, peanuts and black beans. All beans samples were exposed to irradiation at doses of 0, 0.5, 1, and 2 kGy before evaluated for their microbiological quality using AOAC method and protein analysis by gel electrophoresis. Results showed that the amount of bacteria, yeast and mold of irradiated mung beans and peanuts were reduced, whereas these microbiological quality values remained relatively the same for irradiated soy beans and black beans compared to non-irradiated samples. In terms of protein analysis, the protein patterns of the irradiated beans were of the same quality as the non-irradiated samples. To further tested the effect of irradiation on the bean's protein at higher doses, all four selected beans were exposed to gamma ray at 10, 50, 100, 150 and 200 kGy. We found that the protein patterns of mung beans, peanuts and black beans were altered at doses above 50 kGy.

Protein Adsorption Patterns and Analysis on IV Nanoemulsions—The Key Factor Determining the Organ Distribution

Directory of Open Access Journals (Sweden)

Mirko Jansch

2012-12-01

Full Text Available Intravenous nanoemulsions have been on the market for parenteral nutrition since the 1950s; meanwhile, they have also been used successfully for IV drug delivery. To be well tolerable, the emulsions should avoid uptake by the MPS cells of the body; for drug delivery, they should be target-specific. The organ distribution is determined by the proteins adsorbing them after injection from the blood (protein adsorption pattern, typically analyzed by two-dimensional polyacrylamide gel electrophoresis, 2-D PAGE. The article reviews the 2-D PAGE method, the analytical problems to be faced and the knowledge available on how the composition of emulsions affects the protein adsorption patterns, e.g., the composition of the oil phase, stabilizer layer and drug incorporation into the interface or oil core. Data were re-evaluated and compared, and the implications for the in vivo distribution are discussed. Major results are that the interfacial composition of the stabilizer layer is the main determining factor and that this composition can be modulated by simple processes. Drug incorporation affects the pattern depending on the localization of the drug (oil core versus interface. The data situation regarding in vivo effects is very limited; mainly, it has to be referred to in the in vivo data of polymeric nanoparticles. As a conclusion, determination of the protein adsorption patterns can accelerate IV nanoemulsion formulation development regarding optimized organ distribution and related pharmacokinetics.

Lipid recognition propensities of amino acids in membrane proteins from atomic resolution data

Directory of Open Access Journals (Sweden)

Morita Mizuki

2011-12-01

Full Text Available Abstract Background Protein-lipid interactions play essential roles in the conformational stability and biological functions of membrane proteins. However, few of the previous computational studies have taken into account the atomic details of protein-lipid interactions explicitly. Results To gain an insight into the molecular mechanisms of the recognition of lipid molecules by membrane proteins, we investigated amino acid propensities in membrane proteins for interacting with the head and tail groups of lipid molecules. We observed a common pattern of lipid tail-amino acid interactions in two different data sources, crystal structures and molecular dynamics simulations. These interactions are largely explained by general lipophilicity, whereas the preferences for lipid head groups vary among individual proteins. We also found that membrane and water-soluble proteins utilize essentially an identical set of amino acids for interacting with lipid head and tail groups. Conclusions We showed that the lipophilicity of amino acid residues determines the amino acid preferences for lipid tail groups in both membrane and water-soluble proteins, suggesting that tightly-bound lipid molecules and lipids in the annular shell interact with membrane proteins in a similar manner. In contrast, interactions between lipid head groups and amino acids showed a more variable pattern, apparently constrained by each protein's specific molecular function.

Lipid recognition propensities of amino acids in membrane proteins from atomic resolution data

International Nuclear Information System (INIS)

Morita, Mizuki; Katta, AVSK Mohan; Ahmad, Shandar; Mori, Takaharu; Sugita, Yuji; Mizuguchi, Kenji

2011-01-01

Protein-lipid interactions play essential roles in the conformational stability and biological functions of membrane proteins. However, few of the previous computational studies have taken into account the atomic details of protein-lipid interactions explicitly. To gain an insight into the molecular mechanisms of the recognition of lipid molecules by membrane proteins, we investigated amino acid propensities in membrane proteins for interacting with the head and tail groups of lipid molecules. We observed a common pattern of lipid tail-amino acid interactions in two different data sources, crystal structures and molecular dynamics simulations. These interactions are largely explained by general lipophilicity, whereas the preferences for lipid head groups vary among individual proteins. We also found that membrane and water-soluble proteins utilize essentially an identical set of amino acids for interacting with lipid head and tail groups. We showed that the lipophilicity of amino acid residues determines the amino acid preferences for lipid tail groups in both membrane and water-soluble proteins, suggesting that tightly-bound lipid molecules and lipids in the annular shell interact with membrane proteins in a similar manner. In contrast, interactions between lipid head groups and amino acids showed a more variable pattern, apparently constrained by each protein's specific molecular function

‘Gas Syndrome’ - A Culture Bound Syndrome

Directory of Open Access Journals (Sweden)

Anil Kakunje

2014-02-01

Full Text Available Culture refers to the shared patterns of feelings, beliefs and behaviour that reflect in the way of living in a society. Culture uniquely influence the role functioning or psychosoical wellbeing of people living in a given society by exerting influence on their mind by their traditional health beliefs. Cultural factors influence understanding, presentation, diagnosis, management, course and outcome of many diseases, especially psychiatric disorders. Culture-bound syndromes seem to be episodic, dramatic and discrete patterns of behavioral reactions specific to a particular community that articulate both personal predicament and public concerns. Every culture provides explanations and causal attributions for somatic symptoms. One of the common complaints of persons coming to medical attention is ‘Gas’ or similar terminologies like ‘vayu’ etc. People attribute varied symptoms from abdominal discomfort, chest pain, headache, joint pains, back pain, somatic complaints to ‘Gas’. ‘Gas’ is reported to be the cause for the distress and the primary duty of the treating clinician is to relieve them of the gas. The problem of troubling Gas or vayu has been influencing Indian culture/tradition since ancient days. We do see a significant proportion of patients visiting varied specialists attributing all their problems to Gas. 'Gas Syndrome’ is proposed as a culture bound syndrome.

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

MAMP (microbe-associated molecular pattern)-induced changes in plasma membrane-associated proteins.

Science.gov (United States)

Uhlíková, Hana; Solanský, Martin; Hrdinová, Vendula; Šedo, Ondrej; Kašparovský, Tomáš; Hejátko, Jan; Lochman, Jan

2017-03-01

Plant plasma membrane associated proteins play significant roles in Microbe-Associated Molecular Pattern (MAMP) mediated defence responses including signal transduction, membrane transport or energetic metabolism. To elucidate the dynamics of proteins associated with plasma membrane in response to cryptogein, a well-known MAMP of defence reaction secreted by the oomycete Phytophthora cryptogea, 2D-Blue Native/SDS gel electrophoresis of plasma membrane fractions was employed. This approach revealed 21 up- or down-regulated protein spots of which 15 were successfully identified as proteins related to transport through plasma membrane, vesicle trafficking, and metabolic enzymes including cytosolic NADP-malic enzyme and glutamine synthetase. Observed changes in proteins were also confirmed on transcriptional level by qRT-PCR analysis. In addition, a significantly decreased accumulation of transcripts observed after employment of a mutant variant of cryptogein Leu41Phe, exhibiting a conspicuous defect in induction of resistance, sustains the contribution of identified proteins in cryptogein-triggered cellular responses. Our data provide further evidence for dynamic MAMP-induced changes in plasma membrane associated proteins. Copyright © 2016 Elsevier GmbH. All rights reserved.

Changes in the serum protein electrophoretic pattern in lambs during the first month of life

Directory of Open Access Journals (Sweden)

Oskar Nagy

2014-01-01

Full Text Available Studies of the changes in serum protein pattern in the neonatal period in animals are still limited. Therefore, the objective of this study was to evaluate the changes in the concentrations of serum protein fractions in 7 clinically healthy merino lambs (4 males, 3 females during their first month of life. The first blood sampling was performed before the colostrum intake and then at 1, 2, 7, 14 and 30 days of age. Blood serum was analysed for total serum protein concentrations and for the relative and absolute values of serum protein fractions - albumin, alpha1- (α1, alpha2- (α2, beta- (β, and gamma- (γ globulins. The results showed a significant effect of age on the serum total protein concentrations and for all the protein fractions. The concentrations of total proteins and γ-globulins increased significantly 1 day after the colostrum intake (P P 1-globulins significantly decreased during the first month of life (P 2- and β-globulins increased significantly from birth till the end of the monitored period (P < 0.001. Our results suggest that the serum protein electrophoretic pattern in growing lambs is significantly influenced by the age of the evaluated animal, and this should be taken into consideration when interpreting the serum protein profile. Our findings extend existing knowledge about significant changes in the protein profile associated with the physiological adaptation process in the neonatal period in young animals.

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.

Study on dissolution behavior of polymer-bound and polymer-blended photo-acid generator (PAG) resists

Science.gov (United States)

Yamamoto, Hiroki; Kozawa, Takahiro; Tagawa, Seiichi

2013-03-01

The requirements for the next generation resist materials are so challenging that it is indispensable for feasibility of EUV lithography to grasp basic chemistry of resist matrices in all stage of resist processes. Under such circumstances, it is very important to know dissolution characteristics of the resist film into alkaline developer though the dissolution of exposed area of resist films in alkaline developer to form a pattern is a complex reactive process. In this study, the influence of EUV and KrF exposure on the dissolution behavior of polymer bound PAG and polymer blended PAG was studied in detail using quartz crystal microbalance (QCM) methods. The difference in swelling formation between KrF and EUV exposure was observed. It is likely that difference of reaction mechanism induces the difference of these swelling. Also, it is observed that the swelling of polymer-bound PAG is less than that of polymer blended PAG in both KrF and EUV exposure. This result indicates that polymer-bound PAG suppresses swelling very well and showed an excellent performance. Actually, the developed polymer bound-PAG resist showed an excellent performance (half pitch 50 nm line and space pattern). Thus, polymer bound PAG is one of the promising candidate for 16 nm EUV resist.

Radiation induced formation of giant cells in Saccharomyces uvarum. Pt. 4. Macromolecular synthesis and protein patterns

Energy Technology Data Exchange (ETDEWEB)

Rink, H; Baumstark-Khan, C; Partke, H J

1986-08-01

X-irradiated (1.0 kGy) yeast cells (Saccharomyces uvarum, ATCC 9080), grown in liquid medium stop their mitotic activities and form giant cells by development of several buds which do not separate from mother cells. Depending on the time in culture, wet and dry weights per cell, protein- RNA- and DNA- contents per cell as well as incorporation rates of /sup 14/C-leucine per cell and per hour and patterns (isoelectric focusing) of water soluble proteins were studied. Weights per cell, RNA and protein contents per cell and /sup 14/C-leucine incorporation rates increase markedly in giant cells, whereas DNA content per cell is only duplicated. Protein patterns in isoelectric focusing show one interesting difference. In samples from giant cells one protein band (IP=6.63) decreases after 8 h in culture and later on disappears completely. This finding is not due to primary damage in X-irradiated DNA but seems to be related to the control of cell cycle events.

UV lithography-based protein patterning on silicon: Towards the integration of bioactive surfaces and CMOS electronics

Energy Technology Data Exchange (ETDEWEB)

Lenci, S., E-mail: silvia.lenci@iet.unipi.it [Dipartimento di Ingegneria dell' Informazione, via G.Caruso 16, Pisa I-56122 (Italy); Tedeschi, L. [Istituto di Fisiologia Clinica - CNR, via G. Moruzzi 1, Pisa I-56124 (Italy); Pieri, F. [Dipartimento di Ingegneria dell' Informazione, via G.Caruso 16, Pisa I-56122 (Italy); Domenici, C. [Istituto di Fisiologia Clinica - CNR, via G. Moruzzi 1, Pisa I-56124 (Italy)

2011-08-01

A simple and fast methodology for protein patterning on silicon substrates is presented, providing an insight into possible issues related to the interaction between biological and microelectronic technologies. The method makes use of standard photoresist lithography and is oriented towards the implementation of biosensors containing Complementary Metal-Oxide-Semiconductor (CMOS) conditioning circuitry. Silicon surfaces with photoresist patterns were prepared and hydroxylated by means of resist- and CMOS backend-compatible solutions. Subsequent aminosilane deposition and resist lift-off in organic solvents resulted into well-controlled amino-terminated geometries. The discussion is focused on resist- and CMOS-compatibility problems related to the used chemicals. Some samples underwent gold nanoparticle (Au NP) labeling and Scanning Electron Microscopy (SEM) observation, in order to investigate the quality of the silane layer. Antibodies were immobilized on other samples, which were subsequently exposed to a fluorescently labeled antigen. Fluorescence microscopy observation showed that this method provides spatially selective immobilization of protein layers onto APTES-patterned silicon samples, while preserving protein reactivity inside the desired areas and low non-specific adsorption elsewhere. Strong covalent biomolecule binding was achieved, giving stable protein layers, which allows stringent binding conditions and a good binding specificity, really useful for biosensing.

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.

Culture-bound syndromes in Hispanic primary care patients.

Science.gov (United States)

Bayles, Bryan P; Katerndahl, David A

2009-01-01

We sought to document Hispanic primary care patients' knowledge and experience of five culture-bound syndromes (CBS), as well as the basic socio-cultural correlates of these disorders. A convenience sample of 100 adult Hispanic patients presenting in an urban South Texas primary care clinic was recruited to complete a brief cross-sectional survey, presented in an oral format. Interviews sought information concerning five culture-bound syndromes--susto, empacho, nervios, mal de ojo, and ataques de nervios. Additional demographic, socio-economic, and acculturation data was collected. Descriptive and bivariate statistics (chi square, Fisher's) were used to assess relationships among variables and experience with each CBS. A multivariate logistic analysis was conducted to determine the possible contributions of age, gender, acculturation, and education to the personal experience of a culture-bound syndrome. Results indicate that 77% of respondents had knowledge of all five syndromes, with 42% reporting having personally experienced at least one CBS. Nervios was the most commonly suffered disorder, being reported by 30 respondents. This was followed, in declining order ofprevalence, by susto, mal de ojo, empacho, and ataques de nervios. Multivariate logistic regression analysis found that higher education beyond high school was associated with a slightly decreased likelihood of reporting having suffered from any culture-bound syndrome. While co-occurrence among these disorders occurred, the patterns of predictors suggest that the co-occurrence is not a reflection of mislabeling of one common syndrome. Knowledge of and experience with culture-bound syndromes is common among Hispanic primary care patients in South Texas. Healthcare providers ought to consider discussing these illnesses in a non-judgmental manner with patients who present with symptoms that are consistent with these syndromes. Future studies, with larger sample sizes, are warranted to elucidate the nature

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 ...

Protein secretory patterns of rat Sertoli and peritubular cells are influenced by culture conditions

International Nuclear Information System (INIS)

Kierszenbaum, A.L.; Crowell, J.A.; Shabanowitz, R.B.; DePhilip, R.M.; Tres, L.L.

1986-01-01

An approach combining two-dimensional gel electrophoresis and autoradiography was used to correlate patterns of secretory proteins in cultures of Sertoli and peritubular cells with those observed in the incubation medium from segments of seminiferous tubules. Sertoli cells in culture and in seminiferous tubules secreted three proteins designated S70 (Mr 72,000-70,000), S45 (Mr 45,000), and S35 (Mr 35,000). Cultured Sertoli and peritubular cells and incubated seminiferous tubules secreted two proteins designated SP1 (Mr 42,000) and SP2 (Mr 50,000). SP1 and S45 have similar Mr but differ from each other in isoelectric point (pI). Cultured peritubular cells secreted a protein designated P40 (Mr 40,000) that was also seen in intact seminiferous tubules but not in seminiferous tubules lacking the peritubular cell wall. However, a large number of high-Mr proteins were observed only in the medium of cultured peritubular cells but not in the incubation medium of intact seminiferous tubules. Culture conditions influence the morphology and patterns of protein secretion of cultured peritubular cells. Peritubular cells that display a flat-stellate shape transition when placed in culture medium free of serum (with or without hormones and growth factors), accumulate various proteins in the medium that are less apparent when these cells are maintained in medium supplemented with serum. Two secretory proteins stimulated by follicle-stimulating hormone (FSH) (designated SCm1 and SCm2) previously found in the medium of cultured Sertoli cells, were also observed in the incubation medium of seminiferous tubular segments stimulated by FSH. Results of this study show that, although cultured Sertoli and peritubular cells synthesize and secrete proteins also observed in segments of incubated seminiferous tubules anther group of proteins lacks seminiferous tubular correlates

Protein and alkaloid patterns of the floral nectar in some solanaceous species.

Science.gov (United States)

Kerchner, András; Darók, Judit; Bacskay, Ivett; Felinger, Attila; Jakab, Gábor; Farkas, Ágnes

2015-09-01

The family Solanaceae includes several melliferous plants, which tend to produce copious amounts of nectar. Floral nectar is a chemically complex aqueous solution, dominated by sugars, but minor components such as amino acids, proteins, flavonoids and alkaloids are present as well. This study aimed at analysing the protein and alkaloid profile of the nectar in seven solanaceous species. Proteins were examined with SDS-PAGE and alkaloids were analyzed with HPLC. The investigation of protein profile revealed significant differences in nectar-protein patterns not only between different plant genera, but also between the three Nicotiana species investigated. SDS-PAGE suggested the presence of several Nectarin proteins with antimicrobial activity in Nicotiana species. The nectar of all tobacco species contained the alkaloid nicotine, N. tabacum having the highest nicotine content. The nectar of Brugmansia suaveolens, Datura stramonium, Hyoscyamus niger and Lycium barbarum contained scopolamine, the highest content of which was measured in B. suaveolens. The alkaloid concentrations in the nectars of most solanaceous species investigated can cause deterrence in honeybees, and the nectar of N. rustica and N. tabacum can be considered toxic for honeybees.

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.

Upper bound of abutment scour in laboratory and field data

Science.gov (United States)

Benedict, Stephen

2016-01-01

The U.S. Geological Survey, in cooperation with the South Carolina Department of Transportation, conducted a field investigation of abutment scour in South Carolina and used those data to develop envelope curves that define the upper bound of abutment scour. To expand on this previous work, an additional cooperative investigation was initiated to combine the South Carolina data with abutment scour data from other sources and evaluate upper bound patterns with this larger data set. To facilitate this analysis, 446 laboratory and 331 field measurements of abutment scour were compiled into a digital database. This extensive database was used to evaluate the South Carolina abutment scour envelope curves and to develop additional envelope curves that reflected the upper bound of abutment scour depth for the laboratory and field data. The envelope curves provide simple but useful supplementary tools for assessing the potential maximum abutment scour depth in the field setting.

Effects of salt on the pattern of protein synthesis in barley roots

International Nuclear Information System (INIS)

Hurkman, W.J.; Tanaka, C.K.

1987-01-01

The effect of salt stress on the incorporation of [ 3 5 S]methionine into protein was examined in roots of barley (Hordeum vulgare L.cv California Mariout 72). Plants were grown in nutrient solution with or without 200 millimolar NaCl. Roots of intact plants were labeled in vivo and proteins were extracted and analyzed by fluorography of two-dimensional gels. Although the protein patterns for control and salt-stressed plants were qualitatively similar, the net synthesis of a number of proteins was quantitatively changed. The most striking change was a significant increase of label in two protein pairs that had pls of approximately 6.3 and 6.5. Each pair consisted of proteins of approximately 26 and 27 kilodaltons (kD). In roots of control plants, the 27-kD proteins were more heavily labeled in the microsomal fraction relative to the 26-kD proteins, whereas the 26-kD proteins were enriched in the post 178,000g supernatant fraction; in roots of salt treated plants, the 26- and 27-kD proteins were more intensely labeled in both fractions. Labeling of the 26- and 27-kD proteins returned to control levels when salt-stressed plants were transferred to nutrient solution without NaCl. No cross-reaction was detected between the antibody to the 26-kD protein from salt-adapted tobacco cells and the 26- and 27-kD proteins of barley

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

Inheritance of Protein Patterns in a Synthetic Allopolyploid of Triticum Monococcum (AA) and Aegilops Ventricosa (DDMvMv)

DEFF Research Database (Denmark)

Siddiqui, K. A.; Ingversen, J.; Køie, B.

1972-01-01

Patterns of seed proteins in Triticum monococcum (2n = 2x = 14 = AA), Aegilops ventricosa (2n = 4x = 28 = DDMVMV), and their synthetic amphiploid were studied. The distribution of proteins in the individual Osborne protein fractions of the amphiploid was characterized by a 14 per cent increase...

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

Directory of Open Access Journals (Sweden)

Barbara Thiem

2014-01-01

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

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.

Authentication of Whey Protein Powders by Portable Mid-Infrared Spectrometers Combined with Pattern Recognition Analysis.

Science.gov (United States)

Wang, Ting; Tan, Siow Ying; Mutilangi, William; Aykas, Didem P; Rodriguez-Saona, Luis E

2015-10-01

The objective of this study was to develop a simple and rapid method to differentiate whey protein types (WPC, WPI, and WPH) used for beverage manufacturing by combining the spectral signature collected from portable mid-infrared spectrometers and pattern recognition analysis. Whey protein powders from different suppliers are produced using a large number of processing and compositional variables, resulting in variation in composition, concentration, protein structure, and thus functionality. Whey protein powders including whey protein isolates, whey protein concentrates and whey protein hydrolysates were obtained from different suppliers and their spectra collected using portable mid-infrared spectrometers (single and triple reflection) by pressing the powder onto an Attenuated Total Reflectance (ATR) diamond crystal with a pressure clamp. Spectra were analyzed by soft independent modeling of class analogy (SIMCA) generating a classification model showing the ability to differentiate whey protein types by forming tight clusters with interclass distance values of >3, considered to be significantly different from each other. The major bands centered at 1640 and 1580 cm(-1) were responsible for separation and were associated with differences in amide I and amide II vibrations of proteins, respectively. Another important band in whey protein clustering was associated with carboxylate vibrations of acidic amino acids (∼1570 cm(-1)). The use of a portable mid-IR spectrometer combined with pattern recognition analysis showed potential for discriminating whey protein ingredients that can help to streamline the analytical procedure so that it is more applicable for field-based screening of ingredients. A rapid, simple and accurate method was developed to authenticate commercial whey protein products by using portable mid-infrared spectrometers combined with chemometrics, which could help ensure the functionality of whey protein ingredients in food applications. © 2015

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

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.

Discovering approximate-associated sequence patterns for protein-DNA interactions

KAUST Repository

Chan, Tak Ming

2010-12-30

Motivation: The bindings between transcription factors (TFs) and transcription factor binding sites (TFBSs) are fundamental protein-DNA interactions in transcriptional regulation. Extensive efforts have been made to better understand the protein-DNA interactions. Recent mining on exact TF-TFBS-associated sequence patterns (rules) has shown great potentials and achieved very promising results. However, exact rules cannot handle variations in real data, resulting in limited informative rules. In this article, we generalize the exact rules to approximate ones for both TFs and TFBSs, which are essential for biological variations. Results: A progressive approach is proposed to address the approximation to alleviate the computational requirements. Firstly, similar TFBSs are grouped from the available TF-TFBS data (TRANSFAC database). Secondly, approximate and highly conserved binding cores are discovered from TF sequences corresponding to each TFBS group. A customized algorithm is developed for the specific objective. We discover the approximate TF-TFBS rules by associating the grouped TFBS consensuses and TF cores. The rules discovered are evaluated by matching (verifying with) the actual protein-DNA binding pairs from Protein Data Bank (PDB) 3D structures. The approximate results exhibit many more verified rules and up to 300% better verification ratios than the exact ones. The customized algorithm achieves over 73% better verification ratios than traditional methods. Approximate rules (64-79%) are shown statistically significant. Detailed variation analysis and conservation verification on NCBI records demonstrate that the approximate rules reveal both the flexible and specific protein-DNA interactions accurately. The approximate TF-TFBS rules discovered show great generalized capability of exploring more informative binding rules. © The Author 2010. Published by Oxford University Press. All rights reserved.

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.

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.

Alteration of protein patterns in black rock inhabiting fungi as a response to different temperatures

Science.gov (United States)

Tesei, Donatella; Marzban, Gorji; Zakharova, Kristina; Isola, Daniela; Selbmann, Laura; Sterflinger, Katja

2012-01-01

Rock inhabiting fungi are among the most stress tolerant organisms on Earth. They are able to cope with different stressors determined by the typical conditions of bare rocks in hot and cold extreme environments. In this study first results of a system biological approach based on two-dimensional protein profiles are presented. Protein patterns of extremotolerant black fungi – Coniosporium perforans, Exophiala jeanselmei – and of the extremophilic fungus – Friedmanniomyces endolithicus – were compared with the cosmopolitan and mesophilic hyphomycete Penicillium chrysogenum in order to follow and determine changes in the expression pattern under different temperatures. The 2D protein gels indicated a temperature dependent qualitative change in all the tested strains. Whereas the reference strain P. chrysogenum expressed the highest number of proteins at 40 °C, thus exhibiting real signs of temperature induced reaction, black fungi, when exposed to temperatures far above their growth optimum, decreased the number of proteins indicating a down-regulation of their metabolism. Temperature of 1 °C led to an increased number of proteins in all of the analysed strains, with the exception of P. chrysogenum. These first results on temperature dependent reactions in rock inhabiting black fungi indicate a rather different strategy to cope with non-optimal temperature than in the mesophilic hyphomycete P. chrysogenum. PMID:22862921

Disneyization: A framework for understanding illicit drug use in bounded play spaces.

Science.gov (United States)

Turner, Tim

2018-05-17

This paper combines evidence from an ethnographic study of illicit drug use amongst tourists in Ibiza with Bryman's (2004) theoretical model of Disneyization. The principal aim was to construct a new conceptual framework that may help scholars, practitioners and policy makers make sense of dynamic patterns of illegal drug use across bounded play spaces such as tourist resorts, music festivals and nightclubs. Ethnographic fieldwork employing a grounded theory design was undertaken over three summers in tourist locations on the Balearic island of Ibiza, including nightclubs, bars, cafes, beaches, airports and hotels. Field notes from participant observation were supplemented with data from semi-structured interviews (n = 56) and secondary sources gathered from tourist marketing. The framework of Disneyization has been discussed in terms of 5 constructs: theming, hybrid consumption, branding, performative labour and atmospheres; each having a specific role in relation to understanding illicit drug use in bounded play spaces. Thus: Theming sets the stage, by physically and symbolically demarcating space with indelible themes of hedonism that open up the possibility of illicit drug use. Hybrid-consumption blurs the distinction between legal and illegal forms of intoxication, making the trading and consumption of illegal drugs appear like a natural feature of the consumer space. Branding demonstrates how participants construct intricate hierarchies of taste and credibility related to drug of choice. Performative labour re-enforces hybrid consumption, with participants working in the bounded play spaces of Ibiza immersed within the illicit drug market. Atmospheres represents the alchemic synergy of bounded play space and is important to understanding illicit drug use as a sensorial, deeply immersive but transitory experience. This research offers Disneyization as a new conceptual framework for making sense of deeply complex spatial, socio-cultural, psychological and

THE UNCOVERING OF A NOVEL REGULATORY MECHANISM FOR PLD2: FORMATION OF A TERNARY COMPLEX WITH PROTEIN TYROSINE PHOSPHATASE PTP1B AND GROWTH FACTOR RECEPTOR-BOUND PROTEIN GRB2

Science.gov (United States)

Horn, Jeff; Lopez, Isabel; Miller, Mill; Gomez-Cambronero, Julian

2011-01-01

The regulation of PLD2 activation is poorly understood at present. Transient transfection of COS-7 with a mycPLD2 construct results in elevated levels of PLD2 enzymatic activity and tyrosyl phosphorylation. To investigate whether this phosphorylation affects PLD2 enzymatic activity, anti-myc immunoprecipitates were treated with recombinant protein tyrosine phosphatase PTP1B. Surprisingly, lipase activity and PY levels both increased over a range of PTP1B concentrations. These increases occurred in parallel to a measurable PTP1B-associated phosphatase activity. Inhibitor studies demonstrated that an EGF-receptor type kinase is involved in phosphorylation. In a COS-7 cell line created in the laboratory that stably expressed myc-PLD2, PTP1B induced a robust (>6-fold) augmentation of myc-PLD2 phosphotyrosine content. The addition of growth factor receptor-bound protein 2 (Grb2) to cell extracts also elevated PY levels of myc-PLD (>10-fold). Systematic co-immunoprecipitation-immunoblotting experiments pointed at a physical association between PLD2, Grb2 and PTP1B in both physiological conditions and in overexpressed cells. This is the first report of a demonstration of the mammalian isoform PLD2 existing in a ternary complex with a protein tyrosine phosphatase, PTP1b, and the docking protein Grb2 which greatly enhances tyrosyl phosphorylation of the lipase. PMID:15896299

Bound and free waves in non-collinear second harmonic generation.

Science.gov (United States)

Larciprete, M C; Bovino, F A; Belardini, A; Sibilia, C; Bertolotti, M

2009-09-14

We analyze the relationship between the bound and the free waves in the noncollinear SHG scheme, along with the vectorial conservation law for the different components arising when there are two pump beams impinging on the sample with two different incidence angles. The generated power is systematically investigated, by varying the polarization state of both fundamental beams, while absorption is included via the Herman and Hayden correction terms. The theoretical simulations, obtained for samples which are some coherence length thick show that the resulting polarization mapping is an useful tool to put in evidence the interference between bound and free waves, as well as the effect of absorption on the interference pattern.

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...

The duck hepatitis B virus polymerase and core proteins accumulate in different patterns from their common mRNA

International Nuclear Information System (INIS)

Yao Ermei; Schaller, Heinz; Tavis, John E.

2003-01-01

Hepadnaviral reverse transcription occurs in capsids in which the core (C) protein surrounds the reverse transcriptase (P) and pregenomic RNA (pgRNA). We analyzed the accumulation patterns of duck hepatitis B virus P, C, and pgRNA in transfected LMH cells, infected primary duck hepatocytes (PDH), and infected duck liver. In all three systems, P accumulated over time in a different pattern compared with C, despite translation of both proteins from the pgRNA. Although the accumulation patterns of the proteins varied between the systems, in each case P became detectable at the same time or earlier than C and the ratio of P relative to C dropped with time. These accumulation patterns were consistent with the translation rates and half-lives of P and C. Comparing the translation rates of P and C with the pgRNA level over time revealed that translation of P and C was negatively regulated in LMH cells. These data provide a framework for comparing replication studies performed in LMH cells, PDHs and ducks

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.

Transcriptome of E. coli K1 bound to human brain microvascular endothelial cells

OpenAIRE

Xie, Yi; Parthasarathy, Geetha; Di Cello, Francescopaolo; Teng, Ching-Hao; Paul-Satyaseela, Maneesh; Kim, Kwang Sik

2007-01-01

Escherichia coli K1 is the most common Gram-negative organism causing neonatal meningitis. Binding to human brain microvascdular endothelial cells (HBMEC) is an essential step for E. coli K1 traversal of the blood-brain barrier. In this study, we examined expression profiles of E. coli K1 strain RS218 during its binding to HBMEC. Comparison of HBMEC-bound E. coli K1 with collagen-bound E. coli revealed more than one hundred genes whose expression patterns were significantly changed in HBMEC-b...

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

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.

Complement activating soluble pattern recognition molecules with collagen-like regions, mannan-binding lectin, ficolins and associated proteins

DEFF Research Database (Denmark)

Thiel, Steffen

2007-01-01

Mannan-binding lectin (MBL), L-ficolin, M-ficolin and H-ficolin are all complement activating soluble pattern recognition molecules with recognition domains linked to collagen-like regions. All four may form complexes with four structurally related proteins, the three MBL-associated serine...... proteases (MASPs), MASP-1, MASP-2 and MASP-3, and a smaller MBL-associated protein (MAp19). The four recognition molecules recognize patterns of carbohydrate or acetyl-group containing ligands. After binding to the relevant targets all four are able to activate the complement system. We thus have a system...... where four different and/or overlapping patterns of microbial origin or patterns of altered-self may be recognized, but in all cases the signalling molecules, the MASPs, are shared. MASP-1 and MASP-3 are formed from one gene, MASP1/3, by alternative splicing generating two different mRNAs from a single...

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.

Flexible method for fabricating protein patterns on superhydrophobic platforms controlled by magnetic field.

Science.gov (United States)

Wang, Jian; Li, Hao; Zou, Haoyang; Wang, Chenmiao; Zhang, Hao; Mano, João F; Song, Wenlong

2017-02-28

Inspired by the rolling of water droplets on lotus leaves, we developed a novel, magnetic field-controlled patterning method for water-soluble proteins and other functional materials on superhydrophobic platforms. This simple method can be used to fabricate biochips and open micro-fluidic devices in a simple way.

A SIMPLE FLUORESCENT LABELING METHOD FOR STUDIES OF PROTEIN OXIDATION, PROTEIN MODIFICATION, AND PROTEOLYSIS

Science.gov (United States)

Pickering, Andrew. M.; Davies, Kelvin. J. A.

2014-01-01

Proteins are sensitive to oxidation, and oxidized proteins are excellent substrates for degradation by proteolytic enzymes such as the Proteasome and the mitochondrial Lon protease. Protein labeling is required for studies of protein turnover. Unfortunately, most labeling techniques involve 3H or 14C methylation which is expensive, exposes researchers to radioactivity, generates large amounts of radioactive waste, and allows only single-point assays because samples require acid-precipitation. Alternative labeling methods, have largely proven unsuitable, either because the probe itself is modified by the oxidant(s) being studied, or because the alternative labeling techniques are too complex or too costly for routine use. What is needed is a simple, quick, and cheap labeling technique that uses a non-radioactive marker, that binds strongly to proteins, is resistant to oxidative modification, and emits a strong signal. We have devised a new reductive method for labeling free carboxyl groups of proteins with the small fluorophore 7-amino-4-methycoumarin (AMC). When bound to target proteins, AMC fluoresces very weakly but when AMC is released by proteinases, proteases, or peptidases, it fluoresces strongly. Thus, without acid-precipitation, the proteolysis of any target protein can be studied continuously, in multiwell plates. In direct comparisons, 3H-labeled proteins and AMC-labeled proteins exhibited essentially identical degradation patterns during incubation with trypsin, cell extracts, and purified proteasome. AMC-labeled proteins are well-suited to study increased proteolytic susceptibility following protein modification, since the AMC-protein bond is resistant to oxidizing agents such as hydrogen peroxide and peroxynitrite, and is stable over time and to extremes of pH, temperature (even boiling), freeze-thawing, mercaptoethanol, and methanol. PMID:21988844

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

Protein kinase and phosphatase activities of thylakoid membranes

International Nuclear Information System (INIS)

Michel, H.; Shaw, E.K.; Bennett, J.

1987-01-01

Dephosphorylation of the 25 and 27 kDa light-harvesting Chl a/b proteins (LHCII) of the thylakoid membranes is catalyzed by a phosphatase which differs from previously reported thylakoid-bound phosphatases in having an alkaline pH optimum (9.0) and a requirement for Mg 2+ ions. Dephosphorylation of the 8.3 kDa psb H gene product requires a Mg 2+ ion concentration more than 200 fold higher than that for dephosphorylation of LHC II. The 8.3 kDa and 27 kDa proteins appear to be phosphorylated by two distinct kinases, which differ in substrate specificity and sensitivity to inhibitors. The plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB) inhibits phosphorylation of the 27 kDa LHC II much more readily than phosphorylation of the 8.3 kDa protein. A similar pattern of inhibition is seen for two synthetic oligopeptides (MRKSATTKKAVC and ATQTLESSSRC) which are analogs of the phosphorylation sites of the two proteins. Possible modes of action of DBMIB are discussed. 45 refs., 7 figs., 3 tabs

Short-term effects of salt exposure on the maize chloroplast protein pattern.

Science.gov (United States)

Zörb, Christian; Herbst, Ramona; Forreiter, Christoph; Schubert, Sven

2009-09-01

It is of fundamental importance to understand the physiological differences leading to salt resistance and to get access to the molecular mechanisms underlying this physiological response. The aim of this work was to investigate the effects of short-term salt exposure on the proteome of maize chloroplasts in the initial phase of salt stress (up to 4 h). It could be shown that sodium ions accumulate quickly and excessively in chloroplasts in the initial phase of moderate salt stress. A change in the chloroplast protein pattern was observed without a change in water potential of the leaves. 2-DE revealed that 12 salt-responsive chloroplast proteins increased while eight chloroplast proteins decreased. Some of the maize chloroplast proteins such as CF1e and a Ca(2+)-sensing receptor show a rather transient response for the first 4 h of salt exposure. The enhanced abundance of the ferredoxin NADPH reductase, the 23 kDa polypeptide of the photosystem II, and the FtsH-like protein might reflect mechanism to attenuate the detrimental effects of Na(+) on the photosynthetic machinery. The observed transient increase and subsequent decrease of selected proteins may exhibit a counterbalancing effect of target proteins in this context. Intriguingly, several subunits of the CF1-CF0 complex are unequally affected, whereas others do not respond at all.

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

Bound Maxima as a Traffic Feature under DDOS Flood Attacks

Directory of Open Access Journals (Sweden)

Jie Xue

2012-01-01

Full Text Available This paper gives a novel traffic feature for identifying abnormal variation of traffic under DDOS flood attacks. It is the histogram of the maxima of the bounded traffic rate on an interval-by-interval basis. We use it to experiment on the traffic data provided by MIT Lincoln Laboratory under Defense Advanced Research Projects Agency (DARPA in 1999. The experimental results profitably enhance the evidences that traffic rate under DDOS attacks is statistically higher than that of normal traffic considerably. They show that the pattern of the histogram of the maxima of bounded rate of attack-contained traffic greatly differs from that of attack-free traffic. Besides, the present traffic feature is simple in mathematics and easy to use in practice.

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

Proteins labelling with 125I and experimental determination of their specific activity

International Nuclear Information System (INIS)

Caro, R.A.; Ciscato, V.A.; Giacomini, S.M.V. de; Quiroga, S.; Radicella, R.

1975-11-01

A standardization of the labelling technique of proteins with 125 I and the control of the obtained products, principally their specific activities was performed, in order to utilize them correctly in radioimmunoassays. The quantities of chloramine-T and sodium metabisulphite were lowered, with regard to the original method, to 3.6 and 9.6 μg respectively. Under these conditions, optimal yields and radioiodinated proteins with good immunological activities were obtained. It was found that the specific activity calculated, as usual, from the yield obtained by electrophoresis, is higher than the real value. For these reasons the yields and the corresponding specific activities were determined from ascending chromatographies performed with 70 per cent methanol as solvent, during two hours in darkness. The radioimmunoassay displacement curves obtained with proteins labelled which the proposed method and the specific activities of which were calculated from their radiochromatographic patterns, were reproducible and gave a percentage of bound radioiodinated protein in the absence of cold protein of 50 +- 4. (author) [es

HOXA1 and TALE proteins display cross-regulatory interactions and form a combinatorial binding code on HOXA1 targets.

Science.gov (United States)

De Kumar, Bony; Parker, Hugo J; Paulson, Ariel; Parrish, Mark E; Pushel, Irina; Singh, Narendra Pratap; Zhang, Ying; Slaughter, Brian D; Unruh, Jay R; Florens, Laurence; Zeitlinger, Julia; Krumlauf, Robb

2017-09-01

Hoxa1 has diverse functional roles in differentiation and development. We identify and characterize properties of regions bound by HOXA1 on a genome-wide basis in differentiating mouse ES cells. HOXA1-bound regions are enriched for clusters of consensus binding motifs for HOX, PBX, and MEIS, and many display co-occupancy of PBX and MEIS. PBX and MEIS are members of the TALE family and genome-wide analysis of multiple TALE members (PBX, MEIS, TGIF, PREP1, and PREP2) shows that nearly all HOXA1 targets display occupancy of one or more TALE members. The combinatorial binding patterns of TALE proteins define distinct classes of HOXA1 targets, which may create functional diversity. Transgenic reporter assays in zebrafish confirm enhancer activities for many HOXA1-bound regions and the importance of HOX-PBX and TGIF motifs for their regulation. Proteomic analyses show that HOXA1 physically interacts on chromatin with PBX, MEIS, and PREP family members, but not with TGIF, suggesting that TGIF may have an independent input into HOXA1-bound regions. Therefore, TALE proteins appear to represent a wide repertoire of HOX cofactors, which may coregulate enhancers through distinct mechanisms. We also discover extensive auto- and cross-regulatory interactions among the Hoxa1 and TALE genes, indicating that the specificity of HOXA1 during development may be regulated though a complex cross-regulatory network of HOXA1 and TALE proteins. This study provides new insight into a regulatory network involving combinatorial interactions between HOXA1 and TALE proteins. © 2017 De Kumar et al.; Published by Cold Spring Harbor Laboratory Press.

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.

Expression of the Ly-6 family proteins Lynx1 and Ly6H in the rat brain is compartmentalized, cell-type specific, and developmentally regulated

DEFF Research Database (Denmark)

Thomsen, Morten Skøtt; Cinar, Betül; Jensen, Majbrit Myrup

2014-01-01

regarding the distribution and developmental regulation of these proteins in the brain. We use protein cross-linking and synaptosomal fractions to demonstrate that the Ly-6 proteins Lynx1 and Ly6H are membrane-bound proteins in the brain, which are present on the cell surface and localize to synaptic...... demonstrate that Lynx1 and Ly6H are expressed in cultured neurons, but not cultured micro- or astroglial cultures. In addition, Lynx1, but not Ly6H was detected in the CSF. Finally, we show that the Ly-6 proteins Lynx1, Lynx2, Ly6H, and PSCA, display distinct expression patterns during postnatal development...

Characterizing genes with distinct methylation patterns in the context of protein-protein interaction network: application to human brain tissues.

Science.gov (United States)

Li, Yongsheng; Xu, Juan; Chen, Hong; Zhao, Zheng; Li, Shengli; Bai, Jing; Wu, Aiwei; Jiang, Chunjie; Wang, Yuan; Su, Bin; Li, Xia

2013-01-01

DNA methylation is an essential epigenetic mechanism involved in transcriptional control. However, how genes with different methylation patterns are assembled in the protein-protein interaction network (PPIN) remains a mystery. In the present study, we systematically dissected the characterization of genes with different methylation patterns in the PPIN. A negative association was detected between the methylation levels in the brain tissues and topological centralities. By focusing on two classes of genes with considerably different methylation levels in the brain tissues, namely the low methylated genes (LMGs) and high methylated genes (HMGs), we found that their organizing principles in the PPIN are distinct. The LMGs tend to be the center of the PPIN, and attacking them causes a more deleterious effect on the network integrity. Furthermore, the LMGs express their functions in a modular pattern and substantial differences in functions are observed between the two types of genes. The LMGs are enriched in the basic biological functions, such as binding activity and regulation of transcription. More importantly, cancer genes, especially recessive cancer genes, essential genes, and aging-related genes were all found more often in the LMGs. Additionally, our analysis presented that the intra-classes communications are enhanced, but inter-classes communications are repressed. Finally, a functional complementation was revealed between methylation and miRNA regulation in the human genome. We have elucidated the assembling principles of genes with different methylation levels in the context of the PPIN, providing key insights into the complex epigenetic regulation mechanisms.

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)

Serum Protein Electrophoretic Pattern in Neonatal Calves Treated with Clinoptilolite

Directory of Open Access Journals (Sweden)

Simona Marc

2018-05-01

Full Text Available The objective of our study was to determine the effects of clinoptilolite supplemented in colostrum on the blood serum protein electrophoretic pattern of new-born calves. Methods: Romanian Black and White new-born calves involved in the study were divided into 3 groups: the control group (C that received colostrum without clinoptilolite, and experimental groups I (E1 and II (E2 that received colostrum supplemented with 0.5% and 2% clinoptilolite, respectively. The concentration of total protein and protein fractions (albumin, α1-globulin, α2-globulin, β-globulin and γ-globulin were analyzed by electrophoresis on cellulose acetate. Results: At hour 30 after birth, concentrations of γ-globulins, β-globulin and total protein in E1 group of calves were higher than in control group by 42.11% (p < 0.05, 28.48% (p > 0.05 and 18.52% (p > 0.05, respectively, and were higher, but not significantly, in group E2 compared to the control group. This was in accordance with a significant lower albumin/globulin ratio in groups E1 and E2 (29.35%, p < 0.05 and 35.87%, p < 0.05, respectively than in control group at 30 h postpartum, which indicates an obvious increase of the globulins fraction in experimental groups. The conclusion: Clinoptilolite was effective in improving passive transfer in new-born calves, but it was more effective if added in colostrum with a dose of 0.5% than with a dose of 2%.

Protein addressing on patterned microchip by coupling chitosan electrodeposition and 'electro-click' chemistry.

Science.gov (United States)

Shi, Xiao-Wen; Qiu, Ling; Nie, Zhen; Xiao, Ling; Payne, Gregory F; Du, Yumin

2013-12-01

Many applications in proteomics and lab-on-chip analysis require methods that guide proteins to assemble at surfaces with high spatial and temporal control. Electrical inputs are particularly convenient to control, and there has been considerable effort to discover simple and generic mechanisms that allow electrical inputs to trigger protein assembly on-demand. Here, we report the electroaddressing of a protein to a patterned surface by coupling two generic electroaddressing mechanisms. First, we electrodeposit the stimuli-responsive film-forming aminopolysaccharide chitosan to form a hydrogel matrix at the electrode surface. After deposition, the matrix is chemically functionalized with alkyne groups. Second, we ''electro-click' an azide-tagged protein to the functionalized matrix using electrical signals to trigger conjugation by Huisgen 1,3-dipolar cycloadditions. Specifically, a cathodic potential is applied to the matrix-coated electrode to reduce Cu(II) to Cu(I) which is required for the click reaction. Using fluorescently-labeled bovine serum albumin as our model, we demonstrate that protein conjugation can be controlled spatially and temporally. We anticipate that the coupling of polysaccharide electrodeposition and electro-click chemistry will provide a simple and generic approach to electroaddress proteins within compatible hydrogel matrices.

RNA-binding protein VICKZ is expressed in a germinal center associated pattern among lymphoma subtypes

DEFF Research Database (Denmark)

Natkunam, Y.; Vainer, G.; Zhao, S.C.

2005-01-01

and tumorigenesis/metastasis. We generated an antibody that recognizes all three isoforms of VICKZ protein and characterized its expression in normal lymphoid tissue and in lymphoma subtypes. In normal tonsils, VICKZ protein showed a germinal center-specific pattern of expression with staining localized...... to the cytoplasm. Among 868 non-Hodgkin and Hodgkin lymphomas tested by immunohistochemistry on tissue microarrays, staining for VICKZ protein was present in 76% (126/165) of follicular lymphoma, 78% (155/200) of DLBCL, 90% (9/10) of mediastinal large B-cell lymphoma, and 100% (2/2) of Burkitt lymphoma. A subset...... protein in lymphoma subtypes suggests a potential utility for VICKZ in the identification of subgroups of DLBCL associated with different prognoses....

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

Speed Controls in Translating Secretory Proteins in Eukaryotes - an Evolutionary Perspective

Science.gov (United States)

Mahlab, Shelly; Linial, Michal

2014-01-01

Protein translation is the most expensive operation in dividing cells from bacteria to humans. Therefore, managing the speed and allocation of resources is subject to tight control. From bacteria to humans, clusters of relatively rare tRNA codons at the N′-terminal of mRNAs have been implicated in attenuating the process of ribosome allocation, and consequently the translation rate in a broad range of organisms. The current interpretation of “slow” tRNA codons does not distinguish between protein translations mediated by free- or endoplasmic reticulum (ER)-bound ribosomes. We demonstrate that proteins translated by free- or ER-bound ribosomes exhibit different overall properties in terms of their translation efficiency and speed in yeast, fly, plant, worm, bovine and human. We note that only secreted or membranous proteins with a Signal peptide (SP) are specified by segments of “slow” tRNA at the N′-terminal, followed by abundant codons that are considered “fast.” Such profiles apply to 3100 proteins of the human proteome that are composed of secreted and signal peptide (SP)-assisted membranous proteins. Remarkably, the bulks of the proteins (12,000), or membranous proteins lacking SP (3400), do not have such a pattern. Alternation of “fast” and “slow” codons was found also in proteins that translocate to mitochondria through transit peptides (TP). The differential clusters of tRNA adapted codons is not restricted to the N′-terminal of transcripts. Specifically, Glycosylphosphatidylinositol (GPI)-anchored proteins are unified by clusters of low adapted tRNAs codons at the C′-termini. Furthermore, selection of amino acids types and specific codons was shown as the driving force which establishes the translation demands for the secretory proteome. We postulate that “hard-coded” signals within the secretory proteome assist the steps of protein maturation and folding. Specifically, “speed control” signals for delaying the translation

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)

Cyclic AMP-receptor proteins in heart muscle of rats flown on Cosmos 1887

Science.gov (United States)

Mednieks, Maija I.; Popova, Irina A.; Grindeland, Richard E.

1991-01-01

The cellular compartmentalization of the cyclic AMP-receptor proteins in heart ventricular tissue obtained from rats flown on the Cosmos 1887 is determined. Photoaffinity labeling of soluble and particular cell fractions with a (32P)-8-azido analog of cyclic AMP is followed by electrophoretic separation of the proteins and by autoradiographic identification of the labeled isoforms of cAPK R subunits. It is shown that RII in the particulate subcellular fraction was significantly decreased in heart cells from rats in the flight group when compared to controls. Protein banding patterns in both the cytoplasmic fraction and in a fraction enriched in chromatin-bound proteins exhibited some variability in tissues of individual animals, but showed no changes that could be directly attributed to flight conditions. No significant change was apparent in the distribution of RI or RII cyclic AMP binding in the soluble fractions. It is inferred that the cardiac cell integrity or its protein content is not compromised under flight conditions.

Nonadditive protein accumulation patterns in Maize (Zea mays L.) hybrids during embryo development.

Science.gov (United States)

Marcon, Caroline; Schützenmeister, André; Schütz, Wolfgang; Madlung, Johannes; Piepho, Hans-Peter; Hochholdinger, Frank

2010-12-03

Heterosis describes the superior performance of heterozygous F(1)-hybrid plants compared to their homozygous parental inbred lines. In the present study, heterosis was detected for length, weight, and the time point of seminal root primordia initiation in maize (Zea mays L.) embryos of the reciprocal F(1)-hybrids UH005xUH250 and UH250xUH005. A two-dimensional gel electrophoresis (2-DE) proteome survey of the most abundant proteins of the reciprocal hybrids and their parental inbred lines 25 and 35 days after pollination revealed that 141 of 597 detected proteins (24%) exhibited nonadditive accumulation in at least one hybrid. Approximately 44% of all nonadditively accumulated proteins displayed an expression pattern that was not distinguishable from the low parent value. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) analyses and subsequent functional classification of the 141 proteins revealed that development, protein metabolism, redox-regulation, glycolysis, and amino acid metabolism were the most prominent functional classes among nonadditively accumulated proteins. In 35-day-old embryos of the hybrid UH250xUH005, a significant up-regulation of enzymes related to glucose metabolism which often exceeded the best parent values was observed. A comparison of nonadditive protein accumulation between rice and maize embryo data sets revealed a significant overlap of nonadditively accumulated proteins suggesting conserved organ- or tissue-specific regulatory mechanisms in monocots related to heterosis.

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

Bayesian analysis of repairable systems showing a bounded failure intensity

International Nuclear Information System (INIS)

Guida, Maurizio; Pulcini, Gianpaolo

2006-01-01

The failure pattern of repairable mechanical equipment subject to deterioration phenomena sometimes shows a finite bound for the increasing failure intensity. A non-homogeneous Poisson process with bounded increasing failure intensity is then illustrated and its characteristics are discussed. A Bayesian procedure, based on prior information on model-free quantities, is developed in order to allow technical information on the failure process to be incorporated into the inferential procedure and to improve the inference accuracy. Posterior estimation of the model-free quantities and of other quantities of interest (such as the optimal replacement interval) is provided, as well as prediction on the waiting time to the next failure and on the number of failures in a future time interval is given. Finally, numerical examples are given to illustrate the proposed inferential procedure

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

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.

The MUC4 membrane-bound mucin regulates esophageal cancer cell proliferation and migration properties: Implication for S100A4 protein

International Nuclear Information System (INIS)

Bruyere, Emilie; Jonckheere, Nicolas; Frenois, Frederic; Mariette, Christophe; Van Seuningen, Isabelle

2011-01-01

Highlights: → Loss of MUC4 reduces proliferation of esophageal cancer cells. → MUC4 inhibition impairs migration of esophageal cancer cells but not their invasion. → Loss of MUC4 significantly reduces in vivo tumor growth. → Decrease of S100A4 induced by MUC4 inhibition impairs proliferation and migration. -- Abstract: MUC4 is a membrane-bound mucin known to participate in tumor progression. It has been shown that MUC4 pattern of expression is modified during esophageal carcinogenesis, with a progressive increase from metaplastic lesions to adenocarcinoma. The principal cause of development of esophageal adenocarcinoma is the gastro-esophageal reflux, and MUC4 was previously shown to be upregulated by several bile acids present in reflux. In this report, our aim was thus to determine whether MUC4 plays a role in biological properties of human esophageal cancer cells. For that stable MUC4-deficient cancer cell lines (shMUC4 cells) were established using a shRNA approach. In vitro (proliferation, migration and invasion) and in vivo (tumor growth following subcutaneous xenografts in SCID mice) biological properties of shMUC4 cells were analyzed. Our results show that shMUC4 cells were less proliferative, had decreased migration properties and did not express S100A4 protein when compared with MUC4 expressing cells. Absence of MUC4 did not impair shMUC4 invasiveness. Subcutaneous xenografts showed a significant decrease in tumor size when cells did not express MUC4. Altogether, these data indicate that MUC4 plays a key role in proliferative and migrating properties of esophageal cancer cells as well as is a tumor growth promoter. MUC4 mucin appears thus as a good therapeutic target to slow-down esophageal tumor progression.

The MUC4 membrane-bound mucin regulates esophageal cancer cell proliferation and migration properties: Implication for S100A4 protein

Energy Technology Data Exchange (ETDEWEB)

Bruyere, Emilie; Jonckheere, Nicolas; Frenois, Frederic [Inserm, UMR837, Jean-Pierre Aubert Research Center, Team 5 ' Mucins, Epithelial Differentiation and Carcinogenesis' , rue Polonovski, 59045 Lille Cedex (France); Universite Lille-Nord de France, 1 place de Verdun, 59045 Lille Cedex (France); Mariette, Christophe [Inserm, UMR837, Jean-Pierre Aubert Research Center, Team 5 ' Mucins, Epithelial Differentiation and Carcinogenesis' , rue Polonovski, 59045 Lille Cedex (France); Universite Lille-Nord de France, 1 place de Verdun, 59045 Lille Cedex (France); Department of Digestive and Oncological Surgery, University Hospital Claude Huriez, 1 place de Verdun, 59045 Lille Cedex (France); Van Seuningen, Isabelle, E-mail: isabelle.vanseuningen@inserm.fr [Inserm, UMR837, Jean-Pierre Aubert Research Center, Team 5 ' Mucins, Epithelial Differentiation and Carcinogenesis' , rue Polonovski, 59045 Lille Cedex (France); Universite Lille-Nord de France, 1 place de Verdun, 59045 Lille Cedex (France)

2011-09-23

Highlights: {yields} Loss of MUC4 reduces proliferation of esophageal cancer cells. {yields} MUC4 inhibition impairs migration of esophageal cancer cells but not their invasion. {yields} Loss of MUC4 significantly reduces in vivo tumor growth. {yields} Decrease of S100A4 induced by MUC4 inhibition impairs proliferation and migration. -- Abstract: MUC4 is a membrane-bound mucin known to participate in tumor progression. It has been shown that MUC4 pattern of expression is modified during esophageal carcinogenesis, with a progressive increase from metaplastic lesions to adenocarcinoma. The principal cause of development of esophageal adenocarcinoma is the gastro-esophageal reflux, and MUC4 was previously shown to be upregulated by several bile acids present in reflux. In this report, our aim was thus to determine whether MUC4 plays a role in biological properties of human esophageal cancer cells. For that stable MUC4-deficient cancer cell lines (shMUC4 cells) were established using a shRNA approach. In vitro (proliferation, migration and invasion) and in vivo (tumor growth following subcutaneous xenografts in SCID mice) biological properties of shMUC4 cells were analyzed. Our results show that shMUC4 cells were less proliferative, had decreased migration properties and did not express S100A4 protein when compared with MUC4 expressing cells. Absence of MUC4 did not impair shMUC4 invasiveness. Subcutaneous xenografts showed a significant decrease in tumor size when cells did not express MUC4. Altogether, these data indicate that MUC4 plays a key role in proliferative and migrating properties of esophageal cancer cells as well as is a tumor growth promoter. MUC4 mucin appears thus as a good therapeutic target to slow-down esophageal tumor progression.

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...

Steric exclusion and protein conformation determine the localization of plasma membrane transporters.

Science.gov (United States)

Bianchi, Frans; Syga, Łukasz; Moiset, Gemma; Spakman, Dian; Schavemaker, Paul E; Punter, Christiaan M; Seinen, Anne-Bart; van Oijen, Antoine M; Robinson, Andrew; Poolman, Bert

2018-02-05

The plasma membrane (PM) of Saccharomyces cerevisiae contains membrane compartments, MCC/eisosomes and MCPs, named after the protein residents Can1 and Pma1, respectively. Using high-resolution fluorescence microscopy techniques we show that Can1 and the homologous transporter Lyp1 are able to diffuse into the MCC/eisosomes, where a limited number of proteins are conditionally trapped at the (outer) edge of the compartment. Upon addition of substrate, the immobilized proteins diffuse away from the MCC/eisosomes, presumably after taking a different conformation in the substrate-bound state. Our data indicate that the mobile fraction of all integral plasma membrane proteins tested shows extremely slow Brownian diffusion through most of the PM. We also show that proteins with large cytoplasmic domains, such as Pma1 and synthetic chimera of Can1 and Lyp1, are excluded from the MCC/eisosomes. We hypothesize that the distinct localization patterns found for these integral membrane proteins in S. cerevisiae arises from a combination of slow lateral diffusion, steric exclusion, and conditional trapping in membrane compartments.

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.

Multivariate data analysis of two-dimensional gel electrophoresis protein patterns from few samples

DEFF Research Database (Denmark)

Jensen, Kristina Nedenskov; Jessen, Flemming; Jørgensen, Bo

2008-01-01

One application of 2D gel electrophoresis is to reveal differences in protein pattern between two or more groups of individuals, attributable to their group membership. Multivariate data analytical methods are useful in pinpointing the spots relevant for discrimination by focusing not only...... on single spot differences, but on the covariance structure between proteins. However, their outcome is dependent on data scaling, and they may fail in producing valid multivariate models due to the much higher number of "irrelevant" spots present in the gels. The case where only few gels are available...... and where the aim is to find as many as possible of the group-dependent proteins seems particularly difficult to handle. The present paper investigates such a case regarding the effect of scaling and of prefiltering by univariate nonparametric statistics on the selection of spots. Besides, a modified...

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.)

Bounding species distribution models

Directory of Open Access Journals (Sweden)

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

Directory of Open Access Journals (Sweden)

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 and Glycoprotein Patterns Related to Morphogenesis in Mammillaria gracillis Pfeiff. Tissue Culture

Directory of Open Access Journals (Sweden)

Biljana Balen

2002-01-01

Full Text Available As plants with Crassulacean Acid Metabolism (CAM, cacti are highly affected by artificial environmental conditions in tissue culture. Plants of Mammillaria gracillis Pfeiff. (Cactaceae propagated in vitro produced callus spontaneously. This habituated callus regenerated normal and hyperhydric shoots without the addition of growth regulators. In order to compare habituated callus with the tumorous one, cactus cells were transformed with two strains of Agrobacterium tumefaciens: the wild strain B6S3 (tumour line TW and the rooty mutant GV3101 (tumour line TR. Gene expression in cactus plants, habituated callus, regenerated shoots and two tumour lines was analysed at the level of cellular and extracellular protein and glycoprotein profiles. Proteins were separated by SDS-polyacrylamide gel electrophoresis and 2-D PAGE electrophoresis and silver stained. Concavalin A-peroxidase staining detected glycoproteins with D-manose in their glycan component on protein blots. Developmentally specific protein patterns of Mammillaria gracillis tissue lines were detected. The 2-D PAGE electrophoresis revealed some tissue specific protein groups. The cellular glycoprotein of 42 kDa detected by ConA was highly expressed in undifferentiated tissues (habituated callus, TW and TR tumours and in hyperhydric regenerants. Tumours produced extracellular proteins of 33, 23 and 22 kDa. The N glycosylation of cellular and extracellular proteins was related to specific developmental stage of cactus tissue.

A Drosophila protein family implicated in pheromone perception is related to Tay-Sachs GM2-activator protein.

Science.gov (United States)

Starostina, Elena; Xu, Aiguo; Lin, Heping; Pikielny, Claudio W

2009-01-02

Low volatility, lipid-like cuticular hydrocarbon pheromones produced by Drosophila melanogaster females play an essential role in triggering and modulating mating behavior, but the chemosensory mechanisms involved remain poorly understood. Recently, we showed that the CheB42a protein, which is expressed in only 10 pheromone-sensing taste hairs on the front legs of males, modulates progression to late stages of male courtship behavior in response to female-specific cuticular hydrocarbons. Here we report that expression of all 12 genes in the CheB gene family is predominantly or exclusively gustatory-specific, and occurs in many different, often non-overlapping patterns. Only the Gr family of gustatory receptor genes displays a comparable variety of gustatory-specific expression patterns. Unlike Grs, however, expression of all but one CheB gene is sexually dimorphic. Like CheB42a, other CheBs may therefore function specifically in gustatory perception of pheromones. We also show that CheBs belong to the ML superfamily of lipid-binding proteins, and are most similar to human GM2-activator protein (GM2-AP). In particular, GM2-AP residues involved in ligand binding are conserved in CheBs but not in other ML proteins. Finally, CheB42a is specifically secreted into the inner lumen of pheromone-sensing taste hairs, where pheromones interact with membrane-bound receptors. We propose that CheB proteins interact directly with lipid-like Drosophila pheromones and modulate their detection by the gustatory signal transduction machinery. Furthermore, as loss of GM2-AP in Tay-Sachs disease prevents degradation of GM2 gangliosides and results in neurodegeneration, the function of CheBs in pheromone response may involve biochemical mechanisms critical for lipid metabolism in human neurons.

A Drosophila Protein Family Implicated in Pheromone Perception Is Related to Tay-Sachs GM2-Activator Protein*

Science.gov (United States)

Starostina, Elena; Xu, Aiguo; Lin, Heping; Pikielny, Claudio W.

2009-01-01

Low volatility, lipid-like cuticular hydrocarbon pheromones produced by Drosophila melanogaster females play an essential role in triggering and modulating mating behavior, but the chemosensory mechanisms involved remain poorly understood. Recently, we showed that the CheB42a protein, which is expressed in only 10 pheromone-sensing taste hairs on the front legs of males, modulates progression to late stages of male courtship behavior in response to female-specific cuticular hydrocarbons. Here we report that expression of all 12 genes in the CheB gene family is predominantly or exclusively gustatory-specific, and occurs in many different, often non-overlapping patterns. Only the Gr family of gustatory receptor genes displays a comparable variety of gustatory-specific expression patterns. Unlike Grs, however, expression of all but one CheB gene is sexually dimorphic. Like CheB42a, other CheBs may therefore function specifically in gustatory perception of pheromones. We also show that CheBs belong to the ML superfamily of lipid-binding proteins, and are most similar to human GM2-activator protein (GM2-AP). In particular, GM2-AP residues involved in ligand binding are conserved in CheBs but not in other ML proteins. Finally, CheB42a is specifically secreted into the inner lumen of pheromone-sensing taste hairs, where pheromones interact with membrane-bound receptors. We propose that CheB proteins interact directly with lipid-like Drosophila pheromones and modulate their detection by the gustatory signal transduction machinery. Furthermore, as loss of GM2-AP in Tay-Sachs disease prevents degradation of GM2 gangliosides and results in neurodegeneration, the function of CheBs in pheromone response may involve biochemical mechanisms critical for lipid metabolism in human neurons. PMID:18952610

Common and distinctive localization patterns of Crumbs polarity complex proteins in the mammalian eye.

Science.gov (United States)

Kim, Jin Young; Song, Ji Yun; Karnam, Santi; Park, Jun Young; Lee, Jamie J H; Kim, Seonhee; Cho, Seo-Hee

2015-01-01

Crumbs polarity complex proteins are essential for cellular and tissue polarity, and for adhesion of epithelial cells. In epithelial tissues deletion of any of three core proteins disrupts localization of the other proteins, indicating structural and functional interdependence among core components. Despite previous studies of function and co-localization that illustrated the properties that these proteins share, it is not known whether an individual component of the complex plays a distinct role in a unique cellular and developmental context. In order to investigate this question, we primarily used confocal imaging to determine the expression and subcellular localization of the core Crumbs polarity complex proteins during ocular development. Here we show that in developing ocular tissues core Crumbs polarity complex proteins, Crb, Pals1 and Patj, generally appear in an overlapping pattern with some exceptions. All three core complex proteins localize to the apical junction of the retinal and lens epithelia. Pals1 is also localized in the Golgi of the retinal cells and Patj localizes to the nuclei of the apically located subset of progenitor cells. These findings suggest that core Crumbs polarity complex proteins exert common and independent functions depending on cellular context. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Overlapping functions of argonaute proteins in patterning and morphogenesis of Drosophila embryos.

Directory of Open Access Journals (Sweden)

Wibke J Meyer

2006-08-01

Full Text Available Argonaute proteins are essential components of the molecular machinery that drives RNA silencing. In Drosophila, different members of the Argonaute family of proteins have been assigned to distinct RNA silencing pathways. While Ago1 is required for microRNA function, Ago2 is a crucial component of the RNA-induced silencing complex in siRNA-triggered RNA interference. Drosophila Ago2 contains an unusual amino-terminus with two types of imperfect glutamine-rich repeats (GRRs of unknown function. Here we show that the GRRs of Ago2 are essential for the normal function of the protein. Alleles with reduced numbers of GRRs cause specific disruptions in two morphogenetic processes associated with the midblastula transition: membrane growth and microtubule-based organelle transport. These defects do not appear to result from disruption of siRNA-dependent processes but rather suggest an interference of the mutant Ago2 proteins in an Ago1-dependent pathway. Using loss-of-function alleles, we further demonstrate that Ago1 and Ago2 act in a partially redundant manner to control the expression of the segment-polarity gene wingless in the early embryo. Our findings argue against a strict separation of Ago1 and Ago2 functions and suggest that these proteins act in concert to control key steps of the midblastula transition and of segmental patterning.

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...

Correlation of acidic and basic carrier ampholyte and immobilized pH gradient two-dimensional gel electrophoresis patterns based on mass spectrometric protein identification

DEFF Research Database (Denmark)

Nawrocki, A; Larsen, Martin Røssel; Podtelejnikov, A V

1998-01-01

Separation of proteins on either carrier ampholyte-based or immobilized pH gradient-based two-dimensional (2-D) gels gives rise to electrophoretic patterns that are difficult to compare visually. In this paper we have used matrix-assisted laser desorption/ionization mass spectrometry (MALDI......-MS) to determine the identities of 335 protein spots in these two 2-D gel systems, including a substantial number of basic proteins which had never been identified before. Proteins that were identified in both gel systems allowed us to cross-reference the gel patterns. Vector analysis of these cross...

A comparative study of cell cycle mediator protein expression patterns in anaplastic and papillary thyroid carcinoma.

Science.gov (United States)

Evans, Juanita J; Crist, Henry S; Durvesh, Saima; Bruggeman, Richard D; Goldenberg, David

2012-07-01

Anaplastic thyroid carcinoma (ATC) is an extremely aggressive and rapidly fatal neoplasm. The aim of this study was to identify a limited cell cycle associated protein expression pattern unique to ATC and to correlate that pattern with clinical outcome. This represents one of the largest tissue micro-array projects comparing the cell cycle protein expression data of ATC to other well-differentiated tumors in the literature. Tissue microarrays were created from 21 patients with ATC and an age and gender matched cohort of patients with papillary thyroid carcinoma (PTC). Expression of epidermal growth factor receptor, cyclin D1, cyclin E, p53, p21, p16, aurora kinase A, opioid growth factor (OGF), OGF-receptor, thyroglobulin and Ki-67 was evaluated in a semi-quantitative fashion. Differences in protein expression between the cohorts were evaluated using chi-square tests with Bonferroni adjustments. Survival time and presence of metastasis at presentation were collected. The ATC cohort showed a statistically significant decrease (p cycle with aberrant expression of multiple protein markers suggesting increased proliferative activity and loss of control of cell cycle progression to G₁ phase. These findings support the assertion that ATC may represent the furthest end of a continuum of thyroid carcinoma dedifferentiation.

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

Inhibition of chloroplast protein synthesis following light chilling of tomato

International Nuclear Information System (INIS)

Kent, J.; Ort, D.

1989-01-01

In the present study we looked at the effects of a high light chill on the pulsed incorporation of 35 S methionine into total, stromal, and thylakoid proteins of lightly abraded leaflets of 18-21 day old tomato (Lycopersicon esculentum Mill ca. Floramerica) seedlings. Based on gel fluorographic patterns of marker proteins that are indicative of the net rates of chloroplast and cytoplasmic protein synthesis, there appears to be a nearly complete cessation of chloroplastic protein synthesis. No labeling is observed for either the stromal large subunit of Rubisco or the thylakoid-bound alpha and beta subunits of the coupling factor. One notable exception, however, appears to be the 32 kd, D1 protein. Its net synthetic rate remains high despite the inhibition of other chloroplastically synthesized proteins. The small subunit of Rubicso, LHCP-II, as well as several other proteins of known cytoplasmic origin, were still synthesized, albeit, at lower than control rates. Light chilling of chill-insensitive spinach produced a similar, but less dramatic differential behavior between chloroplastic and cytoplasmic protein synthesis. It appears, in chilling-sensitive plants, that chloroplast protein synthesis exhibits a greater sensitivity to low temperature inhibition than does cytoplasmic protein synthesis and that recovery of chloroplast protein synthesis may play an important role in recovery of photosynthetic activity following chilling

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.

Binding of Gallic Acid and Epigallocatechin Gallate to Heat-Unfolded Whey Proteins at Neutral pH Alters Radical Scavenging Activity of in Vitro Protein Digests.

Science.gov (United States)

Cao, Yanyun; Xiong, Youling L

2017-09-27

Preheated (80 °C for 9 min) whey protein isolate (HWPI) was reacted with 20, 120, and 240 μmol/g (protein basis) gallic acid (GA) or epigallocatechin gallate (EGCG) at neutral pH and 25 °C. Isothermal titration calorimetry and fluorometry showed a similar trend that GA binding to HWPI was moderate but weaker than EGCG binding. However, the shift of maximal fluorescence emission wavelength in opposite directions in response to GA (blue) and EGCG (red) suggests discrepant binding patterns. Electrophoresis results showed that EGCG induced formation of HWPI complexes while GA only had a marginal effect. Both free and phenolic-bound HWPI exhibited mild antiradical activity. However, when subjected to in vitro digestion, synergistic radical-scavenging activity was produced between the phenolics and peptides with the highest synergism being observed on 120 μmol/g phenolics.

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 (�

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.

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...

Dietary pattern, serum magnesium, ferritin, C-reactive protein and anaemia among older people.

Science.gov (United States)

Xu, Xiaoyue; Hall, John; Byles, Julie; Shi, Zumin

2017-04-01

Epidemiological data of dietary patterns and anaemia among older Chinese remains extremely scarce. We examined the association between dietary patterns and anaemia in older Chinese, and to assess whether biomarkers of serum magnesium, C-reactive protein (CRP) and serum ferritin can mediate these associations. We analysed the 2009 China Health and Nutrition Survey data (2401 individuals aged ≥60 years for whom both dietary and biomarker data are available). Dietary data was obtained using 24 h-recall over three consecutive days. Fasting blood samples and anthropometry measurement were also collected. Factor analysis was used to identify dietary patterns. Factor scores representing dietary patterns were used in Poisson regression models to explore the association between each dietary pattern and anaemia. Of the 2401 participants, 18.9% had anaemia, 1.9% had anaemia related to inflammation (AI), and 1.3% had iron-deficiency anaemia (IDA). A traditional dietary pattern (high intake of rice, pork and vegetables) was positively associated with anaemia; a modern dietary pattern (high intake of fruit and fast food) was inversely associated with anaemia. Progressively lower magnesium and BMI levels were associated with increasing traditional dietary quartiles; while a progressively higher magnesium and BMI levels were associated with increasing modern dietary quartiles (p  0.05) in CRP and serum ferritin across quartiles for either dietary pattern. In the fully adjusted model, the prevalence ratio (PR) of anaemia, comparing the fourth quartile to the first quartile, was 1.75 (95% CI: 1.33; 2.29) for a traditional dietary pattern, and 0.89 (95% CI: 0.68; 1.16) for a modern dietary pattern. The association between dietary patterns and anaemia is mediated by serum magnesium. Traditional dietary pattern is associated with a higher prevalence of anaemia among older Chinese. Future studies need to examine whether correcting micronutrient deficiency (e.g. magnesium) by

α-Catenin localization and sarcomere self-organization on N-cadherin adhesive patterns are myocyte contractility driven.

Directory of Open Access Journals (Sweden)

Anant Chopra

Full Text Available The N-cadherin (N-cad complex plays a crucial role in cardiac cell structure and function. Cadherins are adhesion proteins linking adjacent cardiac cells and, like integrin adhesions, are sensitive to force transmission. Forces through these adhesions are capable of eliciting structural and functional changes in myocytes. Compared to integrins, the mechanisms of force transduction through cadherins are less explored. α-catenin is a major component of the cadherin-catenin complex, thought to provide a link to the cell actin cytoskeleton. Using N-cad micropatterned substrates in an adhesion constrainment model, the results from this study show that α-catenin localizes to regions of highest internal stress in myocytes. This localization suggests that α-catenin acts as an adaptor protein associated with the cadherin mechanosensory apparatus, which is distinct from mechanosensing through integrins. Myosin inhibition in cells bound by integrins to fibronectin-coated patterns disrupts myofibiril organization, whereas on N-cad coated patterns, myosin inhibition leads to better organized myofibrils. This result indicates that the two adhesion systems provide independent mechanisms for regulating myocyte structural organization.

The E4 protein; structure, function and patterns of expression

Energy Technology Data Exchange (ETDEWEB)

Doorbar, John, E-mail: jdoorba@nimr.mrc.ac.uk

2013-10-15

��}E4, these kinases regulate one of the E1{sup ∧}E4 proteins main functions, the association with the cellular keratin network, and eventually also its cleavage by the protease calpain which allows assembly into amyloid-like fibres and reorganisation of the keratin network. Although the E4 proteins of different HPV types appear divergent at the level of their primary amino acid sequence, they share a recognisable modular organisation and pattern of expression, which may underlie conserved functions and regulation. Assembly into higher-order multimers and suppression of cell proliferation are common to all E4 proteins examined. Although not yet formally demonstrated, a role in virus release and transmission remains a likely function for E4. - Highlights: • E4 gene products have a modular structure, and are expressed from the E1{sup ∧}E4 spliced mRNA. • E4 proteins are modified during epithelial differentiation by phosphorylation and proteolysis. • The E4 proteins contribute to genome amplification-efficiency and virus synthesis. • E4 proteins are abundantly expressed and may facilitate efficient virus release and transmission. • High-risk E4 proteins are deposited as amyloid fibres and can be used as infection biomarkers.

A Repeated Signal Difference for Recognising Patterns

Directory of Open Access Journals (Sweden)

Kieran Greer

2016-08-01

Full Text Available This paper describes a new mechanism that might help with defining pattern sequences, by the fact that it can produce an upper bound on the ensemble value that can persistently oscillate with the actual values produced from each pattern. With every firing event, a node also receives an on/off feedback switch. If the node fires then it sends a feedback result depending on the input signal strength. If the input signal is positive or larger, it can store an ‘on’ switch feedback for the next iteration. If the signal is negative or smaller it can store an ‘off’ switch feedback for the next iteration. If the node does not fire, then it does not affect the current feedback situation and receives the switch command produced by the last active pattern event for the same neuron. The upper bound therefore also represents the largest or most enclosing pattern set and the lower value is for the actual set of firing patterns. If the pattern sequence repeats, it will oscillate between the two values, allowing them to be recognised and measured more easily, over time. Tests show that changing the sequence ordering produces different value sets, which can also be measured.

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)

Classification, expression pattern and comparative analysis of sugarcane expressed sequences tags (ESTs encoding glycine-rich proteins (GRPs

Directory of Open Access Journals (Sweden)

Fusaro Adriana

2001-01-01

Full Text Available Since the isolation of the first glycine-rich proteins (GRPs in plants a wealth of new GRPs have been identified. The highly specific but diverse expression pattern of grp genes, taken together with the distinct sub-cellular localization of some GRP groups, clearly indicate that these proteins are involved in several independent physiological processes. Notwithstanding the absence of a clear definition of the role of GRPs in plant cells, studies conducted with these proteins have provided new and interesting insights into the molecular biology and cell biology of plants. Complexly regulated promoters and distinct mechanisms for the regulation of gene expression have been demonstrated and new protein targeting pathways, as well as the exportation of GRPs from different cell types have been discovered. These data show that GRPs can be useful as markers and/or models to understand distinct aspects of plant biology. In this paper, the structural and functional features of these proteins in sugarcane (Saccharum officinarum L. are summarized. Since this is the first description of GRPs in sugarcane, special emphasis has been given to the expression pattern of these GRP genes by studying their abundance and prevalence in the different cDNA-libraries of the Sugarcane Expressed Sequence Tag (SUCEST project . The comparison of sugarcane GRPs with GRPs from other species is also discussed.

A statistical approach to the estimation of mechanical unfolding parameters from the unfolding patterns of protein heteropolymers

International Nuclear Information System (INIS)

Beddard, G S; Brockwell, D J

2010-01-01

A statistical calculation is described with which the saw-tooth-like unfolding patterns of concatenated heteropolymeric proteins can be used to estimate the forced unfolding parameters of a previously uncharacterized protein. The chance of observing the various sequences of unfolding events, such as ABAABBB or BBAAABB etc, for two proteins of types A and B is calculated using proteins with various ratios of A and B and at different values of effective unfolding rate constants. If the experimental rate constant for forced unfolding, k 0 , and distance to the transition state x u are known for one protein, then the calculation allows an estimation of values for the other. The predictions are compared with Monte Carlo simulations and experimental data. (communication)

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

Salt stress-induced protein pattern associated with photosynthetic parameters and andrographolide content in Andrographis paniculata Nees.

Science.gov (United States)

Talei, Daryush; Valdiani, Alireza; Maziah, Mahmood; Sagineedu, Sreenivasa Rao; Abiri, Rambod

2015-01-01

Andrographis paniculata is a multifunctional medicinal plant and a potent source of bioactive compounds. Impact of environmental stresses such as salinity on protein diversification, as well as the consequent changes in the photosynthetic parameters and andrographolide content (AG) of the herb, has not yet been thoroughly investigated. The present study showed that the salinity affects the protein pattern, and subsequently, it decreased the photosynthetic parameters, protein content, total dry weight, and total crude extract. Exceptionally, the AG content was increased (p ≤ 0.01). Moreover, it was noticed that the salinity at 12 dS m(-1) led to the maximum increase in AG content in all accessions. Interestingly, the leaf protein analysis revealed that the two polymorphic protein bands as low- and medium-sized of 17 and 45 kDa acted as the activator agents for the photosynthetic parameters and AG content. Protein sequencing and proteomic analysis can be conducted based on the present findings in the future.

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.

Changes in the pattern of protein synthesis of prosopis chilensis induced by high temperatures

Energy Technology Data Exchange (ETDEWEB)

Medina, C.; Cardemil, L. (Univ. de Chile, Santiago (USA))

1989-04-01

Seeds of Prosopis chilensis, a leguminous tree from semi-arid regions of Central Chile, were germinated at temperatures of 25-30-35-40-45 and 50{degree}C. Germination was 100% between 25 and 40{degree}C, being faster at 35{degree}C. The best temperature for root growth was also 35{degree}C. There was not germination at 50{degree}C. However, seedlings coming from seeds germinated at 35{degree}C were capable of growing at higher temperatures of 45 and 50{degree}C. Pattern of protein synthesis was followed in roots incubated with {sup 35}S-methionine at increasing temperatures between 35 and 50{degree}C. SDS-PAGE of the proteins followed by fluorography shows that at temperatures above 35{degree}C, new protein bands appear while others become thicker. Most of the protein bands have decreased at 50{degree}C, with the exception of the new bands. A band of 70 KD, that is present at 35{degree}C, is more prominent at 50{degree}C. These proteins may have an important role in the thermotolerance of Prosopis chilensis to stressing temperatures.

Changes in the pattern of protein synthesis of prosopis chilensis induced by high temperatures

International Nuclear Information System (INIS)

Medina, C.; Cardemil, L.

1989-01-01

Seeds of Prosopis chilensis, a leguminous tree from semi-arid regions of Central Chile, were germinated at temperatures of 25-30-35-40-45 and 50 degree C. Germination was 100% between 25 and 40 degree C, being faster at 35 degree C. The best temperature for root growth was also 35 degree C. There was not germination at 50 degree C. However, seedlings coming from seeds germinated at 35 degree C were capable of growing at higher temperatures of 45 and 50 degree C. Pattern of protein synthesis was followed in roots incubated with 35 S-methionine at increasing temperatures between 35 and 50 degree C. SDS-PAGE of the proteins followed by fluorography shows that at temperatures above 35 degree C, new protein bands appear while others become thicker. Most of the protein bands have decreased at 50 degree C, with the exception of the new bands. A band of 70 KD, that is present at 35 degree C, is more prominent at 50 degree C. These proteins may have an important role in the thermotolerance of Prosopis chilensis to stressing temperatures

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.

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

Directory of Open Access Journals (Sweden)

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.

Different zinc sensitivity of Brassica organs is accompanied by distinct responses in protein nitration level and pattern.

Science.gov (United States)

Feigl, Gábor; Kolbert, Zsuzsanna; Lehotai, Nóra; Molnár, Árpád; Ördög, Attila; Bordé, Ádám; Laskay, Gábor; Erdei, László

2016-03-01

Zinc is an essential microelement, but its excess exerts toxic effects in plants. Heavy metal stress can alter the metabolism of reactive oxygen (ROS) and nitrogen species (RNS) leading to oxidative and nitrosative damages; although the participation of these processes in Zn toxicity and tolerance is not yet known. Therefore this study aimed to evaluate the zinc tolerance of Brassica organs and the putative correspondence of it with protein nitration as a relevant marker for nitrosative stress. Both examined Brassica species (B. juncea and B. napus) proved to be moderate Zn accumulators; however B. napus accumulated more from this metal in its organs. The zinc-induced damages (growth diminution, altered morphology, necrosis, chlorosis, and the decrease of photosynthetic activity) were slighter in the shoot system of B. napus than in B. juncea. The relative zinc tolerance of B. napus shoot was accompanied by moderate changes of the nitration pattern. In contrast, the root system of B. napus suffered more severe damages (growth reduction, altered morphology, viability loss) and slighter increase in nitration level compared to B. juncea. Based on these, the organs of Brassica species reacted differentially to excess zinc, since in the shoot system modification of the nitration pattern occurred (with newly appeared nitrated protein bands), while in the roots, a general increment in the nitroproteome could be observed (the intensification of the same protein bands being present in the control samples). It can be assumed that the significant alteration of nitration pattern is coupled with enhanced zinc sensitivity of the Brassica shoot system and the general intensification of protein nitration in the roots is attached to relative zinc endurance. Copyright © 2015 Elsevier Inc. All rights reserved.

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.

Directory of Open Access Journals (Sweden)

Peer Schrapers

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

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.

Modularity in protein structures: study on all-alpha proteins.

Science.gov (United States)

Khan, Taushif; Ghosh, Indira

2015-01-01

Modularity is known as one of the most important features of protein's robust and efficient design. The architecture and topology of proteins play a vital role by providing necessary robust scaffolds to support organism's growth and survival in constant evolutionary pressure. These complex biomolecules can be represented by several layers of modular architecture, but it is pivotal to understand and explore the smallest biologically relevant structural component. In the present study, we have developed a component-based method, using protein's secondary structures and their arrangements (i.e. patterns) in order to investigate its structural space. Our result on all-alpha protein shows that the known structural space is highly populated with limited set of structural patterns. We have also noticed that these frequently observed structural patterns are present as modules or "building blocks" in large proteins (i.e. higher secondary structure content). From structural descriptor analysis, observed patterns are found to be within similar deviation; however, frequent patterns are found to be distinctly occurring in diverse functions e.g. in enzymatic classes and reactions. In this study, we are introducing a simple approach to explore protein structural space using combinatorial- and graph-based geometry methods, which can be used to describe modularity in protein structures. Moreover, analysis indicates that protein function seems to be the driving force that shapes the known structure space.

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.

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.

The ordering of low-lyiing bound states of three identical particles

International Nuclear Information System (INIS)

Richard, J.M.; Taxil, P.

1990-01-01

New results are presented on the ordering of bound states of three identical particles, a problem inspired by baryon spectroscopy. We first study the case of a perturbed harmonic oscillator and relate the splitting pattern to the level spacings in the two-body problem. We also obtain much more general results, valid for almost any symmetric potential, not necessarily pairwise. The proof is given in the framework of the hyperspherical formalism. (orig.)

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

PASA - A Program for Automated Protein NMR Backbone Signal Assignment by Pattern-Filtering Approach

International Nuclear Information System (INIS)

Xu Yizhuang; Wang Xiaoxia; Yang Jun; Vaynberg, Julia; Qin Jun

2006-01-01

We present a new program, PASA (Program for Automated Sequential Assignment), for assigning protein backbone resonances based on multidimensional heteronuclear NMR data. Distinct from existing programs, PASA emphasizes a per-residue-based pattern-filtering approach during the initial stage of the automated 13 C α and/or 13 C β chemical shift matching. The pattern filter employs one or multiple constraints such as 13 C α /C β chemical shift ranges for different amino acid types and side-chain spin systems, which helps to rule out, in a stepwise fashion, improbable assignments as resulted from resonance degeneracy or missing signals. Such stepwise filtering approach substantially minimizes early false linkage problems that often propagate, amplify, and ultimately cause complication or combinatorial explosion of the automation process. Our program (http://www.lerner.ccf.org/moleccard/qin/) was tested on four representative small-large sized proteins with various degrees of resonance degeneracy and missing signals, and we show that PASA achieved the assignments efficiently and rapidly that are fully consistent with those obtained by laborious manual protocols. The results demonstrate that PASA may be a valuable tool for NMR-based structural analyses, genomics, and proteomics

On the analysis and comparison of conformer-specific essential dynamics upon ligand binding to a protein

International Nuclear Information System (INIS)

Grosso, Marcos; Kalstein, Adrian; Parisi, Gustavo; Fernandez-Alberti, Sebastian; Roitberg, Adrian E.

2015-01-01

The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use in combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data

On the analysis and comparison of conformer-specific essential dynamics upon ligand binding to a protein

Energy Technology Data Exchange (ETDEWEB)

Grosso, Marcos; Kalstein, Adrian; Parisi, Gustavo; Fernandez-Alberti, Sebastian, E-mail: sfalberti@gmail.com [Universidad Nacional de Quilmes, Roque Saenz Peña 352, B1876BXD Bernal (Argentina); Roitberg, Adrian E. [Departments of Physics and Chemistry, University of Florida, Gainesville, Florida 32611 (United States)

2015-06-28

The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use in combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data.

A novel magnet focusing plate for matrix-assisted laser desorption/ionization analysis of magnetic bead-bound analytes.

Science.gov (United States)

Gode, David; Volmer, Dietrich A

2013-05-15

Magnetic beads are often used for serum profiling of peptide and protein biomarkers. In these assays, the bead-bound analytes are eluted from the beads prior to mass spectrometric analysis. This study describes a novel matrix-assisted laser desorption/ionization (MALDI) technique for direct application and focusing of magnetic beads to MALDI plates by means of dedicated micro-magnets as sample spots. Custom-made MALDI plates with magnetic focusing spots were made using small nickel-coated neodymium micro-magnets integrated into a stainless steel plate in a 16 × 24 (384) pattern. For demonstrating the proof-of-concept, commercial C-18 magnetic beads were used for the extraction of a test compound (reserpine) from aqueous solution. Experiments were conducted to study focusing abilities, the required laser energies, the influence of a matrix compound, dispensing techniques, solvent choice and the amount of magnetic beads. Dispensing the magnetic beads onto the micro-magnet sample spots resulted in immediate and strong binding to the magnetic surface. Light microscope images illustrated the homogeneous distribution of beads across the surfaces of the magnets, when the entire sample volume containing the beads was pipetted onto the surface. Subsequent MALDI analysis of the bead-bound analyte demonstrated excellent and reproducible ionization yields. The surface-assisted laser desorption/ionization (SALDI) properties of the strongly light-absorbing γ-Fe2O3-based beads resulted in similar ionization efficiencies to those obtained from experiments with an additional MALDI matrix compound. This feasibility study successfully demonstrated the magnetic focusing abilities for magnetic bead-bound analytes on a novel MALDI plate containing small micro-magnets as sample spots. One of the key advantages of this integrated approach is that no elution steps from magnetic beads were required during analyses compared with conventional bead experiments. Copyright © 2013 John Wiley

Tyrosine phosphorylation switching of a G protein.

Science.gov (United States)

Li, Bo; Tunc-Ozdemir, Meral; Urano, Daisuke; Jia, Haiyan; Werth, Emily G; Mowrey, David D; Hicks, Leslie M; Dokholyan, Nikolay V; Torres, Matthew P; Jones, Alan M

2018-03-30

Heterotrimeric G protein complexes are molecular switches relaying extracellular signals sensed by G protein-coupled receptors (GPCRs) to downstream targets in the cytoplasm, which effect cellular responses. In the plant heterotrimeric GTPase cycle, GTP hydrolysis, rather than nucleotide exchange, is the rate-limiting reaction and is accelerated by a receptor-like regulator of G signaling (RGS) protein. We hypothesized that posttranslational modification of the Gα subunit in the G protein complex regulates the RGS-dependent GTPase cycle. Our structural analyses identified an invariant phosphorylated tyrosine residue (Tyr 166 in the Arabidopsis Gα subunit AtGPA1) located in the intramolecular domain interface where nucleotide binding and hydrolysis occur. We also identified a receptor-like kinase that phosphorylates AtGPA1 in a Tyr 166 -dependent manner. Discrete molecular dynamics simulations predicted that phosphorylated Tyr 166 forms a salt bridge in this interface and potentially affects the RGS protein-accelerated GTPase cycle. Using a Tyr 166 phosphomimetic substitution, we found that the cognate RGS protein binds more tightly to the GDP-bound Gα substrate, consequently reducing its ability to accelerate GTPase activity. In conclusion, we propose that phosphorylation of Tyr 166 in AtGPA1 changes the binding pattern with AtRGS1 and thereby attenuates the steady-state rate of the GTPase cycle. We coin this newly identified mechanism "substrate phosphoswitching." © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

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.

Differential expression patterns of metastasis suppressor proteins in basal cell carcinoma.

Science.gov (United States)

Bozdogan, Onder; Yulug, Isik G; Vargel, Ibrahim; Cavusoglu, Tarik; Karabulut, Ayse A; Karahan, Gurbet; Sayar, Nilufer

2015-08-01

Basal cell carcinomas (BCCs) are common malignant skin tumors. Despite having a significant invasion capacity, they metastasize only rarely. Our aim in this study was to detect the expression patterns of the NM23-H1, NDRG1, E-cadherin, RHOGDI2, CD82/KAI1, MKK4, and AKAP12 metastasis suppressor proteins in BCCs. A total of 96 BCC and 10 normal skin samples were included for the immunohistochemical study. Eleven frozen BCC samples were also studied by quantitative real time polymerase chain reaction (qRT-PCR) to detect the gene expression profile. NM23-H1 was strongly and diffusely expressed in all types of BCC. Significant cytoplasmic expression of NDRG1 and E-cadherin was also detected. However, AKAP12 and CD82/KAI1 expression was significantly decreased. The expressions of the other proteins were somewhere between the two extremes. Similarly, qRT-PCR analysis showed down-regulation of AKAP12 and up-regulation of NM23-H1 and NDRG1 in BCC. Morphologically aggressive BCCs showed significantly higher cytoplasmic NDRG1 expression scores and lower CD82/KAI1 scores than non-aggressive BCCs. The relatively preserved levels of NM23-H1, NDRG1, and E-cadherin proteins may have a positive effect on the non-metastasizing features of these tumors. © 2014 The International Society of Dermatology.

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

Transferred nuclear Overhauser effect analyses of membrane-bound enkephalin analogues by sup 1 H nuclear magnetic resonance: Correlation between activities and membrane-bound conformations

Energy Technology Data Exchange (ETDEWEB)

Milon, Alain; Miyazawa, Tatsuo; Higashijima, Tsutomu (Univ. of Tokyo (Japan))

1990-01-09

Leu-enkephalin, (D-Ala{sup 2})Leu-enkephalin, and (D-Ala{sup 2})Leu-enkephalinamide (agonists) and (L-Ala{sup 2})Leu-enkephalin (inactive analogue) bind to lipid bilayer consisting of phosphatidylcholine and phosphatidylserine. The conformations that these compounds assume, once bound to perdeuterated phospholipid bilayer, have been shown to be unique, as shown by the transferred nuclear Overhauser effect (TRNOE) of {sup 1}H NMR spectroscopy. In addition, their location in the bilayer was analyzed by TRNOE in the presence of spin-labeled phospholipids. These analyses showed a clear relationship between the activity and the peptide-membrane interaction. The three active peptides, when bound to membranes, adopt the same conformation, characterized by a type II{prime} {beta}-turn around Gly{sup 3}-Phe and a {gamma}-turn around Gly{sup 2} (or D-Ala{sup 2}). The inactive analogue, (L-Ala{sup 2})Leu-enkephalin, displayed a completely different TRNOE pattern corresponding to a different conformation in the membrane-bound state. The tyrosine residue of the active compounds is not inserted into the interior of membrane, but it is inserted into the bilayer for the L-Ala{sup 2} analogue. According to these results, (L-Ala{sup 2})Leu-enkephalin may be explained to be inactive because the mode of binding to the membranes is different from that of active compounds.

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.

Nutritional Status and Daytime Pattern of Protein Intake on Match, Post-Match, Rest and Training Days in Senior Professional and Youth Elite Soccer Players.

Science.gov (United States)

Bettonviel A, E O; Brinkmans N, Y J; Russcher, Kris; Wardenaar, Floris C; Witard, Oliver C

2016-06-01

The nutritional status of elite soccer players across match, postmatch, training and rest days has not been defined. Recent evidence suggests the pattern of dietary protein intake impacts the daytime turnover of muscle proteins and, as such, influences muscle recovery. We assessed the nutritional status and daytime pattern of protein intake in senior professional and elite youth soccer players and compared findings against published recommendations. Fourteen senior professional (SP) and 15 youth elite (YP) soccer players from the Dutch premier division completed nutritional assessments using a 24-hr web-based recall method. Recall days consisted of a match, postmatch, rest, and training day. Daily energy intake over the 4-day period was similar between SP (2988 ± 583 kcal/day) and YP (2938 ± 465 kcal/day; p = .800). Carbohydrate intake over the combined 4-day period was lower in SP (4.7 ± 0.7 g·kg-1 BM·day-1) vs. YP (6.0 ± 1.5 g·kg-1 BM·day-1, p = .006) and SP failed to meet recommended carbohydrate intakes on match and training days. Conversely, recommended protein intakes were met for SP (1.9 ± 0.3 g·kg-1 BM·day-1) and YP (1.7 ± 0.4 g·kg-1 BM·day-1), with no differences between groups (p = .286). Accordingly, both groups met or exceeded recommended daily protein intakes on individual match, postmatch, rest and training days. A similar "balanced" daytime pattern of protein intake was observed in SP and YP. To conclude, SP increased protein intake on match and training days to a greater extent than YP, however at the expense of carbohydrate intake. The daytime distribution of protein intake for YP and SP aligned with current recommendations of a balanced protein meal pattern.

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.

Optimal recall from bounded metaplastic synapses: predicting functional adaptations in hippocampal area CA3.

Directory of Open Access Journals (Sweden)

Cristina Savin

2014-02-01

Full Text Available A venerable history of classical work on autoassociative memory has significantly shaped our understanding of several features of the hippocampus, and most prominently of its CA3 area, in relation to memory storage and retrieval. However, existing theories of hippocampal memory processing ignore a key biological constraint affecting memory storage in neural circuits: the bounded dynamical range of synapses. Recent treatments based on the notion of metaplasticity provide a powerful model for individual bounded synapses; however, their implications for the ability of the hippocampus to retrieve memories well and the dynamics of neurons associated with that retrieval are both unknown. Here, we develop a theoretical framework for memory storage and recall with bounded synapses. We formulate the recall of a previously stored pattern from a noisy recall cue and limited-capacity (and therefore lossy synapses as a probabilistic inference problem, and derive neural dynamics that implement approximate inference algorithms to solve this problem efficiently. In particular, for binary synapses with metaplastic states, we demonstrate for the first time that memories can be efficiently read out with biologically plausible network dynamics that are completely constrained by the synaptic plasticity rule, and the statistics of the stored patterns and of the recall cue. Our theory organises into a coherent framework a wide range of existing data about the regulation of excitability, feedback inhibition, and network oscillations in area CA3, and makes novel and directly testable predictions that can guide future experiments.

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.

Alteration of protein expression pattern of vascular endothelial growth factor (VEGF) from soluble to cell-associated isoform during tumourigenesis

International Nuclear Information System (INIS)

Cressey, Ratchada; Wattananupong, Onusa; Lertprasertsuke, Nirush; Vinitketkumnuen, Usanee

2005-01-01

Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells, and its expression has been correlated with increased tumour angiogenesis. Although numerous publications dealing with the measurement of circulating VEGF for diagnostic and therapeutic monitoring have been published, the relationship between the production of tissue VEGF and its concentration in blood is still unclear. The aims of this study were to determine: 1) The expression pattern of VEGF isoforms at the protein level in colorectal and lung adenocarcinoma in comparison to the pattern in corresponding adjacent normal tissues 2) The relationship between the expression pattern of VEGF and total level of circulating VEGF in the blood to clarify whether the results of measuring circulating VEGF can be used to predict VEGF expression in tumour tissues. Ninety-four tissue samples were obtained from patients, 76 colorectal tumour tissues and 18 lung tumour tissues. VEGF protein expression pattern and total circulating VEGF were examined using western blot and capture ELISA, respectively. Three major protein bands were predominately detected in tumour samples with an apparent molecular mass under reducing conditions of 18, 23 and 26 kDa. The 18 kDa VEGF protein was expressed equally in both normal and colorectal tumour tissues and predominately expressed in normal tissues of lung, whereas the 23 and 26 kDa protein was only detected at higher levels in tumour tissues. The 18, 23 and 26 kDa proteins are believed to represent the VEGF 121 , the VEGF 165 and the VEGF 189 , respectively. There was a significant correlation of the expression of VEGF 165 with a smaller tumour size maximum diameter <5 cm (p < 0.05), and there was a significant correlation of VEGF 189 with advanced clinical stage of colorectal tumours. The measurement of total circulating VEGF in serum revealed that cancer patients significantly (p < 0.001) possessed a higher level of circulating VEGF (1081 ± 652 pg/ml in

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…

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.

Genomic sequencing and in vivo footprinting of an expression-specific DNase I-hypersensitive site of avian vitellogenin II promoter reveal a demethylation of a mCpG and a change in specific interactions of proteins with DNA.

Science.gov (United States)

Saluz, H P; Feavers, I M; Jiricny, J; Jost, J P

1988-01-01

Genomic sequencing was used to study the in vivo methylation pattern of two CpG sites in the promoter region of the avian vitellogenin gene. The CpG at position +10 was fully methylated in DNA isolated from tissues that do not express the gene but was unmethylated in the liver of mature hens and estradiol-treated roosters. In the latter tissue, this site became demethylated and DNase I hypersensitive after estradiol treatment. A second CpG (position -52) was unmethylated in all tissues examined. In vivo genomic footprinting with dimethyl sulfate revealed different patterns of DNA protection in silent and expressed genes. In rooster liver cells, at least 10 base pairs of DNA, including the methylated CpG, were protected by protein(s). Gel-shift assays indicated that a protein factor, present in rooster liver nuclear extract, bound at this site only when it was methylated. In hen liver cells, the same unmethylated CpG lies within a protected region of approximately equal to 20 base pairs. In vitro DNase I protection and gel-shift assays indicate that this sequence is bound by a protein, which binds both double- and single-stranded DNA. For the latter substrate, this factor was shown to bind solely the noncoding (i.e., mRNA-like) strand. Images PMID:3413118

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.

Comparison of the ligand binding properties of two homologous rat apocellular retinol-binding proteins expressed in Escherichia coli.

Science.gov (United States)

Levin, M S; Locke, B; Yang, N C; Li, E; Gordon, J I

1988-11-25

Cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBP II) are 132-residue cytosolic proteins which have 56% amino acid sequence identity and bind all-trans-retinol as their endogenous ligand. They belong to a family of cytoplasmic proteins which have evolved to bind distinct hydrophobic ligands. Their patterns of tissue-specific and developmental regulation are distinct. We have compared the ligand binding properties of rat apo-CRBP and apo-CRBP II that have been expressed in Escherichia coli. Several observations indicate that the E. coli-derived apoproteins are structurally similar to the native rat proteins: they co-migrate on isoelectric focusing gels; and when complexed with all-trans-retinol, their absorption and excitation/emission spectra are nearly identical to those of the authentic rat holoproteins. Comparative lifetime and acrylamide quenching studies suggest that there are differences in the conformations of apo-CRBP and apo-CRBP II. The interaction of E. coli-derived apo-CRBP and apo-CRBP II with a variety of retinoids was analyzed using spectroscopic techniques. Both apoproteins formed high affinity complexes with all-trans-retinol (K'd approximately 10 nM). In direct binding assays, all-trans-retinal bound to both apoproteins (K'd approximately 50 nM for CRBP; K'd approximately 90 nM for CRBP II). However, all-trans-retinal could displace all-trans-retinol bound to CRBP II but not to CRBP. These observations suggests that there is a specific yet distinct interaction between these two proteins and all-trans-retinal. Apo-CRBP and apo-CRBP II did not demonstrate significant binding to either retinoic acid or methyl retinoate, an uncharged derivative of all-trans-retinoic acid. This indicates that the carboxymethyl group of methyl retinoate cannot be sterically accommodated in their binding pockets and that failure to bind retinoic acid probably is not simply due to the negative charge of its C-15 carboxylate group

Lipid-rich and protein-poor carbon allocation patterns of phytoplankton in the northern Chukchi Sea, 2011

Science.gov (United States)

Yun, Mi Sun; Joo, Hui Tae; Park, Jung Woo; Kang, Jae Joong; Kang, Sung-Ho; Lee, Sang H.

2018-04-01

The carbon allocations of phytoplankton into different photosynthetic end products (lipids, LMWM, polysaccharides, and proteins) were determined to understand physiological conditions of phytoplankton in the northern Chukchi Sea during the Korean Arctic expedition, 2011, using the 13C isotope tracer technique. The carbon allocation rates of lipids, LMWM, polysaccharides, and proteins were 0.00009-0.00062 h-1, 0.00001-0.00049 h-1, 0.00001-0.00025 h-1, and 0.00001-0.00062 h-1 within the euphotic depths from surface to 1% light depths during our cruise period, respectively. Significant relationships between protein production rates and chlorophyll a concentrations (large and total) were found in this study. Moreover, we found a significant negative relationship between lipid production rates and ammonium concentrations. These relationships match well with the previous results for environmental/physiological conditions for phytoplankton growth. Overall, phytoplankton allocated more photosynthetic carbon into lipids (42.5 ± 17.7%) whereas relatively lower to proteins (20.4 ± 15.5%) in this study. The lipid-rich and protein-poor allocation patterns in this study suggest that phytoplankton in the northern Chukchi Sea were in a stationary growth phase under nutrient deficient condition based on biological and environmental conditions observed during our study period. Based on comparison with the previous studies in the northern Bering Sea and southern Chukchi Sea, we found that the photosynthetic carbon allocation patterns depending on physiological status of phytoplankton under the different growth and/or nutrient conditions could be largely vary at different regions in the Arctic Ocean. More intensive research on the physiological status of phytoplankton is further required to determine how phytoplankton response to the changing environmental conditions and consequently how they impact on higher trophic levels in marine ecosystems in the Arctic Ocean.

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

Non-destructive Patterning of Carbon Electrodes by Using the Direct Mode of Scanning Electrochemical Microscopy.

Science.gov (United States)

Stratmann, Lutz; Clausmeyer, Jan; Schuhmann, Wolfgang

2015-11-16

Patterning of glassy carbon surfaces grafted with a layer of nitrophenyl moieties was achieved by using the direct mode of scanning electrochemical microscopy (SECM) to locally reduce the nitro groups to hydroxylamine and amino functionalities. SECM and atomic force microscopy (AFM) revealed that potentiostatic pulses applied to the working electrode lead to local destruction of the glassy carbon surface, most likely caused by etchants generated at the positioned SECM tip used as the counter electrode. By applying galvanostatic pulses, and thus, limiting the current during structuring, corrosion of the carbon surface was substantially suppressed. After galvanostatic patterning, unambiguous proof of the formation of the anticipated amino moieties was possible by modulation of the pH value during the feedback mode of SECM imaging. This patterning strategy is suitable for the further bio-modification of microstructured surfaces. Alkaline phosphatase, as a model enzyme, was locally bound to the modified areas, thus showing that the technique can be used for the development of protein microarrays. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

General transfer matrix formalism to calculate DNA-protein-drug binding in gene regulation: application to OR operator of phage lambda.

Science.gov (United States)

Teif, Vladimir B

2007-01-01

The transfer matrix methodology is proposed as a systematic tool for the statistical-mechanical description of DNA-protein-drug binding involved in gene regulation. We show that a genetic system of several cis-regulatory modules is calculable using this method, considering explicitly the site-overlapping, competitive, cooperative binding of regulatory proteins, their multilayer assembly and DNA looping. In the methodological section, the matrix models are solved for the basic types of short- and long-range interactions between DNA-bound proteins, drugs and nucleosomes. We apply the matrix method to gene regulation at the O(R) operator of phage lambda. The transfer matrix formalism allowed the description of the lambda-switch at a single-nucleotide resolution, taking into account the effects of a range of inter-protein distances. Our calculations confirm previously established roles of the contact CI-Cro-RNAP interactions. Concerning long-range interactions, we show that while the DNA loop between the O(R) and O(L) operators is important at the lysogenic CI concentrations, the interference between the adjacent promoters P(R) and P(RM) becomes more important at small CI concentrations. A large change in the expression pattern may arise in this regime due to anticooperative interactions between DNA-bound RNA polymerases. The applicability of the matrix method to more complex systems is discussed.

Light at night alters daily patterns of cortisol and clock proteins in female Siberian hamsters.

Science.gov (United States)

Bedrosian, T A; Galan, A; Vaughn, C A; Weil, Z M; Nelson, R J

2013-06-01

Humans and other organisms have adapted to a 24-h solar cycle in response to life on Earth. The rotation of the planet on its axis and its revolution around the sun cause predictable daily and seasonal patterns in day length. To successfully anticipate and adapt to these patterns in the environment, a variety of biological processes oscillate with a daily rhythm of approximately 24 h in length. These rhythms arise from hierarchally-coupled cellular clocks generated by positive and negative transcription factors of core circadian clock gene expression. From these endogenous cellular clocks, overt rhythms in activity and patterns in hormone secretion and other homeostatic processes emerge. These circadian rhythms in physiology and behaviour can be organised by a variety of cues, although they are most potently entrained by light. In recent history, there has been a major change from naturally-occurring light cycles set by the sun, to artificial and sometimes erratic light cycles determined by the use of electric lighting. Virtually every individual living in an industrialised country experiences light at night (LAN) but, despite its prevalence, the biological effects of such unnatural lighting have not been fully considered. Using female Siberian hamsters (Phodopus sungorus), we investigated the effects of chronic nightly exposure to dim light on daily rhythms in locomotor activity, serum cortisol concentrations and brain expression of circadian clock proteins (i.e. PER1, PER2, BMAL1). Although locomotor activity remained entrained to the light cycle, the diurnal fluctuation of cortisol concentrations was blunted and the expression patterns of clock proteins in the suprachiasmatic nucleus and hippocampus were altered. These results demonstrate that chronic exposure to dim LAN can dramatically affect fundamental cellular function and emergent physiology. © 2013 British Society for Neuroendocrinology.

Impact of bounded noise on the formation and instability of spiral wave in a 2D Lattice of neurons

Science.gov (United States)

Yao, Yuangen; Deng, Haiyou; Yi, Ming; Ma, Jun

2017-02-01

Spiral waves in the neocortex may provide a spatial framework to organize cortical oscillations, thus help signal communication. However, noise influences spiral wave. Many previous theoretical studies about noise mainly focus on unbounded Gaussian noise, which contradicts that a real physical quantity is always bounded. Furthermore, non-Gaussian noise is also important for dynamical behaviors of excitable media. Nevertheless, there are no results concerning the effect of bounded noise on spiral wave till now. Based on Hodgkin-Huxley neuron model subjected to bounded noise with the form of Asin[ωt + σW(t)], the influences of bounded noise on the formation and instability of spiral wave in a two-dimensional (2D) square lattice of neurons are investigated in detail by separately adjusting the intensity σ, amplitude A, and frequency f of bounded noise. It is found that the increased intensity σ can facilitate the formation of spiral wave while the increased amplitude A tends to destroy spiral wave. Furthermore, frequency of bounded noise has the effect of facilitation or inhibition on pattern synchronization. Interestingly, for the appropriate intensity, amplitude and frequency can separately induce resonance-like phenomenon.

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.

Hidden Markov model approach for identifying the modular framework of the protein backbone.

Science.gov (United States)

Camproux, A C; Tuffery, P; Chevrolat, J P; Boisvieux, J F; Hazout, S

1999-12-01

The hidden Markov model (HMM) was used to identify recurrent short 3D structural building blocks (SBBs) describing protein backbones, independently of any a priori knowledge. Polypeptide chains are decomposed into a series of short segments defined by their inter-alpha-carbon distances. Basically, the model takes into account the sequentiality of the observed segments and assumes that each one corresponds to one of several possible SBBs. Fitting the model to a database of non-redundant proteins allowed us to decode proteins in terms of 12 distinct SBBs with different roles in protein structure. Some SBBs correspond to classical regular secondary structures. Others correspond to a significant subdivision of their bounding regions previously considered to be a single pattern. The major contribution of the HMM is that this model implicitly takes into account the sequential connections between SBBs and thus describes the most probable pathways by which the blocks are connected to form the framework of the protein structures. Validation of the SBBs code was performed by extracting SBB series repeated in recoding proteins and examining their structural similarities. Preliminary results on the sequence specificity of SBBs suggest promising perspectives for the prediction of SBBs or series of SBBs from the protein sequences.

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...

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.

High-resolution structure of TBP with TAF1 reveals anchoring patterns in transcriptional regulation.

Science.gov (United States)

Anandapadamanaban, Madhanagopal; Andresen, Cecilia; Helander, Sara; Ohyama, Yoshifumi; Siponen, Marina I; Lundström, Patrik; Kokubo, Tetsuro; Ikura, Mitsuhiko; Moche, Martin; Sunnerhagen, Maria

2013-08-01

The general transcription factor TFIID provides a regulatory platform for transcription initiation. Here we present the crystal structure (1.97 Å) and NMR analysis of yeast TAF1 N-terminal domains TAND1 and TAND2 bound to yeast TBP, together with mutational data. We find that yeast TAF1-TAND1, which in itself acts as a transcriptional activator, binds TBP's concave DNA-binding surface by presenting similar anchor residues to TBP as does Mot1 but from a distinct structural scaffold. Furthermore, we show how TAF1-TAND2 uses an aromatic and acidic anchoring pattern to bind a conserved TBP surface groove traversing the basic helix region, and we find highly similar TBP-binding motifs also presented by the structurally distinct TFIIA, Mot1 and Brf1 proteins. Our identification of these anchoring patterns, which can be easily disrupted or enhanced, provides insight into the competitive multiprotein TBP interplay critical to transcriptional regulation.

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.

Twist on protein microarrays: layering wax-patterned nitrocellulose to create customizable and separable arrays of multiplexed affinity columns.

Science.gov (United States)

de Lange, Victoria; Vörös, János

2014-05-06

We developed the simple and inexpensive FoRe microarray to simultaneously test several 1 μL samples for multiple proteins. By combining forward and reverse phase microarrays into an innovative three-dimensional format, the FoRe array exploits the advantages and eliminates several drawbacks of the traditional approaches (i.e., large sample volumes, protein loss, and cross-reactivity between detection antibodies). Samples are pipetted into an array of separable, multiplexed affinity columns. Several nitrocellulose membranes, each functionalized with a different capture antibody, are stacked to create a customizable affinity column. The nitrocellulose is patterned with wax to form 25 isolated microspots on each layer, allowing us to analyze multiple samples in parallel. After running the immunoassay, the stacks are quickly disassembled, revealing 2D microarrays of different fractions from multiple samples. By combining the stack-and-separate technique with wax patterning, we keep the arrays low cost and easily tailored to a variety of applications. We successfully performed 3D multiplexing using a model system with mouse and rabbit IgG. Binding proved to be independent of the position in the stack, and the limit of detection for a mouse IgG sandwich assay was 7.3 pM in BSA and 15 pM in human plasma. The FoRe microarray makes it possible to identify protein expression patterns across several minute volume samples; for example, it could be used to analyze cell lysate in drug response studies or pricks of blood from small animal studies.

Nuclear proteins interacting with the promoter region of the human granulocyte/macrophage colony-stimulating factor gene

International Nuclear Information System (INIS)

Shannon, M.F.; Gamble, J.R.; Vadas, M.A.

1988-01-01

The gene for human granulocyte/macrophage colony-stimulating factor (GM-CSF) is expressed in a tissue-specific as well as an activation-dependent manner. The interaction of nuclear proteins with the promoter region of the GM-CSF gene that is likely to be responsible for this pattern of GM-CSF expression was investigated. The authors show that nuclear proteins interact with DNA fragments from the GM-CSF promoter in a cell-specific manner. A region spanning two cytokine-specific sequences, cytokine 1 (CK-1, 5', GAGATTCCAC 3') and cytokine 2 (CK-2, 5' TCAGGTA 3') bound two nuclear proteins from GM-CSF-expressing cells in gel retardation assays. NF-GMb was inducible with phorbol 12-myristate 13-acetate and accompanied induction of GM-CSF message. NF-GMb was absent in cell lines not producing GM-CSF, some of which had other distinct binding proteins. NF-GMa and NF-GMb eluted from a heparin-Sepharose column at 0.3 and 0.6 M KCl, respectively. They hypothesize that the sequences CK-1 and CK-2 bind specific proteins and regulate GM-CSF transcription

Expression patterns of five polymorphic membrane proteins during the Chlamydia abortus developmental cycle.

Science.gov (United States)

Wheelhouse, Nick; Sait, Michelle; Wilson, Kim; Aitchison, Kevin; McLean, Kevin; Smith, David G E; Longbottom, David

2012-12-07

It has been suggested that polymorphic membrane proteins (Pmps) belonging to the Type V autotransporter protein family play an important role in the pathogenesis of Chlamydia abortus (C. abortus; formerly Chlamydophila abortus) infection. In a previous study we demonstrated the expression of all the pmps at the transcriptional level. The purpose of this study was to measure the number of Pmp positive inclusions throughout the C. abortus developmental cycle to investigate heterogeneity in expression patterns. McCoy cells were infected with C. abortus and analysed for Pmp expression over a 72 h period by fluorescent immunocytochemistry. Pmp18D could be detected at all analysed time points, and could only be accurately quantified from 36 hpi while Pmp10G positive inclusions could be visualised from 36hpi. Expression of Pmps 13G, 16G and 17G could only be visualised later in the cycle and within less than half of visualised inclusions. These results indicate that while expression of specific Pmps is constitutive (Pmp18D), the pattern of expression of other Pmps is more variable. This suggests that different members of the Pmp family may play different roles within the developmental cycle of the organism, with some (Pmps10G and 18D) having roles throughout the cycle, while the heterogeneity of expression of others may aid in antigenic variation. Copyright © 2012 Elsevier B.V. All rights reserved.

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...

Improved plasma amino acids pattern following 12 months of supplemented low-protein diet in peritoneal dialysis patients.

Science.gov (United States)

Jiang, Na; Qian, Jiaqi; Lin, Aiwu; Fang, Wei; Cao, Liou; Wang, Qin; Ni, Zhaohui; Lindholm, Bengt; Axelsson, Jonas; Yao, Qiang

2010-07-01

Decreased plasma essential amino acid (EAA) levels, increased nonessential amino acid (NEAA) levels, and low EAA to NEAA ratio (E/NEAA) are common in peritoneal dialysis (PD) patients and may impact uremic complications. In the present study, we investigate the impact of keto acids-supplemented low-protein (sLP) diet on plasma amino acids (AAs) patterns in stable PD patients. This is a supplemental analysis of a previously published prospective and randomized trial. Thirty-nine PD patients selected from the original population were divided to receive either low (LP: 0.6-0.8 g/kg ideal body weight [IBW]/d, n = 13), keto acids-supplemented low- (sLP: 0.6-0.8 g/kg IBW/d + 0.12 g/kg IBW/d of keto acids, n = 12), or high- (HP: 1.0-1.2 g/kg IBW/d, n = 14) protein diets and followed for 1 year. Plasma AA patterns were assessed at baseline and 12 months using high-performance liquid chromatography. Whereas there were no significant differences between the three groups at baseline, following 12 months, the E/NEAA had increased significantly in group sLP (0.58 +/- 0.16 to 0.83 +/- 0.20, p diet supplemented with keto acids significantly improved the pattern of plasma AA in prevalent PD patients.

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)

Some recent results on μ-transfer in systems with bound hydrogen

International Nuclear Information System (INIS)

Knight, J.D.; Mausner, L.F.; Orth, C.J.; Schillaci, M.E.; Naumann, R.A.; Schmidt, G.

1977-01-01

Preliminary results are given for two sets of experiments conducted in order to obtain information on the role of bound hydrogen in possible μ transfer processes by comparisons of muonic Lyman spectra. Muonic x-ray intensity patterns of Cl in CCl 4 , CH 2 Cl 2 and a CCl 4 + C 8 H 18 mixture. Secondly the same type of experiment was performed but with F as the test element. Muonic x-ray spectra of the normal and deutero forms of a compound were compared

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...