Prevalence of IgG antibodies to human parvovirus B19 in haemophilia children treated with recombinant factor (F)VIII only or with at least one plasma-derived FVIII or FIX concentrate: results from the French haemophilia cohort.

PubMed

Gaboulaud, Valérie; Parquet, Armelle; Tahiri, Cedric; Claeyssens, Ségolène; Potard, Valérie; Faradji, Albert; Peynet, Jocelyne; Costagliola, Dominique

2002-02-01

Human parvovirus B19 (B19) has been transmitted by some brands of virally attenuated plasma-derived factor VIII (FVIII) or IX (FIX) concentrates. To quantify the differences of human parvovirus B19 risk transmission between albumin-stabilized recombinant factor and plasma-derived factor, we studied the prevalence of IgG antibodies to B19 (anti-B19) in 193 haemophiliac children between 1 and 6-years of age who had previously been treated with albumin-stabilized recombinant FVIII only (n = 104), and in children previously treated with solvent/detergent high-purity non-immunopurified and non-nanofiltered FVIII or IX concentrates (n = 89). Association between the prevalence of anti-B19 and the treatment group was analysed using multivariate logistic regression. Age, severity and type of haemophilia, number of cumulative days of exposure to factor VIII or IX, previous history of red blood cells or plasma transfusion were considered as potential confounding variables. A higher prevalence of anti-B19 was found in children previously treated with solvent/detergent high-purity non-immunopurified and non-nanofiltered FVIII or IX concentrates than in children treated with albumin- stabilized recombinant FVIII only (OR: 22.3; CI: 7.9-62.8), independently of the other factors studied.

Comparison of (1->3)-β-D-glucan, mannan/anti-mannan antibodies, and Cand-Tec Candida antigen as serum biomarkers for candidemia.

PubMed

Held, Jürgen; Kohlberger, Isabelle; Rappold, Elfriede; Busse Grawitz, Andrea; Häcker, Georg

2013-04-01

We conducted a case-control study using the Fungitell assay, the novel Platelia Candida Antigen (Ag) Plus and Candida Antibody (Ab) Plus assays, and the Cand-Tec latex agglutination test to evaluate the usefulness of (1→3)-β-D-glucan (BDG), mannan antigen with/without anti-mannan antibody, and Cand-Tec Candida antigen measurement for the diagnosis of candidemia. A total of 56 patients fulfilled the inclusion criteria and were enrolled. One hundred patients with bacteremia and 100 patients with sterile blood cultures served as negative controls. In the candidemia group, median (1→3)-β-D-glucan, mannan antigen, and anti-mannan antibody levels were 427 pg/ml, 190 pg/ml, and 18.6 antibody units (AU)/ml, respectively. All three parameters were significantly elevated in patients with candidemia. The sensitivity and specificity were, respectively, 87.5% and 85.5% for (1→3)-β-D-glucan, 58.9% and 97.5% for mannan antigen, 62.5% and 65.0% for anti-mannan antibody, 89.3% and 63.0% for mannan antigen plus anti-mannan antibody, 89.3% and 85.0% for BDG plus mannan antigen, and 13.0% and 93.9% for Cand-Tec Candida antigen. The low mannan antigen sensitivity was in part caused by Candida parapsilosis and Candida guilliermondii fungemias, which were not detected by the Platelia Candida Ag Plus assay. When the cutoff was lowered from 125 pg/ml to 50 pg/ml, mannan antigen sensitivity increased to 69.6% without severely affecting the specificity (93.5%). Contrary to recently published data, superficial candidiasis was not associated with elevated mannan antigen levels, not even after the cutoff was lowered. Combining procalcitonin (PCT) with (1→3)-β-D-glucan to increase specificity provided a limited advantage because the benefit of the combination did not outweigh the loss of sensitivity. Our results demonstrate that the Cand-Tec Candida antigen and the mannan antigen plus anti-mannan antibody measurements have unacceptably low sensitivity or specificity. Of the four

The Macrophage Mannose Receptor Regulate Mannan-Induced Psoriasis, Psoriatic Arthritis, and Rheumatoid Arthritis-Like Disease Models

PubMed Central

Hagert, Cecilia; Sareila, Outi; Kelkka, Tiina; Jalkanen, Sirpa; Holmdahl, Rikard

2018-01-01

The injection of mannan into mice can result in the development of psoriasis (Ps) and psoriatic arthritis (PsA), whereas co-injection with antibodies toward collagen type II leads to a chronic rheumatoid-like arthritis. The critical event in all these diseases is mannan-mediated activation of macrophages, causing more severe disease if the macrophages are deficient in neutrophil cytosolic factor 1 (Ncf1), i.e., lack the capacity to make a reactive oxygen species (ROS) burst. In this study, we investigated the role of one of the receptors binding mannan; the macrophage mannose receptor (MR, CD206). MR is a C-type lectin present on myeloid cells and lymphatics. We found that mice deficient in MR expression had more severe mannan-induced Ps, PsA as well as rheumatoid-like arthritis. Interestingly, the MR-mediated protection was partly lost in Ncf1 mutated mice and was associated with an type 2 macrophage expansion. In conclusion, these results show that MR protects against a pathogenic inflammatory macrophage response induced by mannan and is associated with induction of ROS. PMID:29467756

Enhanced immunogenicity of a tricomponent mannan tetanus toxoid conjugate vaccine targeted to dendritic cells via Dectin-1 by incorporating β-glucan.

PubMed

Lipinski, Tomasz; Fitieh, Amira; St Pierre, Joëlle; Ostergaard, Hanne L; Bundle, David R; Touret, Nicolas

2013-04-15

In a previous attempt to generate a protective vaccine against Candida albicans, a β-mannan tetanus toxoid conjugate showed poor immunogenicity in mice. To improve the specific activation toward the fungal pathogen, we aimed to target Dectin-1, a pattern-recognition receptor expressed on monocytes, macrophages, and dendritic cells. Laminarin, a β-glucan ligand of Dectin-1, was incorporated into the original β-mannan tetanus toxoid conjugate providing a tricomponent conjugate vaccine. A macrophage cell line expressing Dectin-1 was employed to show binding and activation of Dectin-1 signal transduction pathway by the β-glucan-containing vaccine. Ligand binding to Dectin-1 resulted in the following: 1) activation of Src family kinases and Syk revealed by their recruitment and phosphorylation in the vicinity of bound conjugate and 2) translocation of NF-κB to the nucleus. Treatment of immature bone marrow-derived dendritic cells (BMDCs) with tricomponent or control vaccine confirmed that the β-glucan-containing vaccine exerted its enhanced activity by virtue of dendritic cell targeting and uptake. Immature primary cells stimulated by the tricomponent vaccine, but not the β-mannan tetanus toxoid vaccine, showed activation of BMDCs. Moreover, treated BMDCs secreted increased levels of several cytokines, including TGF-β and IL-6, which are known activators of Th17 cells. Immunization of mice with the novel type of vaccine resulted in improved immune response manifested by high titers of Ab recognizing C. albicans β-mannan Ag. Vaccine containing laminarin also affected distribution of IgG subclasses, showing that vaccine targeting to Dectin-1 receptor can benefit from augmentation and immunomodulation of the immune response.

Comparison of (1→3)-β-d-Glucan, Mannan/Anti-Mannan Antibodies, and Cand-Tec Candida Antigen as Serum Biomarkers for Candidemia

PubMed Central

Kohlberger, Isabelle; Rappold, Elfriede; Busse Grawitz, Andrea; Häcker, Georg

2013-01-01

We conducted a case-control study using the Fungitell assay, the novel Platelia Candida Antigen (Ag) Plus and Candida Antibody (Ab) Plus assays, and the Cand-Tec latex agglutination test to evaluate the usefulness of (1→3)-β-d-glucan (BDG), mannan antigen with/without anti-mannan antibody, and Cand-Tec Candida antigen measurement for the diagnosis of candidemia. A total of 56 patients fulfilled the inclusion criteria and were enrolled. One hundred patients with bacteremia and 100 patients with sterile blood cultures served as negative controls. In the candidemia group, median (1→3)-β-d-glucan, mannan antigen, and anti-mannan antibody levels were 427 pg/ml, 190 pg/ml, and 18.6 antibody units (AU)/ml, respectively. All three parameters were significantly elevated in patients with candidemia. The sensitivity and specificity were, respectively, 87.5% and 85.5% for (1→3)-β-d-glucan, 58.9% and 97.5% for mannan antigen, 62.5% and 65.0% for anti-mannan antibody, 89.3% and 63.0% for mannan antigen plus anti-mannan antibody, 89.3% and 85.0% for BDG plus mannan antigen, and 13.0% and 93.9% for Cand-Tec Candida antigen. The low mannan antigen sensitivity was in part caused by Candida parapsilosis and Candida guilliermondii fungemias, which were not detected by the Platelia Candida Ag Plus assay. When the cutoff was lowered from 125 pg/ml to 50 pg/ml, mannan antigen sensitivity increased to 69.6% without severely affecting the specificity (93.5%). Contrary to recently published data, superficial candidiasis was not associated with elevated mannan antigen levels, not even after the cutoff was lowered. Combining procalcitonin (PCT) with (1→3)-β-d-glucan to increase specificity provided a limited advantage because the benefit of the combination did not outweigh the loss of sensitivity. Our results demonstrate that the Cand-Tec Candida antigen and the mannan antigen plus anti-mannan antibody measurements have unacceptably low sensitivity or specificity. Of the four

Compact and multiple plasmonic nanofilter based on ultra-broad stopband in partitioned semicircle or semiring stub waveguide

NASA Astrophysics Data System (ADS)

Zheng, Mingfei; Li, Hongjian; Chen, Zhiquan; He, Zhihui; Xu, Hui; Zhao, Mingzhuo

2017-11-01

We propose a compact plasmonic nanofilter in partitioned semicircle or semiring stub waveguide, and investigate the transmission characteristics of the two novel systems by using the finite-difference time-domain method. An ultra-broad stopband phenomenon is generated by partitioning a single stub into a double stub with a rectangular metal partition, which is caused by the destructive interference superposition of the reflected and transmitted waves from each stub. A tunable stopband is realized in the multiple plasmonic nanofilter by adjusting the width of the partition and the (outer) radius and inner radius of the stub, whose starting wavelength, ending wavelength, center wavelength, bandwidth and total tunable bandwidth are discussed, and specific filtering waveband and optimum structural parameter are obtained. The proposed structures realize asymmetrical stub and achieve ultra-broad stopband, and have potential applications in band-stop nanofilters and high-density plasmonic integrated optical circuits.

Enhanced mannan-derived fermentable sugars of palm kernel cake by mannanase-catalyzed hydrolysis for production of biobutanol.

PubMed

Shukor, Hafiza; Abdeshahian, Peyman; Al-Shorgani, Najeeb Kaid Nasser; Hamid, Aidil Abdul; Rahman, Norliza A; Kalil, Mohd Sahaid

2016-10-01

Catalytic depolymerization of mannan composition of palm kernel cake (PKC) by mannanase was optimized to enhance the release of mannan-derived monomeric sugars for further application in acetone-butanol-ethanol (ABE) fermentation. Efficiency of enzymatic hydrolysis of PKC was studied by evaluating effects of PKC concentration, mannanase loading, hydrolysis pH value, reaction temperature and hydrolysis time on production of fermentable sugars using one-way analysis of variance (ANOVA). The ANOVA results revealed that all factors studied had highly significant effects on total sugar liberated (P<0.01). The optimum conditions for PKC hydrolysis were 20% (w/v) PKC concentration, 5% (w/w) mannanase loading, hydrolysis pH 4.5, 45°C temperature and 72h hydrolysis time. Enzymatic experiments in optimum conditions revealed total fermentable sugars of 71.54±2.54g/L were produced including 67.47±2.51g/L mannose and 2.94±0.03g/L glucose. ABE fermentation of sugar hydrolysate by Clostridium saccharoperbutylacetonicum N1-4 resulted in 3.27±1.003g/L biobutanol. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nonlinear tuning techniques of plasmonic nano-filters

NASA Astrophysics Data System (ADS)

Kotb, Rehab; Ismail, Yehea; Swillam, Mohamed A.

2015-02-01

In this paper, a fitting model to the propagation constant and the losses of Metal-Insulator-Metal (MIM) plasmonic waveguide is proposed. Using this model, the modal characteristics of MIM plasmonic waveguide can be solved directly without solving Maxwell's equations from scratch. As a consequence, the simulation time and the computational cost that are needed to predict the response of different plasmonic structures can be reduced significantly. This fitting model is used to develop a closed form model that describes the behavior of a plasmonic nano-filter. Easy and accurate mechanisms to tune the filter are investigated and analyzed. The filter tunability is based on using a nonlinear dielectric material with Pockels or Kerr effect. The tunability is achieved by applying an external voltage or through controlling the input light intensity. The proposed nano-filter supports both red and blue shift in the resonance response depending on the type of the used non-linear material. A new approach to control the input light intensity by applying an external voltage to a previous stage is investigated. Therefore, the filter tunability to a stage that has Kerr material can be achieved by applying voltage to a previous stage that has Pockels material. Using this method, the Kerr effect can be achieved electrically instead of varying the intensity of the input source. This technique enhances the ability of the device integration for on-chip applications. Tuning the resonance wavelength with high accuracy, minimum insertion loss and high quality factor is obtained using these approaches.

A neutral mannan from Ceratocystis fagacearum culture filtrate

Treesearch

P. McWain; G.F. Gregory

1972-01-01

The culture filtrate of Ceratocystis fagacearum contains a mannan that produces some symptoms similar to oak wilt in red oak seedlings and cuttings. The mannan has a high molecular weight and a skeleton of α- (1 → 6) linked mannose units with considerable branching. Some similarities to commercial yeast mannan have been observed.

Nanofilter platform based on functionalized carbon nanotubes for adsorption and elimination of Acrolein, a toxicant in cigarette smoke

NASA Astrophysics Data System (ADS)

Yoosefian, Mehdi; Pakpour, Atef; Etminan, Nazanin

2018-06-01

This paper discusses the use of carboxylated single-walled carbon nanotube as a general nanofilter platform for the removal of acrolein carcinogen from cigarette smoke. The analyses carried out in the detailed study of the electronic and structural effects of the adsorption of acrolein onto COOH loaded on single-walled carbon nanotube under the density functional theory framework. The results of Bader theory of atoms in molecules, natural bond orbital, molecular potential electron surface and density of state confirm the potential application of the suggested nanofilter platform.

Deriving an explicit hepatic clearance equation accounting for plasma protein binding and hepatocellular uptake.

PubMed

Yoon, Miyoung; Clewell, Harvey J; Andersen, Melvin E

2013-02-01

High throughput in vitro biochemical and cell-based assays have the promise to provide more mechanism-based assessments of the adverse effects of large numbers of chemicals. One of the most challenging hurdles for interpreting in vitro toxicity findings is the need for reverse dosimetry tools that estimate the exposures that will give concentrations in vivo similar to the active concentrations in vitro. Recent experience using IVIVE approaches to estimate in vivo pharmacokinetics (Wetmore et al., 2012) identified the need to develop a hepatic clearance equation that explicitly accounted for a broader set of protein binding and membrane transport processes and did not depend on a well-mixed description of the liver compartment. Here we derive an explicit steady-state hepatic clearance equation that includes these factors. In addition to the derivation, we provide simple computer code to calculate steady-state extraction for any combination of blood flow, membrane transport processes and plasma protein-chemical binding rates. This expanded equation provides a tool to estimate hepatic clearance for a more diverse array of compounds. Copyright © 2012 Elsevier Ltd. All rights reserved.

Mannan-Binding Lectin Is Involved in the Protection against Renal Ischemia/Reperfusion Injury by Dietary Restriction

PubMed Central

Shushimita, Shushimita; van der Pol, Pieter; W.F. de Bruin, Ron; N. M. Ijzermans, Jan; van Kooten, Cees; Dor, Frank J. M. F.

2015-01-01

Preoperative fasting and dietary restriction offer robust protection against renal ischemia/reperfusion injury (I/RI) in mice. We recently showed that Mannan-binding lectin (MBL), the initiator of the lectin pathway of complement activation, plays a pivotal role in renal I/RI. Based on these findings, we investigated the effect of short-term DR (30% reduction of total food intake) or three days of water only fasting on MBL in 10–12 weeks old male C57/Bl6 mice. Both dietary regimens significantly reduce the circulating levels of MBL as well as its mRNA expression in liver, the sole production site of MBL. Reconstitution of MBL abolished the protection afforded by dietary restriction, whereas in the fasting group the protection persisted. These data show that modulation of MBL is involved in the protection against renal I/RI induced by dietary restriction, and suggest that the mechanisms of protection induced by dietary restriction and fasting may be different. PMID:26367533

Large-scale production and properties of human plasma-derived activated Factor VII concentrate.

PubMed

Tomokiyo, K; Yano, H; Imamura, M; Nakano, Y; Nakagaki, T; Ogata, Y; Terano, T; Miyamoto, S; Funatsu, A

2003-01-01

An activated Factor VII (FVIIa) concentrate, prepared from human plasma on a large scale, has to date not been available for clinical use for haemophiliacs with antibodies against FVIII and FIX. In the present study, we attempted to establish a large-scale manufacturing process to obtain plasma-derived FVIIa concentrate with high recovery and safety, and to characterize its biochemical and biological properties. FVII was purified from human cryoprecipitate-poor plasma, by a combination of anion exchange and immunoaffinity chromatography, using Ca2+-dependent anti-FVII monoclonal antibody. To activate FVII, a FVII preparation that was nanofiltered using a Bemberg Microporous Membrane-15 nm was partially converted to FVIIa by autoactivation on an anion-exchange resin. The residual FVII in the FVII and FVIIa mixture was completely activated by further incubating the mixture in the presence of Ca2+ for 18 h at 10 degrees C, without any additional activators. For preparation of the FVIIa concentrate, after dialysis of FVIIa against 20 mm citrate, pH 6.9, containing 13 mm glycine and 240 mm NaCl, the FVIIa preparation was supplemented with 2.5% human albumin (which was first pasteurized at 60 degrees C for 10 h) and lyophilized in vials. To inactivate viruses contaminating the FVIIa concentrate, the lyophilized product was further heated at 65 degrees C for 96 h in a water bath. Total recovery of FVII from 15 000 l of plasma was approximately 40%, and the FVII preparation was fully converted to FVIIa with trace amounts of degraded products (FVIIabeta and FVIIagamma). The specific activity of the FVIIa was approximately 40 U/ micro g. Furthermore, virus-spiking tests demonstrated that immunoaffinity chromatography, nanofiltration and dry-heating effectively removed and inactivated the spiked viruses in the FVIIa. These results indicated that the FVIIa concentrate had both high specific activity and safety. We established a large-scale manufacturing process of human plasma-derived

Mite allergoids coupled to nonoxidized mannan from Saccharomyces cerevisae efficiently target canine dendritic cells for novel allergy immunotherapy in veterinary medicine.

PubMed

Soria, Irene; Alvarez, Javier; Manzano, Ana I; López-Relaño, Juan; Cases, Bárbara; Mas-Fontao, Ana; Cañada, F Javier; Fernández-Caldas, Enrique; Casanovas, Miguel; Jiménez-Barbero, Jesús; Palomares, Oscar; Viñals-Flórez, Luis M; Subiza, José L

2017-08-01

We have recently reported that grass pollen allergoids conjugated with nonoxidized mannan of Saccharomyces cerevisae using glutaraldehyde results in a novel hypoallergenic mannan-allergen complex with improved properties for allergen vaccination. Using this approach, human dendritic cells show a better allergen uptake and cytokine profile production (higher IL-10/IL-4 ratio) for therapeutic purposes. Here we aim to address whether a similar approach can be extended to dogs using canine dendritic cells. Six healthy Spanish Greyhound dogs were used as blood donors to obtain canine dendritic cells (DC) derived from peripheral blood monocytes. Allergens from Dermatophagoides farinae mite were polymerized and conjugated with nonoxidized mannan. Nuclear magnetic resonance (NMR), gel electrophoresis (SDS-PAGE), immunoblotting and IgE-ELISA inhibition studies were conducted to evaluate the main characteristics of the allergoid obtained. Mannan-allergen conjugate and controls were assayed in vitro for canine DC uptake and production of IL-4 and IL-10. The results indicate that the conjugation of D. farinae allergens with nonoxidized mannan was feasible using glutaraldehyde. The resulting product was a polymerized structure showing a high molecular weight as detected by NMR and SDS-PAGE analysis. The mannan-allergen conjugate was hypoallergenic with a reduced reactivity with specific dog IgE. An increase in both allergen uptake and IL-10/IL-4 ratio was obtained when canine DCs were incubated with the mannan-allergen conjugate, as compared with the control allergen preparations (unmodified D. farinae allergens and oxidized mannan-allergen conjugate). We conclude that hypoallergenic D. farinae allergens coupled to nonoxidized mannan is a novel allergen preparation suitable for canine allergy immunotherapy targeting dendritic cells. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism

PubMed Central

Zhu, Yanping; Cameron, Elizabeth; Pudlo, Nicholas A.; Porter, Nathan T.; Urs, Karthik; Thompson, Andrew J.; Cartmell, Alan; Rogowski, Artur; Hamilton, Brian S.; Chen, Rui; Tolbert, Thomas J.; Piens, Kathleen; Bracke, Debby; Vervecken, Wouter; Hakki, Zalihe; Speciale, Gaetano; Munōz-Munōz, Jose L.; Day, Andrew; Peña, Maria J.; McLean, Richard; Suits, Michael D.; Boraston, Alisdair B.; Atherly, Todd; Ziemer, Cherie J.; Williams, Spencer J.; Davies, Gideon J.; Abbott, D. Wade; Martens, Eric C.; Gilbert, Harry J.

2016-01-01

Yeasts, which have been a component of the human diet for at least 7000 years, possess an elaborate cell wall α-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast α-mannan is a viable food source for Bacteroides thetaiotaomicron (Bt), a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of α-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by Bt presents a ‘selfish’ model for the catabolism of this recalcitrant polysaccharide. This report shows how a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet. PMID:25567280

Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism.

PubMed

Cuskin, Fiona; Lowe, Elisabeth C; Temple, Max J; Zhu, Yanping; Cameron, Elizabeth; Pudlo, Nicholas A; Porter, Nathan T; Urs, Karthik; Thompson, Andrew J; Cartmell, Alan; Rogowski, Artur; Hamilton, Brian S; Chen, Rui; Tolbert, Thomas J; Piens, Kathleen; Bracke, Debby; Vervecken, Wouter; Hakki, Zalihe; Speciale, Gaetano; Munōz-Munōz, Jose L; Day, Andrew; Peña, Maria J; McLean, Richard; Suits, Michael D; Boraston, Alisdair B; Atherly, Todd; Ziemer, Cherie J; Williams, Spencer J; Davies, Gideon J; Abbott, D Wade; Martens, Eric C; Gilbert, Harry J

2015-01-08

Yeasts, which have been a component of the human diet for at least 7,000 years, possess an elaborate cell wall α-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast α-mannan is a viable food source for the Gram-negative bacterium Bacteroides thetaiotaomicron, a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of α-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by B. thetaiotaomicron presents a 'selfish' model for the catabolism of this difficult to breakdown polysaccharide. Genomic comparison with B. thetaiotaomicron in conjunction with cell culture studies show that a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet.

Isolation and characterization of a mannan from mesosomal membrane vesicles of Micrococcus lysodeikticus.

PubMed

Owen, P; Salton, M R

1975-10-06

The carbohydrate content of mesosomal membranes of Micrococcus lysodeikticus has been shown to be consistently higher (about four times) than that of corresponding plasma membrane preparations. Analysis of washed membrane fractions by gas-liquid chromatography indicated that mannose was the major neutral sugar of both types of membrane (accounting for 95 and 89%, respectively, of the mesosomal and plasma membrane carbohydrate). Small amounts of inositol, glucose and ribose were also detected. We have shown by polyacrylamide gel electrophoresis in sodium dodecylsulphate and by precipitation and agar gel diffusion experiments with concanavalin A that a mannan is the major carbohydrate component of both types of membrane. This polymer can be selectively released from mesosomal membranes by a simple procedure involving low ionic strength-shock and heating to 80 degrees C for 1 min, and purified by ultrafiltration and ethanol precipitation. The mannan contains mannose as the only neutral carbohydrate, is not phosphorylated and does not contain significant amounts of amino sugars or uronic acids. Agar gel electrophoresis experiments, however, indicate an anionic polymer whose acidic properties are eliminated upon mild base hydrolysis. Analysis of native mannan by infrared spectroscopy reveals absorption bands attributable to ester carbonyl groups and to carboxylate ions, consistent with the presence of succinyl residues in the polymer (Owen, P. and Salton, M.R.J. (1975) Biochem, Biophys. Res. Commun. 63, 875--800). A sedimentation coefficient of 1.39 S was obtained by analytical ultracentrifugation in 1.0 M NaCl and a value of one reducing equivalent per 50 mannose residues by reduction with NaB3H4. The polysaccharide was only slightly degraded (2%) by jack bean alpha-mannosidase and could precipitate 15 times its own weight of concanavalin A. The acidic polymers was also detected in the cell "periplasm" and was secreted from cells grown in defined media during the

Mannan-binding lectin directly interacts with Toll-like receptor 4 and suppresses lipopolysaccharide-induced inflammatory cytokine secretion from THP-1 cells

PubMed Central

Wang, Mingyong; Chen, Yue; Zhang, Yani; Zhang, Liyun; Lu, Xiao; Chen, Zhengliang

2011-01-01

Mannan-binding lectin (MBL) plays a key role in the lectin pathway of complement activation and can influence cytokine expression. Toll-like receptor 4 (TLR4) is expressed extensively and has been demonstrated to be involved in lipopolysaccharide (LPS)-induced signaling. We first sought to determine whether MBL exposure could modulate LPS-induced inflammatory cytokine secretion and nuclear factor-κB (NF-κB) activity by using the monocytoid cell line THP-1. We then investigated the possible mechanisms underlying any observed regulatory effect. Using ELISA and reverse transcriptase polymerase chain reaction (RT-PCR) analysis, we found that at both the protein and mRNA levels, treatment with MBL suppresses LPS-induced tumor-necrosis factor (TNF)-α and IL-12 production in THP-1 cells. An electrophoretic mobility shift assay and western blot analysis revealed that MBL treatment can inhibit LPS-induced NF-κB DNA binding and translocation in THP-1 cells. While the binding of MBL to THP-1 cells was evident at physiological calcium concentrations, this binding occurred optimally in response to supraphysiological calcium concentrations. This binding can be partly inhibited by treatment with either a soluble form of recombinant TLR4 extracellular domain or anti-TLR4 monoclonal antibody (HTA125). Activation of THP-1 cells by LPS treatment resulted in increased MBL binding. We also observed that MBL could directly bind to the extracellular domain of TLR4 in a dose-dependent manner, and this interaction could attenuate the binding of LPS to cell surfaces. Taken together, these data suggest that MBL may affect cytokine expression through modulation of LPS-/TLR-signaling pathways. These findings suggest that MBL may play an important role in both immune regulation and the signaling pathways involved in cytokine networks. PMID:21383675

Mannan oligosaccharide requires functional ETC and TLR for biological radiation protection to normal cells.

PubMed

Sanguri, Sweta; Gupta, Damodar

2018-06-27

Low LET Ionizing radiation is known to alter intracellular redox balance by inducing free radical generation, which may cause oxidative modification of various cellular biomolecules. The extent of biomolecule-modifications/ damages and changes in vital processes (viz. cellular homeostasis, inter-/intra-cellular signaling, mitochondrial physiology/dynamics antioxidant defence systems) are crucial which in turn determine fate of cells. In the present study, we expended TLR expressing (normal/ transformed) and TLR null cells; and we have shown that mannan pretreatment in TLR expressing normal cells offers survival advantage against lethal doses of ionizing radiation. On the contrary, mannan pretreatment does not offer any protection against radiation to TLR null cells, NKE ρ° cells and transformed cells. In normal cells, abrupt decrease in mitochondrial membrane potential and endogenous ROS levels occurs following treatment with mannan. We intend to irradiate mannan-pretreated cells at a specific stage of perturbed mitochondrial functioning and ROS levels to comprehend if mannan pretreatment offers any survival advantage against radiation exposure to cells. Interestingly, pre-irradiation treatment of cells with mannan activates NFκB, p38 and JNK, alters mitochondrial physiology, increases expression of Cu/ZnSOD and MnSOD, minimizes oxidation of mitochondrial phospholipids and offers survival advantage in comparison to irradiated group, in TLR expressing normal cells. The study demonstrates that TLR and mitochondrial ETC functions are inevitable in radio-protective efficacy exhibited by mannan.

Changes in the levels of mannan-binding lectin and ficolins during head-down tilted bed rest.

PubMed

Kelsen, Jens; Sandahl, Thomas D; Storm, Line; Frings-Meuthen, Petra; Dahlerup, Jens F; Thiel, Steffen

2014-08-01

Spaceflight studies and ground-based analogues of microgravity indicate a weakening of human immunity. Mannan-binding lectin (MBL) and H-, L-, and M-ficolin together constitute the lectin pathway and mediate the clearance of pathogens through complement activation. We hypothesized that simulated microgravity may weaken human innate immune functions and studied the impact of 6° head-down tilted bed rest (HDT) for 21 d on MBL and ficolin levels. Within a 6-mo period, seven men underwent two periods of HDT. Blood samples were analyzed for MBL, H-, L-, and M-ficolin, mannose-binding lectin-associated protein of 44 kDa (MAp44), and collectin liver 1 (CL-L1) by time-resolved immunofluorometric assays (TRIFMA). We observed well-defined individual preintervention levels of MBL and ficolins. Remarkably similar intraindividual changes occurred for MBL and MBL levels decreased (mean 282 ng · ml⁻¹) in the recovery phase. Conversely, CL-L1, a protein with MBL-like properties, increased (mean 102 ng · ml⁻¹) during the recovery phase. M-ficolin increased (mean 79 ng · ml⁻¹) within the first 2 d of HDT, followed by a decrease (mean 112 ng · ml⁻¹) during the recovery phase. L-ficolin increased (mean 304 ng · ml⁻¹) during HDT, while H-ficolin was essentially unaffected. MAp44, a down-regulator of the lectin pathway, decreased initially (mean 78 ng · ml⁻¹) in the recovery phase followed by an increase (mean 131 ng · ml⁻¹). Alterations in MBL and ficolin levels were modest and with our current knowledge do not lead to overt immunodeficiency. Pronounced changes occurred when the subjects resumed the upright position. In selected individuals, these changes appear to be a conserved response to HDT.

Selective binding to transthyretin and tetramer stabilization in serum from patients with familial amyloidotic polyneuropathy by an iodinated diflunisal derivative

PubMed Central

2004-01-01

In familial amyloidotic polyneuropathy, TTR (transthyretin) variants are deposited as amyloid fibrils. It is thought that this process involves TTR tetramer dissociation, which leads to partially unfolded monomers that aggregate and polymerize into amyloid fibrils. This process can be counteracted by stabilization of the tetramer. Several small compounds, such as diclofenac, diflunisal and flufenamic acid, have been reported to bind to TTR in vitro, in the T4 (thyroxine) binding channel that runs through the TTR tetramer, and consequently are considered to stabilize TTR. However, if these agents bind plasma proteins other than TTR, decreased drug availability will occur, compromising their use as therapeutic agents for TTR amyloidosis. In the present work, we compared the action of these compounds and of new derivatives designed to increase both selectivity of binding to TTR and inhibitory potency in relation to TTR amyloid fibril formation. We found two diflunisal derivatives that, in contrast with diclofenac, flufenamic acid and diflunisal, displaced T4 from TTR in plasma preferentially over binding to albumin and thyroxine binding globulin. The same diflunisal derivatives also had a stabilizing effect on TTR tetramers in plasma, as studied by isoelectric focusing of whole plasma under semi-denaturing conditions. In addition, by transmission electron microscopy, we demonstrated that, in contrast with other proposed TTR stabilizers (namely diclofenac, flufenamic acid and diflunisal), one of the diflunisal derivatives tested efficiently inhibited TTR aggregation. Taken together, our ex vivo and in vitro studies present evidence for the selectivity and efficiency of novel diflunisal derivates as TTR stabilizers and as inhibitors of fibril formation. PMID:15080795

Clinical relevance of drug binding to plasma proteins

NASA Astrophysics Data System (ADS)

Ascenzi, Paolo; Fanali, Gabriella; Fasano, Mauro; Pallottini, Valentina; Trezza, Viviana

2014-12-01

Binding to plasma proteins highly influences drug efficacy, distribution, and disposition. Serum albumin, the most abundant protein in plasma, is a monomeric multi-domain macromolecule that displays an extraordinary ligand binding capacity, providing a depot and carrier for many endogenous and exogenous compounds, such as fatty acids and most acidic drugs. α-1-Acid glycoprotein, the second main plasma protein, is a glycoprotein physiologically involved in the acute phase reaction and is the main carrier for basic and neutral drugs. High- and low-density lipoproteins play a limited role in drug binding and are natural drug delivery system only for few lipophilic drugs or lipid-based formulations. Several factors influence drug binding to plasma proteins, such as pathological conditions, concurrent administration of drugs, sex, and age. Any of these factors, in turn, influences drug efficacy and toxicity. Here, biochemical, biomedical, and biotechnological aspects of drug binding to plasma proteins are reviewed.

Genetics Home Reference: mannose-binding lectin deficiency

MedlinePlus

... Nobelprize.org: The Immune System - In More Detail Patient Support and Advocacy Resources (1 link) ... Sources for This Page Arora M, Munoz E, Tenner AJ. Identification of a site on mannan-binding lectin critical ...

[Determination of plasma protein binding rate of arctiin and arctigenin with ultrafiltration].

PubMed

Han, Xue-Ying; Wang, Wei; Tan, Ri-Qiu; Dou, De-Qiang

2013-02-01

To determine the plasma protein binding rate of arctiin and arctigenin. The ultrafiltration combined with HPLC was employed to determine the plasma protein binding rate of arctiin and arctigenin as well as rat plasma and healthy human plasma proteins. The plasma protein binding rate of arctiin with rat plasma at the concentrations of 64. 29, 32.14, 16.07 mg x L(-1) were (71.2 +/- 2.0)%, (73.4 +/- 0.61)%, (78.2 +/- 1.9)%, respectively; while the plasma protein binding rate of arctiin with healthy human plasma at the above concentrations were (64.8 +/- 3.1)%, (64.5 +/- 2.5)%, (77.5 +/- 1.7)%, respectively. The plasma protein binding rate of arctigenin with rat plasma at the concentrations of 77.42, 38.71, 19.36 mg x L(-1) were (96.7 +/- 0.41)%, (96.8 +/- 1.6)%, (97.3 +/- 0.46)%, respectively; while the plasma protein binding rate of arctigenin with normal human plasma at the above concentrations were (94.7 +/- 3.1)%, (96.8 +/- 1.6)%, (97.9 +/- 1.3)%, respectively. The binding rate of arctiin with rat plasma protein was moderate, which is slightly higher than the binding rate of arctiin with healthy human plasma protein. The plasma protein binding rates of arctigenin with both rat plasma and healthy human plasma are very high.

From mannan to bioethanol: cell surface co-display of β-mannanase and β-mannosidase on yeast Saccharomyces cerevisiae.

PubMed

Ishii, Jun; Okazaki, Fumiyoshi; Djohan, Apridah Cameliawati; Hara, Kiyotaka Y; Asai-Nakashima, Nanami; Teramura, Hiroshi; Andriani, Ade; Tominaga, Masahiro; Wakai, Satoshi; Kahar, Prihardi; Yopi; Prasetya, Bambang; Ogino, Chiaki; Kondo, Akihiko

2016-01-01

Mannans represent the largest hemicellulosic fraction in softwoods and also serve as carbohydrate stores in various plants. However, the utilization of mannans as sustainable resources has been less advanced in sustainable biofuel development. Based on a yeast cell surface-display technology that enables the immobilization of multiple enzymes on the yeast cell walls, we constructed a recombinant Saccharomyces cerevisiae strain that co-displays β-mannanase and β-mannosidase; this strain is expected to facilitate ethanol fermentation using mannan as a biomass source. Parental yeast S. cerevisiae assimilated mannose and glucose as monomeric sugars, producing ethanol from mannose. We constructed yeast strains that express tethered β-mannanase and β-mannosidase; co-display of the two enzymes on the cell surface was confirmed by immunofluorescence staining and enzyme activity assays. The constructed yeast cells successfully hydrolyzed 1,4-β-d-mannan and produced ethanol by assimilating the resulting mannose without external addition of enzymes. Furthermore, the constructed strain produced ethanol from 1,4-β-d-mannan continually during the third batch of repeated fermentation. Additionally, the constructed strain produced ethanol from ivory nut mannan; ethanol yield was improved by NaOH pretreatment of the substrate. We successfully displayed β-mannanase and β-mannosidase on the yeast cell surface. Our results clearly demonstrate the utility of the strain co-displaying β-mannanase and β-mannosidase for ethanol fermentation from mannan biomass. Thus, co-tethering β-mannanase and β-mannosidase on the yeast cell surface provides a powerful platform technology for yeast fermentation toward the production of bioethanol and other biochemicals from lignocellulosic materials containing mannan components.

Utilization of Exocellular Mannan from Rhodotorula glutinis as an Immunoreactive Antigen in Diagnosis of Leptospirosis

PubMed Central

Matsuo, Kouki; Isogai, Emiko; Araki, Yoshio

2000-01-01

Previously, Rhodotorula glutinis was reported to produce a large amount of exocellular mannan, having a repeating unit of →3)-d-Manp-(1→4)-d-Manp-(1→. Recently, we found that antigenic polysaccharides of Leptospira biflexa serovar patoc strain Patoc I have the same repeating unit and cross-react with antisera raised against extended strains of other leptospires (K. Matsuo, E. Isogai, and Y. Araki, Carbohydr. Res., in press). This structural identity and the difficulty of producing and isolating antigens led us to confirm the usefulness of Rhodotorula mannan as an immunoreactive antigen in a serological diagnosis of leptospirosis. In the present investigation, we confirmed the structural identity of an exocellular mannan isolated from R. glutinis AHU 3479 and tried to use it as an immunoreactive antigen in a serological diagnosis of leptospirosis. From its chemical analysis and 1H- and 13C-labeled nuclear magnetic resonance spectrometry, the Rhodotorula mannan was confirmed to consist of the same disaccharide units. Furthermore, such a preparation was shown to immunoreact to various sera from patients suffering with leptospirosis as well as to most rabbit antiserum preparations obtained from immunization with various strains of pathogenic leptospires. Therefore, the Rhodotorula mannan preparation is useful as an immunoreactive antigen in the serological diagnosis for leptospirosis. PMID:11015396

Inflammatory markers following acute fuel oil exposure or bacterial lipopolysaccharide in mallard ducks (Anas platyrhynchos).

PubMed

Lee, Kelly A; Tell, Lisa A; Mohr, F Charles

2012-12-01

Adult mallard ducks (Anas platyrhynchos) were orally dosed with bunker C fuel oil for 5 days, and five different inflammatory markers (haptoglobin, mannan-binding lectin, ceruloplasmin, unsaturated iron-binding capacity, and plasma iron) were measured in blood plasma prior to and 8, 24, 48, and 72 hr following exposure. In order to contrast the response to fuel oil with that of a systemic inflammatory response, an additional five ducks were injected intramuscularly with bacterial lipopolysaccharide (LPS). Oil-treated birds had an inflammatory marker profile that was significantly different from control and LPS-treated birds, showing decreases in mannan-binding lectin-dependent hemolysis and unsaturated iron-binding capacity, but no changes in any of the other inflammatory markers. Birds treated with oil also exhibited increased liver weights, decreased body and splenic weights, and decreased packed cell volume.

How actin binds and assembles onto plasma membranes from Dictyostelium discoideum

PubMed Central

1988-01-01

We have shown previously (Schwartz, M. A., and E. J. Luna. 1986. J. Cell Biol. 102: 2067-2075) that actin binds with positive cooperativity to plasma membranes from Dictyostelium discoideum. Actin is polymerized at the membrane surface even at concentrations well below the critical concentration for polymerization in solution. Low salt buffer that blocks actin polymerization in solution also prevents actin binding to membranes. To further explore the relationship between actin polymerization and binding to membranes, we prepared four chemically modified actins that appear to be incapable of polymerizing in solution. Three of these derivatives also lost their ability to bind to membranes. The fourth derivative (EF actin), in which histidine-40 is labeled with ethoxyformic anhydride, binds to membranes with reduced affinity. Binding curves exhibit positive cooperativity, and cross- linking experiments show that membrane-bound actin is multimeric. Thus, binding and polymerization are tightly coupled, and the ability of these membranes to polymerize actin is dramatically demonstrated. EF actin coassembles weakly with untreated actin in solution, but coassembles well on membranes. Binding by untreated actin and EF actin are mutually competitive, indicating that they bind to the same membrane sites. Hill plots indicate that an actin trimer is the minimum assembly state required for tight binding to membranes. The best explanation for our data is a model in which actin oligomers assemble by binding to clustered membrane sites with successive monomers on one side of the actin filament bound to the membrane. Individual binding affinities are expected to be low, but the overall actin-membrane avidity is high, due to multivalency. Our results imply that extracellular factors that cluster membrane proteins may create sites for the formation of actin nuclei and thus trigger actin polymerization in the cell. PMID:3392099

Binding of [51Cr]ethylenediaminetetraacetate to proteins of human plasma.

PubMed Central

Babiker, M M

1986-01-01

Binding of [51Cr]EDTA to human plasma proteins was investigated using chemical and chromatographic techniques of separation of the proteins and protein fractions. Total plasma proteins isolated with ethanol retained 12.95 +/- 0.46% of the initial plasma activity. Proteins separated by other precipitants retained about 16% of the initial radioactivity. Globulins exhibited the highest binding capacity for [51Cr]EDTA and retained about 11.7% of the initial plasma activity following chromatographic separation. This value represents about 70% of the radioactivity bound by the total proteins of the plasma. gamma-Globulins contributed most of the binding attributed to the globulins and retained about 8.7% of the initial [51Cr]EDTA activity. The repeatedly reported underestimation of the renal glomerular filtration rate when estimated as the clearance of [51Cr]EDTA could be adequately accounted for by the extent of binding of this marker to the plasma proteins. PMID:2427701

Mannan-Binding Lectin Inhibits Candida albicans-Induced Cellular Responses in PMA-Activated THP-1 Cells through Toll-Like Receptor 2 and Toll-Like Receptor 4

PubMed Central

Yang, Jianbin; Zhao, Dongfang; Wang, Hongpo; Shao, Feng; Wang, Wenjun; Sun, Ruili; Ling, Mingzhi; Zhai, Jingjing; Song, Shijun

2013-01-01

Background Candida albicans (C. albicans), the most common human fungal pathogen, can cause fatal systemic infections under certain circumstances. Mannan-binding lectin (MBL),a member of the collectin family in the C-type lectin superfamily, is an important serum component associated with innate immunity. Toll-like receptors (TLRs) are expressed extensively, and have been shown to be involved in C. albicans-induced cellular responses. We first examined whether MBL modulated heat-killed (HK) C. albicans-induced cellular responses in phorbol 12-myristate 13-acetate (PMA)-activated human THP-1 macrophages. We then investigated the possible mechanisms of its inhibitory effect. Methodology/Principal Finding Enzyme-linked immunosorbent assay (ELISA) and reverse transcriptasepolymerase chain reaction (RT-PCR) analysis showed that MBL at higher concentrations (10–20 µg/ml) significantly attenuated C. albicans-induced chemokine (e.g., IL-8) and proinflammatory cytokine (e.g., TNF-α) production from PMA-activated THP-1 cells at both protein and mRNA levels. Electrophoretic mobility shift assay (EMSA) and Western blot (WB) analysis showed that MBL could inhibit C. albicans-induced nuclear factor-κB (NF-κB) DNA binding and its translocation in PMA-activated THP-1 cells. MBL could directly bind to PMA-activated THP-1 cells in the presence of Ca2+, and this binding decreased TLR2 and TLR4 expressions in C. albicans-induced THP-1 macrophages. Furthermore, the binding could be partially inhibited by both anti-TLR2 monoclonal antibody (clone TL2.1) and anti-TLR4 monoclonal antibody (clone HTA125). In addition, co-immunoprecipitation experiments and microtiter wells assay showed that MBL could directly bind to the recombinant soluble form of extracellular TLR2 domain (sTLR2) and sTLR4. Conclusions/Significance Our study demonstrates that MBL can affect proinflammatory cytokine and chemokine expressions by modifying C. albicans-/TLR-signaling pathways. This study supports an

Mannanase hydrolysis of spruce galactoglucomannan focusing on the influence of acetylation on enzymatic mannan degradation.

PubMed

Arnling Bååth, Jenny; Martínez-Abad, Antonio; Berglund, Jennie; Larsbrink, Johan; Vilaplana, Francisco; Olsson, Lisbeth

2018-01-01

Galactoglucomannan (GGM) is the most abundant hemicellulose in softwood, and consists of a backbone of mannose and glucose units, decorated with galactose and acetyl moieties. GGM can be hydrolyzed into fermentable sugars, or used as a polymer in films, gels, and food additives. Endo -β-mannanases, which can be found in the glycoside hydrolase families 5 and 26, specifically cleave the mannan backbone of GGM into shorter oligosaccharides. Information on the activity and specificity of different mannanases on complex and acetylated substrates is still lacking. The aim of this work was to evaluate and compare the modes of action of two mannanases from Cellvibrio japonicus ( Cj Man5A and Cj Man26A) on a variety of mannan substrates, naturally and chemically acetylated to varying degrees, including naturally acetylated spruce GGM. Both enzymes were evaluated in terms of cleavage patterns and their ability to accommodate acetyl substitutions. Cj Man5A and Cj Man26A demonstrated different substrate preferences on mannan substrates with distinct backbone and decoration structures. Cj Man5A action resulted in higher amounts of mannotriose and mannotetraose than that of Cj Man26A, which mainly generated mannose and mannobiose as end products. Mass spectrometric analysis of products from the enzymatic hydrolysis of spruce GGM revealed that an acetylated hexotriose was the shortest acetylated oligosaccharide produced by Cj Man5A, whereas Cj Man26A generated acetylated hexobiose as well as diacetylated oligosaccharides. A low degree of native acetylation did not significantly inhibit the enzymatic action. However, a high degree of chemical acetylation resulted in decreased hydrolyzability of mannan substrates, where reduced substrate solubility seemed to reduce enzyme activity. Our findings demonstrate that the two mannanases from C. japonicus have different cleavage patterns on linear and decorated mannan polysaccharides, including the abundant and industrially important

Diverse patterns of cell wall mannan/galactomannan occurrence in seeds of the Leguminosae.

PubMed

Bento, João Francisco; Mazzaro, Irineu; de Almeida Silva, Lia Magalhães; de Azevedo Moreira, Renato; Ferreira, Marília Locatelli Correa; Reicher, Fany; Petkowicz, Carmen Lúcia de Oliveira

2013-01-30

Endosperms from seeds of different subfamilies of Leguminosae were submitted to sequential aqueous and alkaline aqueous extractions. The extractions from species belonging to the Mimosoideae and Faboideae subfamilies yielded galactomannans with constant Man:Gal ratios, whereas the extractions from Caesalpinioideae seeds gave rise to galactomannans with increasing values of the Man:Gal ratio. The presence of a family of galactomannans within the same species may be a trait found only in Caesalpinioideae subfamily. The final insoluble residues that were obtained after the removal of galactomannans from the Caesalpinioideae and Faboideae subfamilies are composed of pure mannans and do not contain cellulose, while those from the Mimosoideae subfamily are composed of cellulose. A mannan was isolated from the unripe endosperm of Caesalpinia pulcherrima, suggesting no developmental relationship between galactomannan and mannan. These results are consistent with the presence of a distinctive cell wall pattern in the endosperms of Leguminosae species. Copyright © 2012 Elsevier Ltd. All rights reserved.

Plasma sex steroid binding in Chiroptera.

PubMed

Kwiecinski, G G; Damassa, D A; Gustafson, A W; Armao, M E

1987-04-01

Plasma steroid binding was examined in samples obtained from seven species of bats representing four different families. A specific sex steroid-binding protein (SBP) was identified by steady-state polyacrylamide gel electrophoresis in representatives of two families, the phyllostomids and the vespertilionids. In these species, as in primates, SBP not only exhibited high affinity for the androgens testosterone and dihydrotestosterone (DHT), but also for estradiol. A specific SBP was not identified in the tropical American vampire bat or in the two species of pteropodids examined. In all species examined, except for the vampire bat, a specific corticosteroid-binding globulin (CBG) was also identified. In addition to binding glucocorticoids, CBG in these species appeared to bind androgens as well.

Conservation of Mannan Synthesis in Fungi of the Zygomycota and Ascomycota Reveals a Broad Diagnostic Target.

PubMed

Burnham-Marusich, Amanda R; Hubbard, Breeana; Kvam, Alexander J; Gates-Hollingsworth, Marcellene; Green, Heather R; Soukup, Eric; Limper, Andrew H; Kozel, Thomas R

2018-01-01

Ascomycetes and zygomycetes account for the majority of (i) fungi responsible for cutaneous, subcutaneous, and invasive human fungal infections, (ii) plant fungal pathogens, (iii) fungi that threaten global biodiversity, (iv) fungal agents of agricultural spoilage, and (v) fungi in water-damaged buildings. Rapid recognition of fungal infection (or contamination) enables early treatment (or remediation). A bioinformatics search found homologues of Saccharomyces cerevisiae Mnn9p present in members of the Zygomycota and Ascomycota phyla and absent in members of the Chytridiomycota and Basidiomycota. Mnn9p is a component of the yeast mannan polymerization complex and is necessary for α-1,6 mannan production. A monoclonal antibody (2DA6) was produced that was reactive with purified mannans of Mucor , Rhizopus , Aspergillus , Fusarium , and Candida species. Experimentation using a 2DA6 antigen capture enzyme-linked immunosorbent assay (ELISA) and extracts of fungi from the four phyla found agreement between the presence or absence of Mnn9p homologues and production or lack of production of mannan reactive with 2DA6. Studies of cell extracts from yeast mannan mutants identified α-1,6 mannan as the epitope recognized by 2DA6. To translate this finding into a point-of-use diagnostic, a 2DA6 lateral flow immunoassay was constructed that detected mannan in (i) extracts of dermatophytes and fungi that produce trauma-related infection and (ii) tissue from plants infected with Grosmannia clavigera or Sclerotium cepivorum These studies (i) revealed that the conservation of α-1,6-linked mannan in fungi of the Zygomycota and Ascomycota can be exploited as a broad diagnostic target and (ii) have provided a means to detect that target in an immunoassay platform that is well suited for clinic or field use. IMPORTANCE A key question asked when faced with an infection, an infestation, or environmental damage is whether it is a fungus. Identification of fungi as the cause of the

Conservation of Mannan Synthesis in Fungi of the Zygomycota and Ascomycota Reveals a Broad Diagnostic Target

PubMed Central

Hubbard, Breeana; Kvam, Alexander J.; Gates-Hollingsworth, Marcellene; Green, Heather R.; Soukup, Eric; Limper, Andrew H.; Kozel, Thomas R.

2018-01-01

ABSTRACT Ascomycetes and zygomycetes account for the majority of (i) fungi responsible for cutaneous, subcutaneous, and invasive human fungal infections, (ii) plant fungal pathogens, (iii) fungi that threaten global biodiversity, (iv) fungal agents of agricultural spoilage, and (v) fungi in water-damaged buildings. Rapid recognition of fungal infection (or contamination) enables early treatment (or remediation). A bioinformatics search found homologues of Saccharomyces cerevisiae Mnn9p present in members of the Zygomycota and Ascomycota phyla and absent in members of the Chytridiomycota and Basidiomycota. Mnn9p is a component of the yeast mannan polymerization complex and is necessary for α-1,6 mannan production. A monoclonal antibody (2DA6) was produced that was reactive with purified mannans of Mucor, Rhizopus, Aspergillus, Fusarium, and Candida species. Experimentation using a 2DA6 antigen capture enzyme-linked immunosorbent assay (ELISA) and extracts of fungi from the four phyla found agreement between the presence or absence of Mnn9p homologues and production or lack of production of mannan reactive with 2DA6. Studies of cell extracts from yeast mannan mutants identified α-1,6 mannan as the epitope recognized by 2DA6. To translate this finding into a point-of-use diagnostic, a 2DA6 lateral flow immunoassay was constructed that detected mannan in (i) extracts of dermatophytes and fungi that produce trauma-related infection and (ii) tissue from plants infected with Grosmannia clavigera or Sclerotium cepivorum. These studies (i) revealed that the conservation of α-1,6-linked mannan in fungi of the Zygomycota and Ascomycota can be exploited as a broad diagnostic target and (ii) have provided a means to detect that target in an immunoassay platform that is well suited for clinic or field use. IMPORTANCE A key question asked when faced with an infection, an infestation, or environmental damage is whether it is a fungus. Identification of fungi as the cause of the

The Nano-filters as the tools for the management of the water imbalance in the human society

NASA Astrophysics Data System (ADS)

Singh, R. P.; Kontar, V.

2011-12-01

The imbalance of water in the human society there is some situation where the water demand is not equivalent to the water supply. We are talking now about the shortage of some clear water which suitable for human use, animals, plants, technologies etc. There are existing some various imbalances of water in the human society, but about this will be other publications. The humanity has have the millennial experience of the water imbalance management. The novelty of the matter is the new nano-materials which offer a lot of the new principles more effective management of the water imbalance in the human society. The nano-materials have typical pore size 0.001 micron (1 nano-meter). There are some metal-containing nano-particles, CNTs, fullerene, graphene, zeolites and dendrimers etc, The nano-materials have unique physicochemical properties due to their large surface areas, size and shape-dependent optical, electronic, and catalytic properties that make them very useful for separation components some various stuff and water also. They have ability to functionalize with various chemical groups to increase their affinity toward a desired compound. The silver nano-wires have established a variety of applications, including transparent conductive electrodes for solar cells and optoelectronic. The salt of silver i.e. bulk silver shows photo-catalytic properties. The gold decorated silver nano-wires film may clean the organic molecule while irradiated with either commercial bulb or sun light. The mat (membrane) papers of nano-wires may clean up spilled oil at sea and organic pollutants in water. Arsenic-poisoned drinking water is a global problem, affecting people in Asia, Africa, North America, South America and Europe. Tiny bits of iron oxide that are smaller than living cells known as nanorust, which naturally binds with arsenic, could be used as a low-cost means of removing arsenic from water. Nano-tea bag purifies water on a small scale. The sachets are made up from the

The N-Terminal Domain of the Flo1 Flocculation Protein from Saccharomyces cerevisiae Binds Specifically to Mannose Carbohydrates ▿

PubMed Central

Goossens, Katty V. Y.; Stassen, Catherine; Stals, Ingeborg; Donohue, Dagmara S.; Devreese, Bart; De Greve, Henri; Willaert, Ronnie G.

2011-01-01

Saccharomyces cerevisiae cells possess a remarkable capacity to adhere to other yeast cells, which is called flocculation. Flocculation is defined as the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended. These cell-cell interactions are mediated by a class of specific cell wall proteins, called flocculins, that stick out of the cell walls of flocculent cells. The N-terminal part of the three-domain protein is responsible for carbohydrate binding. We studied the N-terminal domain of the Flo1 protein (N-Flo1p), which is the most important flocculin responsible for flocculation of yeast cells. It was shown that this domain is both O and N glycosylated and is structurally composed mainly of β-sheets. The binding of N-Flo1p to d-mannose, α-methyl-d-mannoside, various dimannoses, and mannan confirmed that the N-terminal domain of Flo1p is indeed responsible for the sugar-binding activity of the protein. Moreover, fluorescence spectroscopy data suggest that N-Flo1p contains two mannose carbohydrate binding sites with different affinities. The carbohydrate dissociation constants show that the affinity of N-Flo1p for mono- and dimannoses is in the millimolar range for the binding site with low affinity and in the micromolar range for the binding site with high affinity. The high-affinity binding site has a higher affinity for low-molecular-weight (low-MW) mannose carbohydrates and no affinity for mannan. However, mannan as well as low-MW mannose carbohydrates can bind to the low-affinity binding site. These results extend the cellular flocculation model on the molecular level. PMID:21076009

Hydrolysis of (1,4)-β-D-mannans in barley (Hordeum vulgare L.) is mediated by the concerted action of (1,4)-β-D-mannan endohydrolase and β-D-mannosidase

PubMed Central

Hrmova, Maria; Burton, Rachel A.; Biely, Peter; Lahnstein, Jelle; Fincher, Geoffrey B.

2006-01-01

A family GH5 (family 5 glycoside hydrolase) (1,4)-β-D-mannan endohydrolase or β-D-mannanase (EC 3.2.1.78), designated HvMAN1, has been purified 300-fold from extracts of 10-day-old barley (Hordeum vulgare L.) seedlings using ammonium sulfate fractional precipitation, followed by ion exchange, hydrophobic interaction and size-exclusion chromatography. The purified HvMAN1 is a relatively unstable enzyme with an apparent molecular mass of 43 kDa, a pI of 7.8 and a pH optimum of 4.75. The HvMAN1 releases Man (mannose or D-mannopyranose)-containing oligosaccharides of degree of polymerization 2–6 from mannans, galactomannans and glucomannans. With locust-bean galactomannan and mannopentaitol as substrates, the enzyme has Km constants of 0.16 mg·ml−1 and 5.3 mM and kcat constants of 12.9 and 3.9 s−1 respectively. Product analyses indicate that transglycosylation reactions occur during hydrolysis of (1,4)-β-D-manno-oligosaccharides. The complete sequence of 374 amino acid residues of the mature enzyme has been deduced from the nucleotide sequence of a near full-length cDNA, and has allowed a three-dimensional model of the HvMAN1 to be constructed. The barley HvMAN1 gene is a member of a small (1,4)-β-D-mannan endohydrolase family of at least six genes, and is transcribed at low levels in a number of organs, including the developing endosperm, but also in the basal region of young roots and in leaf tips. A second barley enzyme that participates in mannan depolymerization through its ability to hydrolyse (1,4)-β-D-manno-oligosaccharides to Man is a family GH1 β-D-mannosidase, now designated HvβMANNOS1, but previously identified as a β-D-glucosidase [Hrmova, MacGregor, Biely, Stewart and Fincher (1998) J. Biol. Chem. 273, 11134–11143], which hydrolyses 4NP (4-nitrophenyl) β-D-mannoside three times faster than 4NP β-D-glucoside, and has an action pattern typical of a (1,4)-β-D-mannan exohydrolase. PMID:16771710

Cell wall polysaccharides from fern leaves: evidence for a mannan-rich Type III cell wall in Adiantum raddianum.

PubMed

Silva, Giovanna B; Ionashiro, Mari; Carrara, Thalita B; Crivellari, Augusto C; Tiné, Marco A S; Prado, Jefferson; Carpita, Nicholas C; Buckeridge, Marcos S

2011-12-01

Primary cell walls from plants are composites of cellulose tethered by cross-linking glycans and embedded in a matrix of pectins. Cell wall composition varies between plant species, reflecting in some instances the evolutionary distance between them. In this work the monosaccharide compositions of isolated primary cell walls of nine fern species and one lycophyte were characterized and compared with those from Equisetum and an angiosperm dicot. The relatively high abundance of mannose in these plants suggests that mannans may constitute the major cross-linking glycan in the primary walls of pteridophytes and lycophytes. Pectin-related polysaccharides contained mostly rhamnose and uronic acids, indicating the presence of rhamnogalacturonan I highly substituted with galactose and arabinose. Structural and fine-structural analyses of the hemicellulose fraction of leaves of Adiantum raddianum confirmed this hypothesis. Linkage analysis showed that the mannan contains mostly 4-Man with very little 4,6-Man, indicating a low percentage of branching with galactose. Treatment of the mannan-rich fractions with endo-β-mannanase produced characteristic mannan oligosaccharides. Minor amounts of xyloglucan and xylans were also detected. These data and those of others suggest that all vascular plants contain xyloglucans, arabinoxylans, and (gluco)mannans, but in different proportions that define cell wall types. Whereas xyloglucan and pectin-rich walls define Type I walls of dicots and many monocots, arabinoxylans and lower proportion of pectin define the Type II walls of commelinoid monocots. The mannan-rich primary walls with low pectins of many ferns and a lycopod indicate a fundamentally different wall type among land plants, the Type III wall. Copyright © 2011 Elsevier Ltd. All rights reserved.

Strong cellulase inhibition by Mannan polysaccharides in cellulose conversion to sugars.

PubMed

Kumar, Rajeev; Wyman, Charles E

2014-07-01

Cellulase enzymes contribute a major fraction of the total cost for biological conversion of lignocellulosic biomass to fuels and chemicals. Although a several fold reduction in cellulase production costs and enhancement of cellulase activity and stability have been reported in recent years, sugar yields are still lower at low enzyme doses than desired commercially. We recently reported that hemicellulose xylan and its oligomers strongly inhibit cellulase and that supplementation of cellulase with xylanase and β-xylosidase would significantly reduce such inhibition. In this study, mannan polysaccharides and their enzymatically prepared hydrolyzates were discovered to be strongly inhibitory to fungal cellulase in cellulose conversion (>50% drop in % relative conversion), even at a small concentration of 0.1 g/L, and inhibition was much greater than experienced by other known inhibitors such as cellobiose, xylooligomers, and furfural. Furthermore, cellulase inhibition dramatically increased with heteromannan loading and mannan substitution with galactose side units. In general, enzymatically prepared hydrolyzates were less inhibitory than their respective mannan polysaccharides except highly substituted ones. Supplementation of cellulase with commercial accessory enzymes such as xylanase, pectinase, and β-glucosidase was effective in greatly relieving inhibition but only for less substituted heteromannans. However, cellulase supplementation with purified heteromannan specific enzymes relieved inhibition by these more substituted heteromannans as well, suggesting that commercial preparations need to have higher amounts of such activities to realize high sugar yields at the low enzyme protein loadings needed for low cost fuels production. © 2014 Wiley Periodicals, Inc.

Cytochemical Localization of Polysaccharides in Dendrobium officinale and the Involvement of DoCSLA6 in the Synthesis of Mannan Polysaccharides

PubMed Central

He, Chunmei; Wu, Kunlin; Zhang, Jianxia; Liu, Xuncheng; Zeng, Songjun; Yu, Zhenming; Zhang, Xinghua; Teixeira da Silva, Jaime A.; Deng, Rufang; Tan, Jianwen; Luo, Jianping; Duan, Jun

2017-01-01

Dendrobium officinale is a precious traditional Chinese medicinal plant because of its abundant polysaccharides found in stems. We determined the composition of water-soluble polysaccharides and starch content in D. officinale stems. The extracted water-soluble polysaccharide content was as high as 35% (w/w). Analysis of the composition of monosaccharides showed that the water-soluble polysaccharides were dominated by mannose, to a lesser extent glucose, and a small amount of galactose, in a molar ratio of 223:48:1. Although starch was also found, its content was less than 10%. This result indicated that the major polysaccharides in D. officinale stems were non-starch polysaccharides, which might be mannan polysaccharides. The polysaccharides formed granules and were stored in plastids similar to starch grains, were localized in D. officinale stems by semi-thin and ultrathin sections. CELLULOSE SYNTHASE-LIKE A (CSLA) family members encode mannan synthases that catalyze the formation of mannan polysaccharides. To determine whether the CSLA gene from D. officinale was responsible for the synthesis of mannan polysaccharides, 35S:DoCSLA6 transgenic lines were generated and characterized. Our results suggest that the CSLA family genes from D. officinale play an important role in the biosynthesis of mannan polysaccharides. PMID:28261235

Plasma protein binding of an antisense oligonucleotide targeting human ICAM-1 (ISIS 2302).

PubMed

Watanabe, Tanya A; Geary, Richard S; Levin, Arthur A

2006-01-01

In vitro ultrafiltration was used to determine the plasma protein-binding characteristics of phosphorothioate oligonucleotides (PS ODNs). Although there are binding data on multiple PS ODNs presented here, the focus of this research is on the protein-binding characteristics of ISIS 2302, a PS ODN targeting human intercellular adhesion molecule-1 (ICAM-1) mRNA, which is currently in clinical trials for the treatment of ulcerative colitis. ISIS 2302 was shown to be highly bound (> 97%) across species (mouse, rat, monkey, human), with the mouse having the least degree of binding. ISIS 2302 was highly bound to albumin and, to a lesser, extent alpha2-macroglobulin and had negligible binding to alpha1-acid glycoprotein. Ten shortened ODN metabolites (8, 10, and 12-19 nucleotides [nt] in length, truncated from the 3' end) were evaluated in human plasma. The degree of binding was reduced as the ODN metabolite length decreased. Three additional 20-nt (20-mer) PS ODNs (ISIS 3521, ISIS 2503, and ISIS 5132) of varying sequence but similar chemistry were evaluated. Although the tested PS ODNs were highly bound to plasma proteins, suggesting a commonality within the chemical class, these results suggested that the protein-binding characteristics in human plasma may be sequence dependent. Lastly, drug displacement studies with ISIS 2302 and other concomitant drugs with known protein-binding properties were conducted to provide information on potential drug interactions. Coadministered ISIS 2302 and other high-binding drugs evaluated in this study did not displace one another at supraclinical plasma concentrations and, thus, are not anticipated to cause any pharmacokinetic interaction in the clinic as a result of the displacement of binding to plasma proteins.

Adipose-Derived Fatty Acid-Binding Proteins Plasma Concentrations Are Increased in Breast Cancer Patients.

PubMed

Guaita-Esteruelas, Sandra; Saavedra-García, Paula; Bosquet, Alba; Borràs, Joan; Girona, Josefa; Amiliano, Kepa; Rodríguez-Balada, Marta; Heras, Mercedes; Masana, Luís; Gumà, Josep

2017-11-01

Adipose tissue is an endocrine organ that could play a role in tumor progression via its secreted adipokines. The role of adipose-derived fatty acid-binding protein (FABP) 4 and FABP5 in breast cancer is presently under study, but their circulating levels in this pathology are poorly known. We analyzed the blood concentrations of FABP4 and FABP5 in breast cancer patients to determine whether there is an association between them and breast cancer. We studied 294 women in the oncology department with a family history of breast cancer; 198 of the women had breast cancer, and 96 were healthy controls. The levels of FABP4, FABP5, lipid profile, standard biochemical parameter, and high-sensitivity C-reactive protein (hsCRP) were determined. We analyzed the association of FABP4 and FABP5 with breast cancer, while adjusting for demographic, anthropometric, and biochemical parameters. Breast cancer patients had a 24.8% ( p  < .0001) and 11.4% ( p  < .05) higher blood concentration of FABP4 and FABP5, respectively. Fatty acid-binding protein 4 was positively associated with age, body mass index (BMI), FABP5, very-low-density lipoprotein cholesterol (VLDLc), non-high-density lipoprote in cholesterol (non-HDLc), Apolipoprotein B 100 (ApoB100), triglycerides, glycerol, glucose, and hsCRP ( p  < .05), and was negatively associated with HDLc ( p  < .005) in breast cancer patients. Fatty acid-binding protein 5 was positively associated with BMI, FABP4, VLDLc, triglycerides, glycerol, and hsCRP ( p  < .05), and was negatively associated with HDLc and Apolipoprotein AI (ApoAI) ( p  < .05) in breast cancer patients. Using a logistic regression analysis and adjusting for age, BMI, hsCRP, non-HDLc, and triglycerides, FABP4 was independently associated with breast cancer (odds ratio [OR]: 1.091 [95% CI: 1.037-1.149]). Moreover, total cholesterol, VLDLc, non-HDLc, ApoB100, triglycerides, and hsCRP were significantly increased in breast cancer patients ( p �

Programmable calculator software for computation of the plasma binding of ligands.

PubMed

Conner, D P; Rocci, M L; Larijani, G E

1986-01-01

The computation of the extent of plasma binding of a ligand to plasma constituents using radiolabeled ligand and equilibrium dialysis is complex and tedious. A computer program for the HP-41C Handheld Computer Series (Hewlett-Packard) was developed to perform these calculations. The first segment of the program constructs a standard curve for quench correction of post-dialysis plasma and buffer samples, using either external standard ratio (ESR) or sample channels ratio (SCR) techniques. The remainder of the program uses the counts per minute, SCR or ESR, and post-dialysis volume of paired plasma and buffer samples generated from the dialysis procedure to compute the extent of binding after correction for background radiation, counting efficiency, and intradialytic shifts of fluid between plasma and buffer compartments during dialysis. This program greatly simplifies the analysis of equilibrium dialysis data and has been employed in the analysis of dexamethasone binding in normal and uremic sera.

Mannan adjuvants intranasally administered inactivated influenza virus in mice rendering low doses inductive of strong serum IgG and IgA in the lung.

PubMed

Proudfoot, Owen; Esparon, Sandra; Tang, Choon-Kit; Laurie, Karen; Barr, Ian; Pietersz, Geoffrey

2015-02-26

H1N1 influenza viruses mutate rapidly, rendering vaccines developed in any given year relatively ineffective in subsequent years. Thus it is necessary to generate new vaccines every year, but this is time-consuming and resource-intensive. Should a highly virulent influenza strain capable of human-to-human transmission emerge, these factors will severely limit the number of people that can be effectively immunised against that strain in time to prevent a pandemic. An adjuvant and mode of administration capable of rendering ordinarily unprotective vaccine doses protective would thus be highly advantageous. The carbohydrate mannan was conjugated to whole inactivated H1N1 influenza virus at a range of ratios, and mixed with it at a range of ratios, and various doses of the resulting preparations were administered to mice via the intranasal (IN) route. Serum immunity was assessed via antigen-specific IgG ELISA and the haemagglutination-inhibition (HI) assay, and mucosal immunity was assessed via IgA ELISA of bronchio-alveolar lavages. IN-administered inactivated H1N1 mixed with mannan induced higher serum IgG and respiratory-tract IgA than inactivated H1N1 conjugated to mannan, and HIN1 alone. Adjuvantation was mannan-dose-dependent, with 100 μg of mannan adjuvanting 1 μg of H1N1 more effectively than 10 or 50 μg of mannan. Serum samples from mice immunised with 1 μg H1N1 adjuvanted with 10 μg mannan did not inhibit agglutination of red blood cells (RBCs) at a dilution factor of 10 in the HI assay, but samples resulting from adjuvantation with 50 and 100 μg mannan inhibited agglutination at dilution factors of ≥ 40. Both serum IgG1 and IgG2a were induced by IN mannan-adjuvanted H1N1 vaccination, suggesting the induction of humoral and cellular immunity. Mixing 100 μg of mannan with 1 μg of inactivated H1N1 adjuvanted the vaccine in mice, such that IN immunisation induced higher serum IgG and respiratory tract IgA than immunisation with virus alone. The

Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-beta-mannosidase from Aspergillus niger BK01.

PubMed

Do, Bien-Cuong; Dang, Thi-Thu; Berrin, Jean-Guy; Haltrich, Dietmar; To, Kim-Anh; Sigoillot, Jean-Claude; Yamabhai, Montarop

2009-11-13

Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-beta-mannosidases (1,4-beta-D-mannanases) catalyze the random hydrolysis of beta-1,4-mannosidic linkages in the main chain of beta-mannans. Biodegradation of beta-mannans by the action of thermostable mannan endo-1,4-beta-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS). A gene encoding mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannan mannanohydrolase (E.C. 3.2.1.78), commonly termed beta-mannanase, from Aspergillus niger BK01, which belongs to glycosyl hydrolase family 5 (GH5), was cloned and successfully expressed heterologously (up to 243 microg of active recombinant protein per mL) in Pichia pastoris. The enzyme was secreted by P. pastoris and could be collected from the culture supernatant. The purified enzyme appeared glycosylated as a single band on SDS-PAGE with a molecular mass of approximately 53 kDa. The recombinant beta-mannanase is highly thermostable with a half-life time of approximately 56 h at 70 degrees C and pH 4.0. The optimal temperature (10-min assay) and pH value for activity are 80 degrees C and pH 4.5, respectively. The enzyme is not only active towards structurally different mannans but also exhibits low activity towards birchwood xylan. Apparent Km values of the enzyme for konjac glucomannan (low viscosity), locust bean gum galactomannan, carob galactomannan (low viscosity), and 1,4-beta-D-mannan (from carob) are 0.6 mg mL-1, 2.0 mg mL-1, 2.2 mg mL-1 and 1.5 mg mL-1, respectively, while the kcat values for these

Pharmacokinetics, brain distribution and plasma protein binding of carbamazepine and nine derivatives: new set of data for predictive in silico ADME models.

PubMed

Fortuna, Ana; Alves, Gilberto; Soares-da-Silva, Patrício; Falcão, Amílcar

2013-11-01

In silico approaches to predict absorption, distribution, metabolism and excretion (ADME) of new drug candidates are gaining a relevant importance in drug discovery programmes. When considering particularly the pharmacokinetics during the development of oral antiepileptic drugs (AEDs), one of the most prominent goals is designing compounds with good bioavailability and brain penetration. Thus, it is expected that in silico models able to predict these features may be applied during the early stages of AEDs discovery. The present investigation was mainly carried out in order to generate in vivo pharmacokinetic data that can be utilized for development and validation of in silico models. For this purpose, a single dose of each compound (1.4mmol/kg) was orally administered to male CD-1 mice. After quantifying the parent compound and main metabolites in plasma and brain up to 12h post-dosing, a non-compartmental pharmacokinetic analysis was performed and the corresponding brain/plasma ratios were calculated. Moreover the plasma protein binding was estimated in vitro applying the ultrafiltration procedure. The present in vivo pharmacokinetic characterization of the test compounds and corresponding metabolites demonstrated that the metabolism extensively compromised the in vivo activity of CBZ derivatives and their toxicity. Furthermore, it was clearly evidenced that the time to reach maximum peak concentration, bioavailability (given by the area under the curve) and metabolic stability (given by the AUC0-12h ratio of the parent compound and total systemic drug) influenced the in vivo pharmacological activities and must be considered as primary parameters to be investigated. All the test compounds presented brain/plasma ratios lower than 1.0, suggesting that the blood-brain barrier restricts drug entry into the brain. In agreement with in vitro studies already performed within our research group, CBZ, CBZ-10,11-epoxide and oxcarbazepine exhibited the highest brain/plasma

Stereoselective binding of doxazosin enantiomers to plasma proteins from rats, dogs and humans in vitro

PubMed Central

Sun, Jia-an; Kong, De-zhi; Zhen, Ya-qin; Li, Qing; Zhang, Wei; Zhang, Jiang-hua; Yin, Zhi-wei; Ren, Lei-ming

2013-01-01

Aim: (±)Doxazosin is a long-lasting inhibitor of α1-adrenoceptors that is widely used to treat benign prostatic hyperplasia and lower urinary tract symptoms. In this study we investigated the stereoselective binding of doxazosin enantiomers to the plasma proteins of rats, dogs and humans in vitro. Methods: Human, dog and rat plasma were prepared. Equilibrium dialysis was used to determine the plasma protein binding of each enantiomer in vitro. Chiral HPLC with fluorescence detection was used to measure the drug concentrations on each side of the dialysis membrane bag. Results: Both the enantiomers were highly bound to the plasma proteins of rats, dogs and humans [(−)doxazosin: 89.4%–94.3%; (+)doxazosin: 90.9%–95.4%]. (+)Doxazosin exhibited significantly higher protein binding capacities than (−)doxazosin in all the three species, and the difference in the bound concentration (Cb) between the two enantiomers was enhanced as their concentrations were increased. Although the percentage of the plasma protein binding in the dog plasma was significantly lower than that in the human plasma at 400 and 800 ng/mL, the corrected percentage of plasma protein binding was dog>human>rat. Conclusion: (−)Doxazosin and (+)doxazosin show stereoselective plasma protein binding with a significant species difference among rats, dogs and humans. PMID:24241343

Conservation and Divergence in the Candida Species Biofilm Matrix Mannan-Glucan Complex Structure, Function, and Genetic Control

PubMed Central

Dominguez, Eddie; Zarnowski, Robert; Sanchez, Hiram; Covelli, Antonio S.; Westler, William M.; Azadi, Parastoo; Nett, Jeniel

2018-01-01

ABSTRACT Candida biofilms resist the effects of available antifungal therapies. Prior studies with Candida albicans biofilms show that an extracellular matrix mannan-glucan complex (MGCx) contributes to antifungal sequestration, leading to drug resistance. Here we implement biochemical, pharmacological, and genetic approaches to explore a similar mechanism of resistance for the three most common clinically encountered non-albicans Candida species (NAC). Our findings reveal that each Candida species biofilm synthesizes a mannan-glucan complex and that the antifungal-protective function of this complex is conserved. Structural similarities extended primarily to the polysaccharide backbone (α-1,6-mannan and β-1,6-glucan). Surprisingly, biochemical analysis uncovered stark differences in the branching side chains of the MGCx among the species. Consistent with the structural analysis, similarities in the genetic control of MGCx production for each Candida species also appeared limited to the synthesis of the polysaccharide backbone. Each species appears to employ a unique subset of modification enzymes for MGCx synthesis, likely accounting for the observed side chain diversity. Our results argue for the conservation of matrix function among Candida spp. While biogenesis is preserved at the level of the mannan-glucan complex backbone, divergence emerges for construction of branching side chains. Thus, the MGCx backbone represents an ideal drug target for effective pan-Candida species biofilm therapy. PMID:29615504

Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-β-mannosidase from Aspergillus niger BK01

PubMed Central

2009-01-01

Background Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-β-mannosidases (1,4-β-D-mannanases) catalyze the random hydrolysis of β-1,4-mannosidic linkages in the main chain of β-mannans. Biodegradation of β-mannans by the action of thermostable mannan endo-1,4-β-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS). Results A gene encoding mannan endo-1,4-β-mannosidase or 1,4-β-D-mannan mannanohydrolase (E.C. 3.2.1.78), commonly termed β-mannanase, from Aspergillus niger BK01, which belongs to glycosyl hydrolase family 5 (GH5), was cloned and successfully expressed heterologously (up to 243 μg of active recombinant protein per mL) in Pichia pastoris. The enzyme was secreted by P. pastoris and could be collected from the culture supernatant. The purified enzyme appeared glycosylated as a single band on SDS-PAGE with a molecular mass of approximately 53 kDa. The recombinant β-mannanase is highly thermostable with a half-life time of approximately 56 h at 70°C and pH 4.0. The optimal temperature (10-min assay) and pH value for activity are 80°C and pH 4.5, respectively. The enzyme is not only active towards structurally different mannans but also exhibits low activity towards birchwood xylan. Apparent Km values of the enzyme for konjac glucomannan (low viscosity), locust bean gum galactomannan, carob galactomannan (low viscosity), and 1,4-β-D-mannan (from carob) are 0.6 mg mL-1, 2.0 mg mL-1, 2.2 mg mL-1 and 1.5 mg mL-1, respectively, while the kcat values for these substrates are 215 s-1, 330

Parvovirus B19V DNA contamination in Chinese plasma and plasma derivatives

PubMed Central

2012-01-01

Background To ensure the safety of plasma derivatives, screening for human parvovirus B19V genomic DNA in donated plasma using a pooling strategy is performed in some countries. We investigated the prevalence of B19V DNA and anti-B19V antibodies in Chinese plasma pools, plasma derivatives and plasma donations to evaluate the risk posed by B19V. Methods Using a Q-PCR assay developed in-house, we tested for B19V genomic DNA in 142 plasma pools collected between January 2009 and June 2011 from two Chinese blood products manufacturers. Plasma derivatives collected between 1993–1995 (10 batches of albumin, 155 batches of intravenous immunoglobulin, IVIG) and 2009–2011 (50 batches of albumin, 54 batches of IVIG, 35 batches of factor VIII, 7 batches of fibrinogen, and 17 batches of prothrombin complex concentrate, PCC) were also tested for B19V contamination. In addition, B19V genome prevalence in minipools(including 90 individual donations) of 49680 individual plasma samples collected between August 2011 and March 2012 by a single Chinese manufacturer was investigated. IgM/IgG was also investigated in plasma pools/derivatives and in minipools with B19V-DNA titers above 1x104 and 1x106 geq/mL using B19 ELISA IgM/IgG assay(Virion-Serion, Würzburg, Germany), respectively. Results B19V-DNA was detected in 54.2% of plasma pools from two Chinese blood product manufacturers; among recently produced blood products, B19V was detected in 21/54 IVIG samples, 19/35 factor VIII samples, 6/7 fibrinogen samples, and 12/17 PCC samples, but not in albumin samples. The levels of B19V-DNA in these samples varied from 102-107 geq/mL. In samples with >104 geq/mL genome DNA, B19V-specific IgG was also found in all corresponding plasma pools and IVIG, whereas none was detected in the majority of other plasma derivatives. Screening of plasma donations indicated that most minipools were contaminated with B19V-DNA (102-108 geq/mL) and one donation had 1.09 × 1010 geq/mL B19V genomic DNA

Human plasma platelet-derived exosomes: effects of aspirin.

PubMed

Goetzl, Edward J; Goetzl, Laura; Karliner, Joel S; Tang, Norina; Pulliam, Lynn

2016-05-01

Platelet-derived exosomes mediate platelet atherogenic interactions with endothelial cells and monocytes. A new method for isolation of plasma platelet-derived exosomes is described and used to examine effects of aging and aspirin on exosome cargo proteins. Exosome secretion by purified platelets in vitro did not increase after exposure to thrombin or collagen, as assessed by exosome counts and quantification of the CD81 exosome marker. Thrombin and collagen increased exosome content of α-granule chemokines CXCL4 and CXCL7 and cytoplasmic high-mobility group box 1 (HMGB1) protein, but not membrane platelet glycoprotein VI (GPVI), with dependence on extracellular calcium. Aspirin consumption significantly blocked thrombin- and collagen-induced increases in exosome cargo levels of chemokines and HMGB1, without altering total exosome secretion or GPVI cargo. Plasma platelet-derived exosomes, enriched by absorption with mouse antihuman CD42b [platelet glycoprotein Ib (GPIb)] mAb, had sizes and cargo protein contents similar to those of exosomes from purified platelets. The plasma platelet-derived exosome number is lower and its chemokine and HMGB1 levels higher after age 65 yr. Aspirin consumption significantly suppressed cargo protein levels of plasma platelet-derived exosomes without altering total levels of exosomes. Cargo proteins of human plasma platelet-derived exosomes may biomark platelet abnormalities and in vivo effects of drugs.- Goetzl, E. J., Goetzl, L., Karliner, J. S., Tang, N., Pulliam, L. Human plasma platelet-derived exosomes: effects of aspirin. © FASEB.

Identification and properties of steroid-binding proteins in nesting Chelonia mydas plasma.

PubMed

Ikonomopoulou, M P; Bradley, A J; Whittier, J M; Ibrahim, K

2006-11-01

We report for the first time the presence of a sex steroid-binding protein in the plasma of green sea turtles Chelonia mydas, which provides an insight into reproductive status. A high affinity, low capacity sex hormone steroid-binding protein was identified in nesting C. mydas and its thermal profile was established. In nesting C. mydas testosterone and oestradiol bind at 4 degrees C with high affinity (K (a) = 1.49 +/- 0.09 x 10(9) M(-1); 0.17 +/- 0.02 x 10(7) M(-1)) and low binding capacity (B (max) = 3.24 +/- 0.84 x 10(-5) M; 0.33 +/- 0.06 x 10(-4) M). The binding affinity and capacity of testosterone at 23 and 36 degrees C, respectively were similar to those determined at 4 degrees C. However, oestradiol showed no binding activity at 36 degrees C. With competition studies we showed that oestradiol and oestrone do not compete for binding sites. Furthermore, in nesting C. mydas plasma no high-affinity binding was observed for adrenocortical steroids (cortisol and corticosterone) and progesterone. Our results indicate that in nesting C. mydas plasma temperature has a minimal effect on the high-affinity binding of testosterone to sex steroid-binding protein, however, the high affinity binding of oestradiol to sex steroid-binding protein is abolished at a hypothetically high (36 degrees C) sea/ambient/body temperature. This suggests that at high core body temperatures most of the oestradiol becomes biologically available to the tissues rather than remaining bound to a high-affinity carrier.

Competitive Binding of Natural Amphiphiles with Graphene Derivatives

NASA Astrophysics Data System (ADS)

Radic, Slaven; Geitner, Nicholas K.; Podila, Ramakrishna; Käkinen, Aleksandr; Chen, Pengyu; Ke, Pu Chun; Ding, Feng

2013-07-01

Understanding the transformation of graphene derivatives by natural amphiphiles is essential for elucidating the biological and environmental implications of this emerging class of engineered nanomaterials. Using rapid discrete-molecular-dynamics simulations, we examined the binding of graphene and graphene oxide with peptides, fatty acids, and cellulose, and complemented our simulations by experimental studies of Raman spectroscopy, FTIR, and UV-Vis spectrophotometry. Specifically, we established a connection between the differential binding and the conformational flexibility, molecular geometry, and hydrocarbon content of the amphiphiles. Importantly, our dynamics simulations revealed a Vroman-like competitive binding of the amphiphiles for the graphene oxide substrate. This study provides a mechanistic basis for addressing the transformation, evolution, transport, biocompatibility, and toxicity of graphene derivatives in living systems and the natural environment.

Competitive Binding of Natural Amphiphiles with Graphene Derivatives

PubMed Central

Radic, Slaven; Geitner, Nicholas K.; Podila, Ramakrishna; Käkinen, Aleksandr; Chen, Pengyu; Ke, Pu Chun; Ding, Feng

2013-01-01

Understanding the transformation of graphene derivatives by natural amphiphiles is essential for elucidating the biological and environmental implications of this emerging class of engineered nanomaterials. Using rapid discrete-molecular-dynamics simulations, we examined the binding of graphene and graphene oxide with peptides, fatty acids, and cellulose, and complemented our simulations by experimental studies of Raman spectroscopy, FTIR, and UV-Vis spectrophotometry. Specifically, we established a connection between the differential binding and the conformational flexibility, molecular geometry, and hydrocarbon content of the amphiphiles. Importantly, our dynamics simulations revealed a Vroman-like competitive binding of the amphiphiles for the graphene oxide substrate. This study provides a mechanistic basis for addressing the transformation, evolution, transport, biocompatibility, and toxicity of graphene derivatives in living systems and the natural environment. PMID:23881402

Radioligand binding analysis of α 2 adrenoceptors with [11C]yohimbine in brain in vivo: Extended Inhibition Plot correction for plasma protein binding.

PubMed

Phan, Jenny-Ann; Landau, Anne M; Jakobsen, Steen; Wong, Dean F; Gjedde, Albert

2017-11-22

We describe a novel method of kinetic analysis of radioligand binding to neuroreceptors in brain in vivo, here applied to noradrenaline receptors in rat brain. The method uses positron emission tomography (PET) of [ 11 C]yohimbine binding in brain to quantify the density and affinity of α 2 adrenoceptors under condition of changing radioligand binding to plasma proteins. We obtained dynamic PET recordings from brain of Spraque Dawley rats at baseline, followed by pharmacological challenge with unlabeled yohimbine (0.3 mg/kg). The challenge with unlabeled ligand failed to diminish radioligand accumulation in brain tissue, due to the blocking of radioligand binding to plasma proteins that elevated the free fractions of the radioligand in plasma. We devised a method that graphically resolved the masking of unlabeled ligand binding by the increase of radioligand free fractions in plasma. The Extended Inhibition Plot introduced here yielded an estimate of the volume of distribution of non-displaceable ligand in brain tissue that increased with the increase of the free fraction of the radioligand in plasma. The resulting binding potentials of the radioligand declined by 50-60% in the presence of unlabeled ligand. The kinetic unmasking of inhibited binding reflected in the increase of the reference volume of distribution yielded estimates of receptor saturation consistent with the binding of unlabeled ligand.

Conservation and Divergence in the Candida Species Biofilm Matrix Mannan-Glucan Complex Structure, Function, and Genetic Control.

PubMed

Dominguez, Eddie; Zarnowski, Robert; Sanchez, Hiram; Covelli, Antonio S; Westler, William M; Azadi, Parastoo; Nett, Jeniel; Mitchell, Aaron P; Andes, David R

2018-04-03

Candida biofilms resist the effects of available antifungal therapies. Prior studies with Candida albicans biofilms show that an extracellular matrix mannan-glucan complex (MGCx) contributes to antifungal sequestration, leading to drug resistance. Here we implement biochemical, pharmacological, and genetic approaches to explore a similar mechanism of resistance for the three most common clinically encountered non- albicans Candida species (NAC). Our findings reveal that each Candida species biofilm synthesizes a mannan-glucan complex and that the antifungal-protective function of this complex is conserved. Structural similarities extended primarily to the polysaccharide backbone (α-1,6-mannan and β-1,6-glucan). Surprisingly, biochemical analysis uncovered stark differences in the branching side chains of the MGCx among the species. Consistent with the structural analysis, similarities in the genetic control of MGCx production for each Candida species also appeared limited to the synthesis of the polysaccharide backbone. Each species appears to employ a unique subset of modification enzymes for MGCx synthesis, likely accounting for the observed side chain diversity. Our results argue for the conservation of matrix function among Candida spp. While biogenesis is preserved at the level of the mannan-glucan complex backbone, divergence emerges for construction of branching side chains. Thus, the MGCx backbone represents an ideal drug target for effective pan- Candida species biofilm therapy. IMPORTANCE Candida species, the most common fungal pathogens, frequently grow as a biofilm. These adherent communities tolerate extremely high concentrations of antifungal agents, due in large part, to a protective extracellular matrix. The present studies define the structural, functional, and genetic similarities and differences in the biofilm matrix from the four most common Candida species. Each species synthesizes an extracellular mannan-glucan complex (MGCx) which

Mannan-decorated thiolated Eudragit microspheres for targeting antigen presenting cells via nasal vaccination.

PubMed

Li, Hui-Shan; Singh, Bijay; Park, Tae-Eun; Hong, Zhong-Shan; Kang, Sang-Kee; Cho, Chong-Su; Choi, Yun-Jaie

2015-12-01

Mucosal vaccination of protein as an antigen requires appropriate delivery or adjuvant systems to deliver antigen to mucosal immune cells efficiently and generate valid immune responses. For successful nasal immunization, the obstacles imposed by the normal process of mucociliary clearance which limits residence time of applied antigens and low antigen delivery to antigen presenting cells (APCs) in nasal associated lymphoid tissue (NALT) need to be overcome for the efficient vaccination. Here, we prepared mucoadhesive and mannan-decorated thiolated Eudragit microspheres (Man-TEM) as a nasal vaccine carrier to overcome the limitations. Mucoadhesive thiolated Eudragit (TE) were decorated with mannan for targeting mannose receptors (MR) in antigen presenting cells (APCs) to obtain efficient immune responses. The potential adjuvant ability of Man-TEM for intranasal immunization was confirmed by in vitro and in vivo experiments. In mechanistic study using APCs in vitro, we obtained that Man-TEM enhanced the receptor-mediated endocytosis by stimulating the MR receptors of APCs. The nasal vaccination of OVA-loaded Man-TEM in mice showed higher levels of serum IgG and mucosal sIgA than the soluble OVA group due to the specific recognition of MR of APCs by the mannan in the Man-TEM. These results suggest that mucoadhesive and Man-TEM may be a promising candidate for nasal vaccine delivery system to elicit systemic and mucosal immunity. Copyright © 2015 Elsevier B.V. All rights reserved.

Mannans and endo-β-mannanase transcripts are located in different seed compartments during Brassicaceae germination.

PubMed

Carrillo-Barral, Néstor; Matilla, Angel J; Rodríguez-Gacio, María Del Carmen; Iglesias-Fernández, Raquel

2018-03-01

Mannans but not endo-β-mannanases are mainly found in the mucilage layer of two Brassicaceae seeds. Nonetheless, mannanase mobilization from inner to outer seed layers cannot be ruled out. The contribution of endo-β-mannanase (MAN) genes to the germination of the wild-type Sisymbrium officinale and cultivated Brassica rapa (Brassicaceae) species has been explored. In both species, mannans have been localized to the imbibed external seed coat layer (mucilage) by fluorescence immunolocalization and MAN enzymatic activity increases in seeds as imbibition progresses, reaching a peak before 100% germination is achieved. The MAN gene families have been annotated and the expression of their members analyzed in vegetative and reproductive organs. In S. officinale and B. rapa, MAN2, MAN5, MAN6, and MAN7 transcripts accumulate upon seed imbibition. SoMAN7 is the most expressed MAN gene in S. officinale germinating seeds, as occurs with its ortholog in Arabidopsis thaliana, but in B. rapa, the most abundant transcripts are BrMAN2 and BrMAN5. These genes (MAN2, MAN5, MAN6, and MAN7) are localized, by mRNA in situ hybridization, to the micropylar at the endosperm layer and to the radicle in S. officinale, but in B. rapa, these mRNAs are faintly found to the micropylar living seed coat layer and are mainly present at the radicle tip and the vascular bundles. If the domestication process undergone by B. rapa is responsible for these different MAN expression patterns, upon germination remains to be elucidated. Since mannans and MAN genes are not spatially distributed in the same seed tissues, a movement of MAN enzymes that are synthesized with typical signal peptides from the embryo tissues to the mucilage layer (via apoplastic space) is necessary for the mannans to be hydrolyzed.

Sequential microwave superheated water extraction of mannans from spent coffee grounds.

PubMed

Passos, Cláudia P; Moreira, Ana S P; Domingues, M Rosário M; Evtuguin, Dmitry V; Coimbra, Manuel A

2014-03-15

The feasibility of using sequential microwave superheated water extraction (MAE) for the recovery of mannans from spent coffee grounds (SCG) was studied. Due to the high contents of mannose still present in the SCG residue left after two consecutive MAE, the unextracted material was re-suspended in water and submitted to a third microwave irradiation (MAE3) at 200 °C for 3 min. With MAE3, mannose recovery achieved 48%, increasing to 56% by MAE4, and reaching a maximum of 69% with MAE5. Glycosidic-linkage analysis showed that in MAE3 mainly galactomannans were recovered, while debranched galactomannans were recovered with MAE4 and MAE5. With increasing the number of extractions, the average degree of polymerization of the mannans decreased, as observed by size-exclusion chromatography and by methylation analysis. Scanning electron microscopy images showed a decrease on cell walls thickness. After final MAE5, the remaining un-extracted insoluble material, representing 22% of the initial SCG, was composed mainly by cellulose (84%). Copyright © 2013 Elsevier Ltd. All rights reserved.

Elucidation of the binding mechanism of coumarin derivatives with human serum albumin.

PubMed

Garg, Archit; Manidhar, Darla Mark; Gokara, Mahesh; Malleda, Chandramouli; Suresh Reddy, Cirandur; Subramanyam, Rajagopal

2013-01-01

Coumarin is a benzopyrone which is widely used as an anti-coagulant, anti-oxidant, anti-cancer and also to cure arthritis, herpes, asthma and inflammation. Here, we studied the binding of synthesized coumarin derivatives with human serum albumin (HSA) at physiological pH 7.2 by using fluorescence spectroscopy, circular dichroism spectroscopy, molecular docking and molecular dynamics simulation studies. By addition of coumarin derivatives to HSA the maximum fluorescence intensity was reduced due to quenching of intrinsic fluorescence upon binding of coumarin derivatives to HSA. The binding constant and free energy were found to be 1.957±0.01×10(5) M(-1), -7.175 Kcal M(-1) for coumarin derivative (CD) enamide; 0.837±0.01×10(5) M(-1), -6.685 Kcal M(-1) for coumarin derivative (CD) enoate, and 0.606±0.01×10(5) M(-1), -6.49 Kcal M(-1) for coumarin derivative methylprop (CDM) enamide. The CD spectroscopy showed that the protein secondary structure was partially unfolded upon binding of coumarin derivatives. Further, the molecular docking studies showed that coumarin derivatives were binding to HSA at sub-domain IB with the hydrophobic interactions and also with hydrogen bond interactions. Additionally, the molecular dynamics simulations studies contributed in understanding the stability of protein-drug complex system in the aqueous solution and the conformational changes in HSA upon binding of coumarin derivatives. This study will provide insights into designing of the new inspired coumarin derivatives as therapeutic agents against many life threatening diseases.

The connection Between Plasma Protein Binding and Acute Toxicity as Determined by the LD50 Value.

PubMed

Svennebring, Andreas

2016-02-01

Preclinical Research A dataset of three drug classes (acids, bases, and neutrals) with LD50 values in mice was analysed to investigate a possible connection between high plasma protein binding and acute toxicity. Initially, it was found that high plasma protein binding was associated with toxicity for acids and neutrals, but after compensating for differences in lipophilicity, plasma protein binding was found not to be associated with toxicity. The therapeutic index established by the quotient between mouse LD50 and the defined daily dose was unaffected by both lipophilicity and plasma protein binding. © 2015 Wiley Periodicals, Inc.

The effect of tenidap sodium on the disposition and plasma protein binding of phenytoin in healthy male volunteers

PubMed Central

Blum, R. A.; Schentag, J. J.; Gardner, M. J.; Wilner, K. D.

1995-01-01

1 The effects of tenidap sodium 120 mg day-1 at steady state and placebo on the plasma protein binding and pharmacokinetics of phenytoin were compared in this randomised, double-blind, placebo-controlled, parallel-group study, involving 12 healthy young men, conducted over 34 days. 2 Single oral doses of phenytoin 200 mg were given on days 1-3 and 29-31, and intravenous phenytoin, 250 mg infused over 20 min, was given on days 4 and 32. Tenidap (120 mg day-1), or matching placebo, was administered as single oral daily doses from days 8 to 34 inclusive. 3 The plasma protein binding of phenytoin was determined immediately before oral phenytoin administration on days 1 and 29. Pharmacokinetic parameters were estimated from the serum phenytoin concentration-time curves derived on days 4 and 32 following the phenytoin infusions. The differences between the pre- and post-treatment mean percentage of unbound plasma phenytoin and mean pharmacokinetic parameters were compared between treatment groups. 4 Tenidap sodium 120 mg day-1, at steady state, increased the percentage of unbound phenytoin in plasma by approximately 25%, but did not significantly affect AUC(0,48h) or Cmax. 5 Since tenidap increases the percentage of unbound phenytoin in plasma, when monitoring phenytoin plasma concentrations free concentrations of phenytoin should be considered. 6 Tenidap was well tolerated throughout the study. PMID:7547092

Circulating mannan-binding lectin, M-, L-, H-ficolin and collectin-liver-1 levels in patients with acute liver failure.

PubMed

Laursen, Tea L; Sandahl, Thomas D; Støy, Sidsel; Schiødt, Frank V; Lee, William M; Vilstrup, Hendrik; Thiel, Steffen; Grønbaek, Henning

2015-03-01

The complement system is activated in liver diseases including acute liver failure (ALF); however, the role of the lectin pathway of complement has scarcely been investigated in ALF. The pathway is initiated by soluble pattern recognition molecules: mannan-binding lectin (MBL), M-, L-, and H-ficolin and collectin-liver-1 (CL-L1), which are predominantly synthesized in the liver. We aimed to study lectin levels in ALF patients and associations with clinical outcome. Serum samples from 75 patients enrolled by the US ALF Study Group were collected on days 1 and 3. We included 75 healthy blood donors and 20 cirrhosis patients as controls. Analyses were performed using sandwich-type immunoassays (ELISA, TRIFMA). At day 1, the MBL level in ALF patients was 40% lower compared with healthy controls {[median (interquartile range) 0.72 μg/ml(0.91) vs. 1.15 (1.92)(P = 0.02]}, and increased significantly by day 3 [0.83 μg/ml(0.94)(P = 0.01)]. The M-ficolin level was 60% lower [0.54 μg/ml(0.50) vs. 1.48(1.01)(P < 0.0001)]. The CL-L1 level at day 1 was slightly higher compared with healthy controls [3.20 μg/ml(2.37) vs. 2.64(0.72)(P = 0.11)]; this was significant at day 3 [3.35(1.84)(P = 0.006)]. H- and L-ficolin levels were similar to healthy controls. Spontaneous ALF survivors had higher levels of MBL at day 1 [0.96 μg/ml(1.15) vs. 0.60(0.60)(P = 0.02)] and lower levels of L-ficolin by day 3 compared with patients who died or were transplanted [1.61 μg/ml(1.19) vs. 2.17(2.19)(P = 0.02)]. We observed significant dynamics in lectin levels in ALF patients, which may suggest they play a role in ALF pathogenesis. High MBL and low L-ficolin levels are associated with survival. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Beneficial Effects of Prebiotic Saccharomyces cerevisiae Mannan on Allergic Asthma Mouse Models.

PubMed

Lew, D Betty; Michael, Christie F; Overbeck, Tracie; Robinson, W Scout; Rohman, Erin L; Lehman, Jeffrey M; Patel, Jennifer K; Eiseman, Brandi; LeMessurier, Kim S; Samarasinghe, Amali E; Gaber, M Waleed

2017-01-01

One of the unmet needs for asthma management is a new therapeutic agent with both anti-inflammatory and anti-smooth muscle (ASM) remodeling effects. The mannose receptor (MR) family plays an important role in allergen uptake and processing of major allergens Der p 1 and Fel d 1. We have previously reported that ASM cells express a mannose receptor (ASM-MR) and that mannan derived from Saccharomyces cerevisiae (SC-MN) inhibits mannosyl-rich lysosomal hydrolase-induced bovine ASM cell proliferation. Using a humanized transgenic mouse strain (huASM-MRC2) expressing the human MRC2 receptor in a SM tissue-specific manner, we have demonstrated that ASM hyperplasia/hypertrophy can occur as early as 15 days after allergen challenge in this mouse model and this phenomenon is preventable with SC-MN treatment. This proof-of-concept study would facilitate future development of a potential asthma therapeutic agent with dual function of anti-inflammatory and anti-smooth muscle remodeling effects.

Multifunctional cellulase catalysis targeted by fusion to different carbohydrate-binding modules

DOE PAGES

Walker, Johnnie A.; Takasuka, Taichi E.; Deng, Kai; ...

2015-12-21

Carbohydrate binding modules (CBMs) bind polysaccharides and help target glycoside hydrolases catalytic domains to their appropriate carbohydrate substrates. To better understand how CBMs can improve cellulolytic enzyme reactivity, representatives from each of the 18 families of CBM found in Ruminoclostridium thermocellum were fused to the multifunctional GH5 catalytic domain of CelE (Cthe_0797, CelEcc), which can hydrolyze numerous types of polysaccharides including cellulose, mannan, and xylan. Since CelE is a cellulosomal enzyme, none of these fusions to a CBM previously existed. CelEcc_CBM fusions were assayed for their ability to hydrolyze cellulose, lichenan, xylan, and mannan. Several CelEcc_CBM fusions showed enhanced hydrolyticmore » activity with different substrates relative to the fusion to CBM3a from the cellulosome scaffoldin, which has high affinity for binding to crystalline cellulose. Additional binding studies and quantitative catalysis studies using nanostructure-initiator mass spectrometry (NIMS) were carried out with the CBM3a, CBM6, CBM30, and CBM44 fusion enzymes. In general, and consistent with observations of others, enhanced enzyme reactivity was correlated with moderate binding affinity of the CBM. Numerical analysis of reaction time courses showed that CelEcc_CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexose and pentose fractions of ionic-liquid pretreated switchgrass. In conclusion, we have shown that fusions of different CBMs to a single multifunctional GH5 catalytic domain can increase its rate of reaction with different pure polysaccharides and with pretreated biomass. This fusion approach, incorporating domains with broad specificity for binding and catalysis, provides a new avenue to improve reactivity of simple combinations of enzymes within the complexity of plant biomass.« less

Receptor binding kinetics equations: Derivation using the Laplace transform method.

PubMed

Hoare, Sam R J

Measuring unlabeled ligand receptor binding kinetics is valuable in optimizing and understanding drug action. Unfortunately, deriving equations for estimating kinetic parameters is challenging because it involves calculus; integration can be a frustrating barrier to the pharmacologist seeking to measure simple rate parameters. Here, a well-known tool for simplifying the derivation, the Laplace transform, is applied to models of receptor-ligand interaction. The method transforms differential equations to a form in which simple algebra can be applied to solve for the variable of interest, for example the concentration of ligand-bound receptor. The goal is to provide instruction using familiar examples, to enable investigators familiar with handling equilibrium binding equations to derive kinetic equations for receptor-ligand interaction. First, the Laplace transform is used to derive the equations for association and dissociation of labeled ligand binding. Next, its use for unlabeled ligand kinetic equations is exemplified by a full derivation of the kinetics of competitive binding equation. Finally, new unlabeled ligand equations are derived using the Laplace transform. These equations incorporate a pre-incubation step with unlabeled or labeled ligand. Four equations for measuring unlabeled ligand kinetics were compared and the two new equations verified by comparison with numerical solution. Importantly, the equations have not been verified with experimental data because no such experiments are evident in the literature. Equations were formatted for use in the curve-fitting program GraphPad Prism 6.0 and fitted to simulated data. This description of the Laplace transform method will enable pharmacologists to derive kinetic equations for their model or experimental paradigm under study. Application of the transform will expand the set of equations available for the pharmacologist to measure unlabeled ligand binding kinetics, and for other time

Antibodies against glucan, chitin, and Saccharomyces cerevisiae mannan as new biomarkers of Candida albicans infection that complement tests based on C. albicans mannan.

PubMed

Sendid, B; Dotan, N; Nseir, S; Savaux, C; Vandewalle, P; Standaert, A; Zerimech, F; Guery, B P; Dukler, A; Colombel, J F; Poulain, D

2008-12-01

Antibodies against Saccharomyces cerevisiae mannan (ASCA) and antibodies against synthetic disaccharide fragments of glucans (ALCA) and chitin (ACCA) are biomarkers of Crohn's disease (CD). We previously showed that Candida albicans infection generates ASCA. Here, we explored ALCA and ACCA as possible biomarkers of invasive C. albicans infection (ICI). ASCA, ALCA, ACCA, and Candida mannan antigen and antibody detection tests were performed on 69 sera obtained sequentially from 18 patients with ICIs proven by blood culture, 59 sera from CD patients, 47 sera from hospitalized subjects colonized by Candida species (CZ), and 131 sera from healthy controls (HC). ASCA, ALCA, and ACCA levels in CD and ICI patients were significantly different from those in CZ and HC subjects (P<0.0001). In ICI patients, these levels increased as infection developed. Using ASCA, ALCA, ACCA, and Platelia Candida tests, 100% of ICIs were detected, with the kinetics of the antibody response depending on the patient during the time course of infection. A large number of sera presented with more than three positive tests. This is the first evidence that the detection of antibodies against chitin and glucans has diagnostic value in fungal infections and that these tests can complement more specific tests. Future trials are necessary to assess the value of these tests in multiparametric analysis, as well as their pathophysiological relevance.

Enantiomeric cannabidiol derivatives: synthesis and binding to cannabinoid receptors.

PubMed

Hanus, Lumír O; Tchilibon, Susanna; Ponde, Datta E; Breuer, Aviva; Fride, Ester; Mechoulam, Raphael

2005-03-21

(-)-Cannabidiol (CBD) is a major, non psychotropic constituent of cannabis. It has been shown to cause numerous physiological effects of therapeutic importance. We have reported that CBD derivatives in both enantiomeric series are of pharmaceutical interest. Here we describe the syntheses of the major CBD metabolites, (-)-7-hydroxy-CBD and (-)-CBD-7-oic acid and their dimethylheptyl (DMH) homologs, as well as of the corresponding compounds in the enantiomeric (+)-CBD series. The starting materials were the respective CBD enantiomers and their DMH homologs. The binding of these compounds to the CB(1) and CB(2) cannabinoid receptors are compared. Surprisingly, contrary to the compounds in the (-) series, which do not bind to the receptors, most of the derivatives in the (+) series bind to the CB(1) receptor in the low nanomole range. Some of these compounds also bind weakly to the CB(2) receptor.

[Purification of arsenic-binding proteins in hamster plasma after oral administration of arsenite].

PubMed

Wang, Wenwen; Zhang, Min; Li, Chunhui; Qin, Yingjie; Hua, Naranmandura

2013-01-01

To purify the arsenic-binding proteins (As-BP) in hamster plasma after a single oral administration of arsenite (iAs(III)). Arsenite was given to hamsters in a single dose. Three types of HPLC columns, size exclusion, gel filtration and anion exchange columns, combined with an inductively coupled argon plasma mass spectrometer (ICP MS) were used to purify the As-BP in hamster plasma. SDS-PAGE was used to confirm the arsenic-binding proteins at each purification step. The three-step purification process successfully separated As-BP from other proteins (ie, arsenic unbound proteins) in hamster plasma. The molecular mass of purified As-BP in plasma was approximately 40-50 kD on SDS-PAGE. The three-step purification method is a simple and fast approach to purify the As-BP in plasma samples.

Novel coumarin modified GLP-1 derivatives with enhanced plasma stability and prolonged in vivo glucose-lowering ability.

PubMed

Han, Jing; Sun, Lidan; Huang, Xun; Li, Zheng; Zhang, Chenyu; Qian, Hai; Huang, Wenlong

2014-12-01

The short biological half-life limits the therapeutic use of glucagon-like peptide-1 (GLP-1) and chemical modification to improve the interaction of peptides with serum albumin represents an effective strategy to develop long-acting peptide analogues. Coumarin, a natural product, is known to bind tightly to plasma proteins and possesses many biological activities. Therefore, we designed and synthesized a series of coumarin-modified GLP-1 derivatives, hypothesizing that conjugation with coumarin would retain the therapeutic effects and prolong the biological half-life of the conjugates. Four cysteine-modified GLP-1 analogues (1-4) were prepared using Gly8 -GLP-1(7-36)-NH2 peptide as a starting point. These analogues were conjugated with two coumarin maleimides to yield eight compounds (conjugates 6-13) for testing. Activation of human GLP-1 receptors, stability to enzymic inactivation in plasma and binding to human albumin were assessed in vitro. In vivo, effects on oral glucose tolerance tests (OGTT) in rats and on blood glucose levels in db/db mice were studied. Most conjugates showed well preserved receptor activation efficacy, enhanced albumin-binding properties and improved in vitro plasma stability and conjugate 7 was selected to undergo further assessment. In rats, conjugate 7 had a longer circulating t1/2 than exendin-4 or liraglutide. A prolonged antidiabetic effect of conjugate 7 was observed after OGTT in rats and a prolonged hypoglycaemic effect in db/db mice. Cysteine-specific coumarin conjugation with GLP-1 offers a useful approach to the development of long-acting incretin-based antidiabetic agents. Conjugate 7 is a promising long-lasting GLP-1 derivative deserving further investigation. © 2014 The British Pharmacological Society.

ATP-binding cassette-like transporters are involved in the transport of lignin precursors across plasma and vacuolar membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miao, Y.C.; Liu, C.

2010-12-28

Lignin is a complex biopolymer derived primarily from the condensation of three monomeric precursors, the monolignols. The synthesis of monolignols occurs in the cytoplasm. To reach the cell wall where they are oxidized and polymerized, they must be transported across the cell membrane. However, the molecular mechanisms underlying the transport process are unclear. There are conflicting views about whether the transport of these precursors occurs by passive diffusion or is an energized active process; further, we know little about what chemical forms are required. Using isolated plasma and vacuolar membrane vesicles prepared from Arabidopsis, together with applying different transporter inhibitorsmore » in the assays, we examined the uptake of monolignols and their derivatives by these native membrane vesicles. We demonstrate that the transport of lignin precursors across plasmalemma and their sequestration into vacuoles are ATP-dependent primary-transport processes, involving ATP-binding cassette-like transporters. Moreover, we show that both plasma and vacuolar membrane vesicles selectively transport different forms of lignin precursors. In the presence of ATP, the inverted plasma membrane vesicles preferentially take up monolignol aglycones, whereas the vacuolar vesicles are more specific for glucoconjugates, suggesting that the different ATP-binding cassette-like transporters recognize different chemical forms in conveying them to distinct sites, and that glucosylation of monolignols is necessary for their vacuolar storage but not required for direct transport into the cell wall in Arabidopsis.« less

Binding affinities of vascular endothelial growth factor (VEGF) for heparin-derived oligosaccharides

PubMed Central

Zhao, Wenjing; McCallum, Scott A.; Xiao, Zhongping; Zhang, Fuming; Linhardt, Robert J.

2011-01-01

Heparin and heparan sulphate (HS) exert their wide range of biological activities by interacting with extracellular protein ligands. Among these important protein ligands are various angiogenic growth factors and cytokines. HS-binding to vascular endothelial growth factor (VEGF) regulates multiple aspects of vascular development and function through its specific interaction with HS. Many studies have focused on HS-derived or HS-mimicking structures for the characterization of VEGF165 interaction with HS. Using a heparinase 1-prepared small library of heparin-derived oligosaccharides ranging from hexasaccharide to octadecasaccharide, we systematically investigated the heparin-specific structural features required for VEGF binding. We report the apparent affinities for the association between the heparin-derived oligosaccharides with both VEGF165 and VEGF55, a peptide construct encompassing exclusively the heparin-binding domain of VEGF165. An octasaccharide was the minimum size of oligosaccharide within the library to efficiently bind to both forms of VEGF and that a tetradecasaccharide displayed an effective binding affinity to VEGF165 comparable to unfractionated heparin. The range of relative apparent binding affinities among VEGF and the panel of heparin-derived oligosaccharides demonstrate that VEGF binding affinity likely depends on the specific structural features of these oligosaccharides including their degree of sulphation and sugar ring stereochemistry and conformation. Notably, the unique 3-O-sulpho group found within the specific antithrombin binding site of heparin is not required for VEGF165 binding. These findings afford new insight into the inherent kinetics and affinities for VEGF association with heparin and heparin-derived oligosaccharides with key residue specific modifications and may potentially benefit the future design of oligosaccharide-based anti-angiogenesis drugs. PMID:21658003

Resistant starch reduces colonic and urinary p-cresol in rats fed a tyrosine-supplemented diet, whereas konjac mannan does not.

PubMed

Chen, Bixiao; Morioka, Sahya; Nakagawa, Tomoyuki; Hayakawa, Takashi

2016-10-01

The effect of resistant starch (RS) and konjac mannan (KM) to maintain and improve the large intestinal environment was compared. Wistar SPF rats were fed the following diets for 4 weeks: negative control diet (C diet), tyrosine-supplemented positive control diet (T diet), and luminacoid supplemented diets containing either high-molecular konjac mannan A (KMAT diet), low-molecular konjac mannan B (KMBT diet), high-amylose cornstarch (HAST diet), or heat-moisture-treated starch (HMTST diet). The luminacoid-fed group had an increased content of short-chain fatty acids in the cecum. HAS caused a significant decrease in p-cresol content in the cecum, whereas KM did not. Urinary p-cresol was reduced in the HAST group compared with the T group, but not the KM fed groups. Deterioration in the large intestinal environment was only improved completely in the HAST and HMTST groups, suggesting that RS is considerably more effective than KM in maintaining the large intestinal environment.

Plasma sex-steroid binding protein in a seasonally breeding reptile, Alligator mississippiensis.

PubMed

Ho, S M; Lance, V; Megaloudis, M

1987-01-01

The properties of a sex-steroid binding protein (SSBP) in the plasma of the American alligator, Alligator mississippiensis, were partially characterized. Alligator SSBP has a sedimentation coefficient of 4S in a 5-20% sucrose gradient. It binds to estradiol-17 beta (E2) and testosterone (T) with limited capacities and moderate affinities (association constant for [3H]E2 is 4.70 +/- 0.09 X 10(8) M-1 and for [3H]T is 1.05 +/- 0.07 X 10(8) M-1, mean +/- SEM of six determinations). Plasma SSBP level, as measured by plasma [3H]E2 binding capacity, varies from 30 to 140 nmol per liter plasma (nM) and was found to be dependent on the gender, sexual maturity, and reproductive state of the animal. Distinct annual fluctuations in plasma SSBP level were observed in female alligators. In adult females, plasma SSBP levels were high (122 +/- 6 nM) in the fall during the nonbreeding season and low (30-60 nM) in spring and early summer during the breeding season. A minimum (33 +/- 6 nM) was reached in mid-June coinciding with the time of oviposition and rapid decline in circulating estrogen levels. This decline in adult female plasma SSBP levels during the breeding season was not observed in immature females. On the contrary, plasma SSBP levels in immature females increased from 81 +/- 14 nM in April to 134 +/- 9 nM in June. Plasma SSBP levels in male alligators showed little changes throughout the entire breeding season; they remained within the range of 80-100 nM from March to June. We believe that seasonal fluctuations in plasma SSBP levels constitute part of the mechanism involved in the regulation of free steroid delivered to target organs in female alligators and that such a mechanism does not exist in male animals.

Human Plasma-derived Polymeric IgA and IgM Antibodies Associate with Secretory Component to Yield Biologically Active Secretory-like Antibodies*

PubMed Central

Longet, Stéphanie; Miled, Sarah; Lötscher, Marius; Miescher, Sylvia M.; Zuercher, Adrian W.; Corthésy, Blaise

2013-01-01

Immunotherapy with monoclonal and polyclonal immunoglobulin is successfully applied to improve many clinical conditions, including infection, autoimmune diseases, or immunodeficiency. Most immunoglobulin products, recombinant or plasma-derived, are based on IgG antibodies, whereas to date, the use of IgA for therapeutic application has remained anecdotal. In particular, purification or production of large quantities of secretory IgA (SIgA) for potential mucosal application has not been achieved. In this work, we sought to investigate whether polymeric IgA (pIgA) recovered from human plasma is able to associate with secretory component (SC) to generate SIgA-like molecules. We found that ∼15% of plasma pIgA carried J chain and displayed selective SC binding capacity either in a mixture with monomeric IgA (mIgA) or after purification. The recombinant SC associated covalently in a 1:1 stoichiometry with pIgA and with similar efficacy as colostrum-derived SC. In comparison with pIgA, the association with SC delayed degradation of SIgA by intestinal proteases. Similar results were obtained with plasma-derived IgM. In vitro, plasma-derived IgA and SIgA neutralized Shigella flexneri used as a model pathogen, resulting in a delay of bacteria-induced damage targeted to polarized Caco-2 cell monolayers. The sum of these novel data demonstrates that association of plasma-derived IgA or IgM with recombinant/colostrum-derived SC is feasible and yields SIgA- and SIgM-like molecules with similar biochemical and functional characteristics as mucosa-derived immunoglobulins. PMID:23250751

SDS-binding assay based on tyrosine fluorescence as a tool to determine binding properties of human serum albumin in blood plasma

NASA Astrophysics Data System (ADS)

Zhdanova, Nadezda; Shirshin, Evgeny; Fadeev, Victor; Priezzhev, Alexander

2016-04-01

Among all plasma proteins human serum albumin (HSA) is the most studied one as it is the main transport protein and can bind a wide variety of ligands especially fatty acids (FAs). The concentration of FAs bound to HSA in human blood plasma differs by three times under abnormal conditions (fasting, physical exercises or in case of social important diseases). In the present study a surfactant sodium dodecyl sulfate (SDS) was used to simulate FAs binding to HSA. It was shown that the increase of Tyr fluorescence of human blood plasma due to SDS addition can be completely explained by HSA-SDS complex formation. Binding parameters of SDS-HSA complex (average number of sites and apparent constant of complex formation) were determined from titration curves based on tyrosine (Tyr) fluorescence.

Microbiome dynamic modulation through functional diets based on pre- and probiotics (mannan-oligosaccharides and Saccharomyces cerevisiae) in juvenile rainbow trout (Oncorhynchus mykiss).

PubMed

Gonçalves, A T; Gallardo-Escárate, C

2017-05-01

This study used high-throughput sequencing to evaluate the intestinal microbiome dynamics in rainbow trout (Oncorhynchus mykiss) fed commercial diets supplemented with either pre- or probiotics (0·6% mannan-oligosaccharides and 0·5% Saccharomyces cerevisiae respectively) or the mixture of both. A total of 57 fish whole intestinal mucosa and contents bacterial communities were characterized by high-throughput sequencing and analysis of the V3-V4 region of the 16S rRNA gene, as well as the relationship between plasma biochemical health indicators and microbiome diversity. This was performed at 7, 14 and 30 days after start feeding functional diets, and microbiome diversity increased when fish fed functional diets after 7 days and it was positively correlated with plasma cholesterol levels. Dominant phyla were, in descending order, Proteobacteria, Firmicutes, Actinobacteria, Acidobacteria, Bacteroidetes and Fusobacteria. However, functional diets reduced the abundance of Gammaproteobacteria to favour abundances of organisms from Firmicutes and Fusobacteria, two phyla with members that confer beneficial effects. A dynamic shift of the microbiome composition was observed with changes after 7 days of feeding and the modulation by functional diets tend to cluster the corresponding groups apart from CTRL group. The core microbiome showed an overall stability with functional diets, except genus such as Escherichia-Shigella that suffered severe reductions on their abundances when feeding any of the functional diets. Functional diets based on pre- or probiotics dynamically modulate intestinal microbiota of juvenile trout engaging taxonomical abundance shifts that might impact fish physiological performance. This study shows for the first time the microbiome modulation dynamics by functional diets based on mannan-oligosaccharides and S. cerevisiae and their synergy using culture independent high-throughput sequencing technology, revealing the complexity behind the dietary

Fluorescence studies on binding of pyrene and its derivatives to humic acid

NASA Astrophysics Data System (ADS)

Nakashima, K.; Maki, M.; Ishikawa, F.; Yoshikawa, T.; Gong, Y.-K.; Miyajima, T.

2007-07-01

Binding of pyrene (PyH) and its derivatives to humic acid (HA) has been studied by fluorescence spectroscopy. The nature of the interaction between HA and pyrene derivatives are extensively investigated by employing three derivatives ranging from anionic to cationic compounds: 1-pyrenebutylic acid (PyA), 1-pyrenemethanol (PyM), and 1-pyrenebutyltrimethylammonium bromide (PyB). Binding constants between HA and PyX (X = H, A, M, B) are obtained by steady-state fluorescence quenching techniques, and it is found that PyB has a markedly large binding constant among the pyrene family. This is attributed to a strong electrostatic interaction between cationic PyB and anionic HA. The result suggests that an electrostatic interaction plays a dominant role in binding of pyrenes to humic acid. The importance of electrostatic interaction was also confirmed by a salt effect on the binding constant. Influence of collisional quenching on the binding constant, which causes overestimation of the binding constant, was examined by time-resolved fluorescence spectroscopy as well as temperature effect in steady-state fluorescence measurements. It is elucidated that collisional quenching does not much bring overestimation into the binding constants.

Ellipsometric studies of synthetic albumin-binding chitosan-derivatives and selected blood plasma proteins

NASA Astrophysics Data System (ADS)

Sarkar, Sabyasachi

This dissertation summarizes work on the synthesis of chitosan-derivatives and the development of ellipsometric methods to characterize materials of biological origin. Albumin-binding chitosan-derivatives were synthesized via addition reactions that involve amine groups naturally present in chitosan. These surfaces were shown to have an affinity towards human serum albumin via ELISA, UV spectroscopy and SDS PAGE. Modified surfaces were characterized with IR ellipsometry at various stages of their synthesis using appropriate optical models. It was found that spin cast chitosan films were anisotropic in nature. All optical models used for characterizing chitosan-derivatives were thus anisotropic. Chemical signal dependence on molecular structure and composition was illustrated via IR spectroscopic ellipsometry (IRSE). An anisotropic optical model of an ensemble of Lorentz oscillators were used to approximate material behavior. The presence of acetic acid in spin-cast non-neutralized chitosan samples was thus shown. IRSE application to biomaterials was also demonstrated by performing a step-wise chemical characterizations during synthesis stages. Protein adsorbed from single protein solutions on these modified surfaces was monitored by visible in-situ variable wavelength ellipsometry. Based on adsorption profiles obtained from single protein adsorption onto silicon surfaces, lumped parameter kinetic models were developed. These models were used to fit experimental data of immunoglobulin-G of different concentrations and approximate conformational changes in fibrinogen adsorption. Biomaterial characterization by ellipsometry was further extended to include characterization of individual protein solutions in the IR range. Proteins in an aqueous environment were characterized by attenuated total internal reflection (ATR) IR ellipsometry using a ZnSe prism. Parameterized dielectric functions were created for individual proteins using Lorentz oscillators. These

Novel coumarin modified GLP-1 derivatives with enhanced plasma stability and prolonged in vivo glucose-lowering ability

PubMed Central

Han, Jing; Sun, Lidan; Huang, Xun; Li, Zheng; Zhang, Chenyu; Qian, Hai; Huang, Wenlong

2014-01-01

Background and Purpose The short biological half-life limits the therapeutic use of glucagon-like peptide-1 (GLP-1) and chemical modification to improve the interaction of peptides with serum albumin represents an effective strategy to develop long-acting peptide analogues. Coumarin, a natural product, is known to bind tightly to plasma proteins and possesses many biological activities. Therefore, we designed and synthesized a series of coumarin-modified GLP-1 derivatives, hypothesizing that conjugation with coumarin would retain the therapeutic effects and prolong the biological half-life of the conjugates. Experimental Approach Four cysteine-modified GLP-1 analogues (1–4) were prepared using Gly8-GLP-1(7–36)-NH2 peptide as a starting point. These analogues were conjugated with two coumarin maleimides to yield eight compounds (conjugates 6–13) for testing. Activation of human GLP-1 receptors, stability to enzymic inactivation in plasma and binding to human albumin were assessed in vitro. In vivo, effects on oral glucose tolerance tests (OGTT) in rats and on blood glucose levels in db/db mice were studied. Key Results Most conjugates showed well preserved receptor activation efficacy, enhanced albumin-binding properties and improved in vitro plasma stability and conjugate 7 was selected to undergo further assessment. In rats, conjugate 7 had a longer circulating t1/2 than exendin-4 or liraglutide. A prolonged antidiabetic effect of conjugate 7 was observed after OGTT in rats and a prolonged hypoglycaemic effect in db/db mice. Conclusions and Implications Cysteine-specific coumarin conjugation with GLP-1 offers a useful approach to the development of long-acting incretin-based antidiabetic agents. Conjugate 7 is a promising long-lasting GLP-1 derivative deserving further investigation. PMID:25039358

Identification of clam plasma proteins that bind its pathogen Quahog Parasite Unknown.

PubMed

Hartman, Rachel; Pales Espinosa, Emmanuelle; Allam, Bassem

2018-06-01

The hard clam (Mercenaria mercenaria) is among the most economically-important marine species along the east coast of the United States, representing the first marine resource in several Northeastern states. The species is rather resilient to infections and the only important disease of hard clams results from an infection caused by Quahog Parasite Unknown (QPX), a protistan parasite that can lead to significant mortality events in wild and aquacultured clam stocks. Though the presence of QPX disease has been documented since the 1960s, little information is available on cellular and molecular interactions between the parasite and the host. This study examined the interactions between the clam immune system and QPX cells. First, the effect of clam plasma on the binding of hemocytes to parasite cells was evaluated. Second, clam plasma proteins that bind QPX cells were identified through proteomic (LC-MS/MS) analyses. Finally, the effect of prior clam exposure to QPX on the abundance of QPX-reactive proteins in the plasma was evaluated. Results showed that plasma factors enhance the attachment of hemocytes to QPX. Among the proteins that specifically bind to QPX cells, several lectins were identified, as well as complement component proteins and proteolytic enzymes. Furthermore, results showed that some of these lectins and complement-related proteins are inducible as their abundance significantly increased following QPX challenge. These results shed light on plasma proteins involved in the recognition and binding of parasite cells and provide molecular targets for future investigations of factors involved in clam resistance to the disease, and ultimately for the selection of resistant clam stocks. Copyright © 2018 Elsevier Ltd. All rights reserved.

Measurement of Epidermal Growth Factor Receptor-Derived Signals Within Plasma Membrane Clathrin Structures.

PubMed

Lucarelli, Stefanie; Delos Santos, Ralph Christian; Antonescu, Costin N

2017-01-01

The epidermal growth factor (EGF) receptor (EGFR) is an important regulator of cell growth, proliferation, survival, migration, and metabolism. EGF binding to EGFR triggers the activation of the receptor's intrinsic kinase activity, in turn eliciting the recruitment of many secondary signaling proteins and activation of downstream signals, such as the activation of phosphatidylinositol-3-kinase (PI3K) and Akt, a process requiring the phosphorylation of Gab1. While the identity of many signals that can be activated by EGFR has been revealed, how the spatiotemporal organization of EGFR signaling within cells controls receptor outcome remains poorly understood. Upon EGF binding at the plasma membrane, EGFR is internalized by clathrin-mediated endocytosis following recruitment to clathrin-coated pits (CCPs). Further, plasma membrane CCPs, but not EGFR internalization, are required for EGF-stimulated Akt phosphorylation. Signaling intermediates such as phosphorylated Gab1, which lead to Akt phosphorylation, are enriched within CCPs upon EGF stimulation. These findings indicate that some plasma membrane CCPs also serve as signaling microdomains required for certain facets of EGFR signaling and are enriched in key EGFR signaling intermediates. Understanding how the spatiotemporal organization of EGFR signals within CCP microdomains controls receptor signaling outcome requires imaging methods that can systematically resolve and analyze the properties of CCPs, EGFR and key signaling intermediates. Here, we describe methods using total internal reflection fluorescence microscopy imaging and analysis to systematically study the enrichment of EGFR and key EGFR-derived signals within CCPs.

Sustainability of a public system for plasma collection, contract fractionation and plasma-derived medicinal product manufacturing

PubMed Central

Grazzini, Giuliano; Ceccarelli, Anna; Calteri, Deanna; Catalano, Liviana; Calizzani, Gabriele; Cicchetti, Americo

2013-01-01

Background In Italy, the financial reimbursement for labile blood components exchanged between Regions is regulated by national tariffs defined in 1991 and updated in 1993–2003. Over the last five years, the need for establishing standard costs of healthcare services has arisen critically. In this perspective, the present study is aimed at defining both the costs of production of blood components and the related prices, as well as the prices of plasma-derived medicinal products obtained by national plasma, to be used for interregional financial reimbursement. Materials and methods In order to analyse the costs of production of blood components, 12 out 318 blood establishments were selected in 8 Italian Regions. For each step of the production process, driving costs were identified and production costs were. To define the costs of plasma-derived medicinal products obtained by national plasma, industrial costs currently sustained by National Health Service for contract fractionation were taken into account. Results The production costs of plasma-derived medicinal products obtained from national plasma showed a huge variability among blood establishments, which was much lower after standardization. The new suggested plasma tariffs were quite similar to those currently in force. Comparing the overall costs theoretically sustained by the National Health Service for plasma-derived medicinal products obtained from national plasma to current commercial costs, demonstrates that the national blood system could gain a 10% cost saving if it were able to produce plasma for fractionation within the standard costs defined in this study. Discussion Achieving national self-sufficiency through the production of plasma-derived medicinal products from national plasma, is a strategic goal of the National Health Service which must comply not only with quality, safety and availability requirements but also with the increasingly pressing need for economic sustainability. PMID:24333307

Sustainability of a public system for plasma collection, contract fractionation and plasma-derived medicinal product manufacturing.

PubMed

Grazzini, Giuliano; Ceccarelli, Anna; Calteri, Deanna; Catalano, Liviana; Calizzani, Gabriele; Cicchetti, Americo

2013-09-01

In Italy, the financial reimbursement for labile blood components exchanged between Regions is regulated by national tariffs defined in 1991 and updated in 1993-2003. Over the last five years, the need for establishing standard costs of healthcare services has arisen critically. In this perspective, the present study is aimed at defining both the costs of production of blood components and the related prices, as well as the prices of plasma-derived medicinal products obtained by national plasma, to be used for interregional financial reimbursement. In order to analyse the costs of production of blood components, 12 out 318 blood establishments were selected in 8 Italian Regions. For each step of the production process, driving costs were identified and production costs were. To define the costs of plasma-derived medicinal products obtained by national plasma, industrial costs currently sustained by National Health Service for contract fractionation were taken into account. The production costs of plasma-derived medicinal products obtained from national plasma showed a huge variability among blood establishments, which was much lower after standardization. The new suggested plasma tariffs were quite similar to those currently in force. Comparing the overall costs theoretically sustained by the National Health Service for plasma-derived medicinal products obtained from national plasma to current commercial costs, demonstrates that the national blood system could gain a 10% cost saving if it were able to produce plasma for fractionation within the standard costs defined in this study. Achieving national self-sufficiency through the production of plasma-derived medicinal products from national plasma, is a strategic goal of the National Health Service which must comply not only with quality, safety and availability requirements but also with the increasingly pressing need for economic sustainability.

Luteinizing hormone-releasing hormone inactivation by purified pituitary plasma membranes: effects of receptor-binding studies.

PubMed

Clayton, R N; Shakespear, R A; Duncan, J A; Marshall, J C

1979-05-01

Inactivation of LHRH by purified bovine pituitary plasma membranes was studied in vitro. After incubation of [125I]iodo-LHRH with plasma membranes, the amount of tracer bound to the pellet was measured, and the integrity of the unbound tracer in the supernatant was assessed. Reduction in ability to bind to anti-LHRH serum and to rebind to plasma membranes together with altered electrophoretic mobility on polyacrylamide gels showed that the unbound [125I]iodo-LHRH was inactivated. LHRH inactivation occurred rapidly and was dependent upon membrane concentration and incubation temperature. These results indicate that hormone inactivation must be taken into account in the interpretation of LHRH-receptor interactions. During 37 C incubations, the apparent absence of specific LHRH binding can be explained by inactivation of tracer hormone. Significant LHRH inactivation also occurred at 0 C, which in part explains the insensitivity of LHRH receptor assays. Assessment of LHRH inactivation by different particulate subcellular fractions of pituitary tissue showed that the inactivating enzyme was associated with the plasma membranes; other organelles did not alter LHRH. The enzyme appeared to be an integral part of the plasma membrane structure, since enzymic activity could not be removed by washing without reducing specific LHRH binding. Additionally, reduction of LHRH inactivation by the inhibitors Bacitracin and Trasylol and by magnesium was also accompanied by reduced LHRH binding. Previous studies have shown that the majority of LHRH binding to pituitary plasma membranes is to the low affinity site (approximately 10(-6) M), but the significance of this binding has been uncertain. Our findings indicate that low affinity binding probably represents binding of LHRH to the inactivating enzyme. The LHRH analog, D-Ser6(TBu), des Gly10, ethylamide, has greater biological activity than LHRH and is not inactivated to a significant extent by pituitary plasma membranes. The

Nutrition quality of extraction mannan residue from palm kernel cake on brolier chicken

NASA Astrophysics Data System (ADS)

Tafsin, M.; Hanafi, N. D.; Kejora, E.; Yusraini, E.

2018-02-01

This study aims to find out the nutrient residue of palm kernel cake from mannan extraction on broiler chicken by evaluating physical quality (specific gravity, bulk density and compacted bulk density), chemical quality (proximate analysis and Van Soest Test) and biological test (metabolizable energy). Treatment composed of T0 : palm kernel cake extracted aquadest (control), T1 : palm kernel cake extracted acetic acid (CH3COOH) 1%, T2 : palm kernel cake extracted aquadest + mannanase enzyme 100 u/l and T3 : palm kernel cake extracted acetic acid (CH3COOH) 1% + enzyme mannanase 100 u/l. The results showed that mannan extraction had significant effect (P<0.05) in improving the quality of physical and numerically increase the value of crude protein and decrease the value of NDF (Neutral Detergent Fiber). Treatments had highly significant influence (P<0.01) on the metabolizable energy value of palm kernel cake residue in broiler chickens. It can be concluded that extraction with aquadest + enzyme mannanase 100 u/l yields the best nutrient quality of palm kernel cake residue for broiler chicken.

Evaluation of lectin pathway activity and mannan-binding lectin levels in the course of pregnancy complicated by diabetes type 1, based on the genetic background.

PubMed

Pertyńska Marczewska, Magdalena; Cedzyński, Maciej; Swierzko, Anna; Szala, Agnieszka; Sobczak, Małgorzata; Cypryk, Katarzyna; Wilczyński, Jan

2009-01-01

There are numerous indications that either mannan-binding lectin (MBL) deficiency or its excessive activity are associated with adverse pregnancy outcomes. High MBL concentrations and corresponding MBL2 genotypes were shown to be associated with microvascular complications in type 1 diabetes. The aim of this study was to evaluate levels of MBL and MBL-dependent activity of the lectin pathway (LP) of complement in the course of pregnancy in diabetic mothers, based on genetic background. These parameters were determined in samples from healthy non-pregnant (control), diabetic non-pregnant, healthy pregnant, and pregnant diabetic women. No significant differences in median MBL levels or LP activities were found in any study group compared to the control. However, statistically significant differences in MBL levels were noted during pregnancy between the 1st and 3rd trimesters in both healthy controls and pregnant diabetics. With regard to LP values, similar trends were evident, but statistically significant results were obtained only in the healthy pregnant group. When data analysis was confined to patients carrying the A/A (wild-type) MBL2 genotype, an increase in MBL level during pregnancy (in both healthy and diabetic pregnant women) was still observed. Similarly, LP activity increased during both healthy and diabetic pregnancies, significantly so for the former. Diabetes, an autoimmune disease, is a serious complication of pregnancy. Therefore, determination of MBL status might be beneficial in identifying type 1 diabetic patients who are at increased risk of developing both vascular complications and poor pregnancy outcomes.

Inhibition of vincristine binding to plasma membrane vesicles from daunorubicin-resistant Ehrlich ascites cells by multidrug resistance modulators.

PubMed Central

Sehested, M.; Jensen, P. B.; Skovsgaard, T.; Bindslev, N.; Demant, E. J.; Friche, E.; Vindeløv, L.

1989-01-01

The multidrug resistance (MDR) phenotype is presumed to be mostly dependent on changes in the resistant cell plasma membrane, notably the emergence of a 170 kDa glycoprotein called P-glycoprotein, which facilitate increased drug efflux. We have previously demonstrated that ATP-enhanced binding of vincristine (VCR) to plasma membrane vesicles is much greater in MDR than in wild type cells. The present study has shown that VCR binding to MDR Ehrlich ascites tumour cell plasma membrane vesicles is inhibited 50% most efficiently by quinidine (0.5 microM) followed by verapamil (4.1 microM) and trifluoperazine (23.2 microM). This is the reverse order of the effect on whole cells where a ranking of efficiency in terms of enhancement of VCR accumulation, inhibition of VCR efflux, DNA perturbation and modulation of resistance in a clonogenic assay, was trifluoperazine greater than or equal to verapamil much greater than quinidine. The detergent Tween 80 inhibited VCR binding to plasma membrane vesicles at 0.001% v/v which agreed with the level which modulated resistance and increased VCR accumulation in whole cells. No effect was observed on daunorubicin binding to MDR plasma membrane vesicles after incubation with either Tween 80 (up to 0.1% v/v) or verapamil (up to 25 microM). We conclude that the effect of a modulating drug in reversing resistance to VCR correlates with its ability to raise intracellular VCR levels but not with its capability to inhibit VCR binding to the plasma membrane. Thus, enhancement of VCR accumulation in MDR cells is hardly solely due to competition for a drug binding site on P-glycoprotein. Furthermore, the lack of a demonstrable effect on daunorubicin binding to the plasma membrane by modulators points to transport mechanisms which do not utilise specific drug binding to the plasma membrane. PMID:2605092

LH-RH binding to purified pituitary plasma membranes: absence of adenylate cyclase activation.

PubMed

Clayton, R N; Shakespear, R A; Marshall, J C

1978-06-01

Purified bovine pituitary plasma membranes possess two specific LH-RH binding sites. The high affinity site (2.5 X 10(9) l/mol) has low capacity (9 X 10(-15) mol/mg membrane protein) while the low affinity site 6.1 X 10(5) l/mol) has a much higher capacity (1.1 X 10(-10) mol/mg). Specific LH-RH binding to plasma membranes is increased 8.5-fold during purification from homogenate whilst adenylate cyclase activity is enriched 7--8-fold. Distribution of specific LH-RH binding to sucrose density gradient interface fractions parallels that of adenylate cyclase activity. Mg2+ and Ca2+ inhibit specific [125I]LH-RH binding at micromolar concentrations. Synthetic LH-RH, up to 250 microgram/ml, failed to stimulate adenylase cyclase activity of the purified bovine membranes. Using a crude 10,800 g rat pituitary membrane preparation, LH-RH similarly failed to activate adenylate cyclase even in the presence of guanyl nucleotides. These data confirm the presence of LH-RH receptor sites on pituitary plasma membranes and suggest that LH-RH-induced gonadotrophin release may be mediated by mechanisms other than activation of adenylate cyclase.

Economical impact of plasma fractionation project in Iran on affordability of plasma-derived medicines.

PubMed

Cheraghali, A M; Aboofazeli, R

2009-12-01

In Iran all transfusion services are concentrated under authority of one public and centralized transfusion organization which has created the opportunity of using plasma produced in its blood centers for fractionation. In 2008 voluntary and non remunerated Iranian donors donated 1.8 million units of blood. This indicates a 25/1000 donation index. After responding to the needs for fresh plasma and cryoprecipitate each year about 150000 L of recovered plasma are reserved for fractionation. In an attempt to improve both blood safety profile and availability and affordability of plasma derived medicines, Iran's national transfusion service has entered into a contract fractionation agreement for surplus of plasma produced from donated blood by voluntary non remunerated donors. In order to ensure safety of product produced, Iran has chosen to collaborate with international fractionators based in highly regulated countries. The main objective of this study was to evaluate the impact of contract plasma fractionation on the affordability of the plasma derived medicines in Iran. During 2006-2008, Iran's contract fractionation project was able to produce 46%, 18% and 6% of IVIG, Albumin and FVIII consumed in Iran's market, respectively. In contrary to IVIG and Albumin, due to fairly high consumption of FVIII in Iran, the role of fractionation project in meeting the needs to FVIII was not substantial. However, Iran's experience has shown that contract plasma fractionation, through direct and indirect effects on price of plasma derived medicines, could substantially improve availability and affordability of such products in national health care system.

Coevolution of yeast mannan digestion: Convergence of the civilized human diet, distal gut microbiome, and host immunity

PubMed Central

Abbott, D Wade; Martens, Eric C; Gilbert, Harry J; Cuskin, Fiona; Lowe, Elisabeth C

2015-01-01

The complex carbohydrates accessible to the distal gut microbiota (DGM) are key drivers in determining the structure of this ecosystem. Typically, plant cell wall polysaccharides and recalcitrant starch (i.e. dietary fiber), in addition to host glycans are considered the primary nutrients for the DGM; however, we recently demonstrated that α-mannans, highly branched polysaccharides that decorate the surface of yeast, are also nutrients for several members of Bacteroides spp. This relationship suggests that the advent of yeast in contemporary food technologies and the colonization of the intestine by endogenous fungi have roles in microbiome structure and function. Here we discuss the process of yeast mannan metabolism, and the intersection between various sources of intestinal fungi and their roles in recognition by the host innate immune system. PMID:26440374

Seasonal changes in plasma androgens, glucocorticoids and glucocorticoid-binding proteins in the marsupial sugar glider Petaurus breviceps.

PubMed

Bradley, A J; Stoddart, D M

1992-01-01

An investigation spanning two breeding seasons was carried out to examine endocrine changes associated with reproduction in a wild population of the marsupial sugar glider Petaurus breviceps, a small arboreal gliding possum. Using techniques of equilibrium dialysis and polyacrylamide gel electrophoresis at steady-state conditions, a high-affinity, low-capacity glucocorticoid-binding protein was demonstrated in the plasma of Petaurus breviceps. Equilibrium dialysis at 36 degrees C using cortisol gave a high-affinity binding constant of 95 +/- 5.2 litres/mumol for a presumed corticosteroid-binding globulin (CBG) while the binding constant for the cortisol-albumin interaction was 3.5 +/- 0.4 litres/mmol. There was no difference between the sexes in the affinity of binding of cortisol to CBG; however, the cortisol-binding capacity underwent seasonal variation in both sexes. Progesterone was bound strongly to the presumed CBG while neither oestradiol nor aldosterone appeared to be bound with high affinity to P. breviceps plasma. In the males, peaks in the plasma concentration of testosterone coincided with the July-September breeding season in both years. A significant inverse relationship was shown to exist between the plasma testosterone concentration and the CBG-binding capacity. In both sexes an increase occurred in the plasma concentration of free cortisol during the first breeding season, a pattern which was not repeated in the subsequent breeding season, possibly due to a lower population density in that year.

Binding of heparin to plasma proteins and endothelial surfaces is inhibited by covalent linkage to antithrombin.

PubMed

Chan, Anthony K C; Paredes, Nethnapha; Thong, Bruce; Chindemi, Paul; Paes, Bosco; Berry, Leslie R; Monagle, Paul

2004-05-01

Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) are used for prophylaxis and treatment of thrombosis. However, UFH has a short plasma half-life and variable anticoagulant response in vivo due to plasma or vessel wall protein binding and LMWH has a decreased ability to inactivate thrombin, the pivotal enzyme in the coagulation cascade. Covalent linkage of antithrombin to heparin gave a complex (ATH) with superior anticoagulant activity compared to UFH and LMWH, and longer intravenous half-life compared to UFH. We found that plasma proteins bound more to UFH than ATH, and least to LMWH. Also, UFH bound significantly more to endothelial cells than ATH, with 100% of UFH and 94% of ATH binding being on the cell surface and the remainder was endocytosed. Competition studies with UFH confirmed that ATH binding was likely through its heparin moiety. These findings suggest that differences in plasma protein and endothelial cell binding may be due to available heparin chain length. Although ATH is polydisperse, the covalently-linked antithrombin may shield a portion of the heparin chain from association with plasma or endothelial cell surface proteins. This model is consistent with ATH's better bioavailability and more predictable dose response.

The specificity of binding of growth hormone and prolactin to purified plasma membranes from pregnant-rabbit liver.

PubMed Central

Webb, C F; Cadman, H F; Wallis, M

1986-01-01

The binding of 125I-labelled human growth hormone (hGH) to a purified plasma membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction with Triton X-100, was dependent on time, temperature, the cations used and the receptor concentration. Solubilization did not affect the binding properties of the receptors at low concentrations of Triton X-100. Some somatogenic hormones, such as bovine GH, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled hGH from purified plasma membranes and solubilized receptor preparations, but GHs and prolactins from various other species were rather ineffective. The results indicate that although there are binding sites for hGH in these pregnant rabbit liver membranes, few of these are specifically somatogenic or lactogenic. The binding properties of the purified plasma membranes are similar to those of a microsomal preparation studied previously, suggesting that the complex nature of the binding of hGH is not due to the heterogeneity of cellular membranes used to study binding, but is a property of the receptors associated with plasma membranes. PMID:3790086

LHRH-pituitary plasma membrane binding: the presence of specific binding sites in other tissues.

PubMed

Marshall, J C; Shakespear, R A; Odell, W D

1976-11-01

Two specific binding sites for LHRH are present on plasma membranes prepared from rat and bovine anterior pituitary glands. One site is of high affinity (K = 2X108 1/MOL) and the second is of lower affinity (8-5X105 1/mol) and much greater capacity. Studies on membrane fractions prepared from other tissues showed the presence of a single specific site for LHRH. The kinetics and specificity of this site were similar to those of the lower affinity pituitary receptor. These results indicate that only pituitary membranes possess the higher affinity binding site and suggest that the low affinity site is not of physiological importance in the regulation of gonadotrophin secretion. After dissociation from membranes of non-pituitary tissues 125I-LHRH rebound to pituitary membrane preparations. Thus receptor binding per se does not result in degradation of LHRH and the function of these peripheral receptors remains obscure.

Methadone adverse reaction presenting with large increase in plasma methadone binding: a case series

PubMed Central

2011-01-01

Introduction The use of methadone as an analgesic is on the increase, but it is widely recognized that the goal of predictable and reproducible dosing is confounded by considerable variability in methadone pharmacokinetics, and unpredictable side effects that include sedation, respiratory depression and cardiac arrhythmias. The mechanisms underlying these unpredictable effects are frequently unclear. Here, to the best of our knowledge we present the first report of an association between accidental methadone overexposure and increased plasma protein binding, a new potential mechanism for drug interactions with methadone. Case presentation We describe here the cases of two patients who experienced markedly different responses to the same dose of methadone during co-administration of letrozole. Both patients were post-menopausal Caucasian women who were among healthy volunteers participating in a clinical trial. Under the trial protocol both patients received 6 mg of intravenous methadone before and then after taking letrozole for seven days. One woman (aged 59) experienced symptoms consistent with opiate overexposure after the second dose of methadone that were reversed by naloxone, while the other (aged 49) did not. To understand the etiology of this event, we measured methadone pharmacokinetics in both patients. In our affected patient only, a fourfold to eightfold increase in methadone plasma concentrations after letrozole treatment was observed. Detailed pharmacokinetic analysis indicated no change in metabolism or renal elimination in our patient, but the percentage of unbound methadone in the plasma decreased 3.7-fold. As a result, the volume of distribution of methadone decreased approximately fourfold. The increased plasma binding in our affected patient was consistent with observed increases in plasma protein concentrations. Conclusions The marked increase in the total plasma methadone concentration observed in our patient, and the enhanced pharmacodynamic

Effect of anticonvulsants on plasma testosterone and sex hormone binding globulin levels.

PubMed Central

Barragry, J M; Makin, H L; Trafford, D J; Scott, D F

1978-01-01

Plasma sex hormone binding globulin (SHBG) and testosterone levels were measured in 29 patients with epilepsy (16 men and 13 women), most of them on chronic therapy with anticonvulsant drugs. Sex hormone binding globulin concentrations were increased in both sexes and testosterone levels in male patients. It is postulated that anticonvulsants may induce hepatic synthesis of SHBG. PMID:569688

Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets.

PubMed

Prada, Ilaria; Meldolesi, Jacopo

2016-08-09

Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated.

The binding modes of carbazole derivatives with telomere G-quadruplex

NASA Astrophysics Data System (ADS)

Zhang, Xiu-feng; Zhang, Hui-juan; Xiang, Jun-feng; Li, Qian; Yang, Qian-fan; Shang, Qian; Zhang, Yan-xia; Tang, Ya-lin

2010-10-01

It is reported that carbazole derivatives can stabilize G-quadruplex DNA structure formed by human telomeric sequence, and therefore, they have the potential to serve as anti-cancer agents. In this present study, in order to further explore the binding mode between carbazole derivatives and G-quadruplex formed by human telomeric sequence, two carbazole iodides (BMVEC, MVEC) molecules were synthesized and used to investigate the interaction with the human telomeric parallel and antiparallel G-quadruplex structures by NMR, CD and molecular modeling study. Interestingly, it is the pivotal the cationic charge pendant groups of pyridinium rings of carbazole that plays an essential role in the stabilizing and binding mode of the human telomeric sequences G-quadruplex structure. It was found that BMVEC with two cationic charge pendant groups of pyridinium rings of 9-ethylcarbazole cannot only stabilize parallel G-quadruple of Hum6 by groove binding and G-tetrad stacking modes and antiparallel G-quadruplex of Hum22 by groove binding, but also induce the formation of mixed G-quadruplex of Hum22. While MVEC with one cationic charge pendant groups of pyridinium ring only can bind with the parallel G-quadruplex of Hum6 by the stacking onto the G4 G-tetrad and could not interact with the G-quadruplex of Hum22.

Stereoselective HDAC inhibition from cysteine-derived zinc-binding groups.

PubMed

Butler, Kyle V; He, Rong; McLaughlin, Kathryn; Vistoli, Giulio; Langley, Brett; Kozikowski, Alan P

2009-08-01

A series of small-molecule histone deacetylase (HDAC) inhibitors, which feature zinc binding groups derived from cysteine, were synthesized. These inhibitors were tested against multiple HDAC isoforms, and the most potent, compound 10, was determined to have IC(50) values below 1 microM. The compounds were also tested in a cellular assay of oxidative stress-induced neurodegeneration. Many of the inhibitors gave near-complete protection against cell death at 10 microM without the neurotoxicity seen with hydroxamic acid-based inhibitors, and were far more neuroprotective than HDAC inhibitors currently in clinical trials. Both enantiomers of cysteine were used in the synthesis of a variety of novel zinc-binding groups (ZBGs). Derivatives of L-cysteine were active in the HDAC inhibition assays, while the derivatives of D-cysteine were inactive. Notably, the finding that both the D- and L-cysteine derivatives were active in the neuroprotection assays suggests that multiple mechanisms are working to protect the neurons from cell death. Molecular modeling was employed to investigate the differences in inhibitory activity between the HDAC inhibitors generated from the two enantiomeric forms of cysteine.

Surface-functionalized, pH-responsive poly(lactic-co-glycolic acid)-based microparticles for intranasal vaccine delivery: Effect of surface modification with chitosan and mannan.

PubMed

Li, Ze; Xiong, Fangfang; He, Jintian; Dai, Xiaojing; Wang, Gaizhen

2016-12-01

In the present study, surface-functionalized, pH-responsive poly(lactic-co-glycolic acid) (PLGA) microparticles were investigated for nasal delivery of hepatitis B surface Antigen (HBsAg). pH-responsive PLGA, chitosan modified PLGA (CS-PLGA), mannan modified PLGA (MN-PLGA), mannan and chitosan co-modified PLGA (MN-CS-PLGA) microparticles were prepared utilizing a double-emulsion method. Antigen was released rapidly from four types of microparticles at pH5.0 and pH 6.0, but slowly released at pH 7.4. Mannan and chitosan surface modification enhanced intracellular microparticle uptake by macrophages. Following intracellular macrophage antigen uptake, antigen release occurred in three different patterns: fast release from PLGA and MN-PLGA microparticles in endosomes/lysosomes, slow release from CS-PLGA microparticles in cytoplasm and a combination of fast release and slow release patterns from MN-CS-PLGA microparticles. Furthermore, chitosan coating modification increased the residence time of CS-PLGA and MN-CS-PLGA microparticles in the nasal cavity. In vivo immunogenicity studies indicated that MN-CS-PLGA microparticles induced stronger humoral and cell-mediated immune responses compared with PLGA, MN-PLGA and CS-PLGA microparticles. These results suggest that surface modification of pH-responsive PLGA microparticles with mannan and chitosan is a promising tool for nasal delivery of HBsAg. Copyright © 2016. Published by Elsevier B.V.

LC-MS/MS quantification of next-generation biotherapeutics: a case study for an IgE binding Nanobody in cynomolgus monkey plasma.

PubMed

Sandra, Koen; Mortier, Kjell; Jorge, Lucie; Perez, Luis C; Sandra, Pat; Priem, Sofie; Poelmans, Sofie; Bouche, Marie-Paule

2014-05-01

Nanobodies(®) are therapeutic proteins derived from the smallest functional fragments of heavy chain-only antibodies. The development and validation of an LC-MS/MS-based method for the quantification of an IgE binding Nanobody in cynomolgus monkey plasma is presented. Nanobody quantification was performed making use of a proteotypic tryptic peptide chromatographically enriched prior to LC-MS/MS analysis. The validated LLOQ at 36 ng/ml was measured with an intra- and inter-assay precision and accuracy <20%. The required sensitivity could be obtained based on the selectivity of 2D LC combined with MS/MS. No analyte specific tools for affinity purification were used. Plasma samples originating from a PK/PD study were analyzed and compared with the results obtained with a traditional ligand-binding assay. Excellent correlations between the two techniques were obtained, and similar PK parameters were estimated. A 2D LC-MS/MS method was successfully developed and validated for the quantification of a next generation biotherapeutic.

Changes in the transmission properties of multi-tooth plasmonic nano-filters (multi-TPNFs) caused by geometrical imperfection

NASA Astrophysics Data System (ADS)

Khaksar, A.; Fatemi, H.

2012-08-01

To model the filtering behavior of a multi-tooth plasmonic nano-filter (multi-TPNF), an equivalent circuitry composed of a set of serried impedances is considered. The changes caused in its filtering behavior are proposed as a measuring tool to investigate the effect of the geometrical imperfections occurring during the manufacture of the device. Consequently, the effects of changes in the nominal size of each of the geometrical parameters of a multi-TPNF sample, such as its tooth height, d, its tooth width, w, and the separation between two successive teeth, Δ, on its transmittance are investigated. It is observed that each single tooth of the multi-TPNF and also the waveguide between any of its two successive teeth exhibit a very Fabry-Perot interferometer like behavior. The variation of the transmission spectra of a multi-TPNF whose geometrical parameters are imperfect is compared with the desired filter, and also the effect of the number of geometrically imperfect teeth of the multi-TPNF on the filtering spectra is examined.

Topographical analysis of the plasma membrane-associated sucrose binding protein from soybean.

PubMed

Overvoorde, P J; Grimes, H D

1994-05-27

Plasma membranes of soybean cells actively engaged in sucrose transport have a sucrose binding protein (SBP) that does not appear to be an integral membrane protein. Experiments were undertaken to analyze the topographical association of this protein with the membrane. Treatment of purified plasma membrane vesicles with either 1 M KCl or KI released less than 35% of the sucrose binding protein from the membrane whereas treatment with either 4 M urea or 0.1 M Na2CO3, pH 11.5, disassociated between 50 and 70%, respectively, of this protein from the membrane. SDS, at either 0.5x, 1x, or 10x of its critical micelle concentration, effectively solubilized the sucrose binding protein. The nonionic detergents Triton X-100 and CHAPS, at either 0.5x, 1x, or 10x of their critical micelle concentration, solubilized between 65 and 75% of this protein. When either native plasma membrane-associated or in vitro-transcribed and -translated SBP were subjected to Triton X-114 phase separation, 80% partitioned into the detergent-poor aqueous phase. These results indicate that the SBP is a peripheral membrane protein but also suggest that there is a population of this protein that is tethered to the membrane.

Efficient recombinant expression and secretion of a thermostable GH26 mannan endo-1,4-beta-mannosidase from Bacillus licheniformis in Escherichia coli.

PubMed

Songsiriritthigul, Chomphunuch; Buranabanyat, Bancha; Haltrich, Dietmar; Yamabhai, Montarop

2010-04-11

Mannans are one of the key polymers in hemicellulose, a major component of lignocellulose. The Mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannanase (EC 3.2.1.78), commonly named beta-mannanase, is an enzyme that can catalyze random hydrolysis of beta-1,4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans. The enzyme has found a number of applications in different industries, including food, feed, pharmaceutical, pulp/paper industries, as well as gas well stimulation and pretreatment of lignocellulosic biomass for the production of second generation biofuel. Bacillus licheniformis is a Gram-positive endospore-forming microorganism that is generally non-pathogenic and has been used extensively for large-scale industrial production of various enzymes; however, there has been no previous report on the cloning and expression of mannan endo-1,4-beta-mannosidase gene (manB) from B. licheniformis. The mannan endo-1,4-beta-mannosidase gene (manB), commonly known as beta-mannanase, from Bacillus licheniformis strain DSM13 was cloned and overexpressed in Escherichia coli. The enzyme can be harvested from the cell lysate, periplasmic extract, or culture supernatant when using the pFLAG expression system. A total activity of approximately 50,000 units could be obtained from 1-l shake flask cultures. The recombinant enzyme was 6 x His-tagged at its C-terminus, and could be purified by one-step immobilized metal affinity chromatography (IMAC) to apparent homogeneity. The specific activity of the purified enzyme when using locust bean gum as substrate was 1672 +/- 96 units/mg. The optimal pH of the enzyme was between pH 6.0 - 7.0; whereas the optimal temperature was at 50 - 60 degrees C. The recombinant beta-mannanase was stable within pH 5 - 12 after incubation for 30 min at 50 degrees C, and within pH 6 - 9 after incubation at 50 degrees C for 24 h. The enzyme was stable at temperatures up to 50 degrees C with a half-life time of activity (tau1

Plasma-derived microparticles in polycythaemia vera.

PubMed

Ahadon, M; Abdul Aziz, S; Wong, C L; Leong, C F

2018-04-01

Microparticles are membrane bound vesicles, measuring less than 1.0 um, which are released during cellular activation or during apoptosis. Studies have shown that these circulating microparticles play a role in coagulation, cell signaling and cellular interactions. Increased levels of circulating microparticles have been observed in a number of conditions where there is vascular dysfunction, thrombosis and inflammation. The objective of this study was to determine the various plasma-derived microparticles in patients with polycythaemia vera (PV) in Universiti Kebangsaan Malaysia Medical Centre and to compare them with normal control. A total of 15 patients with PV and 15 healthy volunteers were included in this cross-sectional descriptive study. Plasma samples from both patients and healthy volunteers were prepared and further processed for isolation of microparticles. Flow cytometry analyses were then carried out in all samples to determine the cellular origin of the microparticles. Full blood count parameters for both groups were also collected. Data collected were analyzed using SPSS version 12.0. Patients with PV had a significantly higher percentage of platelet derived microparticles compared to healthy controls (P <0.05). The control group had a higher level of endothelial derived microparticles but the differences were not statistically significant (P > 0.05). The median percentage of positive events for platelet derived microparticles was higher in patients with PV compared to normal healthy controls.

Distinct p53 genomic binding patterns in normal and cancer-derived human cells

PubMed Central

McCorkle, Sean R; McCombie, WR; Dunn, John J

2011-01-01

Here, we report genome-wide analysis of the tumor suppressor p53 binding sites in normal human cells. 743 high-confidence ChIP-seq peaks representing putative genomic binding sites were identified in normal IMR90 fibroblasts using a reference chromatin sample. More than 40% were located within 2 kb of a transcription start site (TSS), a distribution similar to that documented for individually studied, functional p53 binding sites and, to date, not observed by previous p53 genome-wide studies. Nearly half of the high-confidence binding sites in the IMR90 cells reside in CpG islands in marked contrast to sites reported in cancer-derived cells. The distinct genomic features of the IMR90 binding sites do not reflect a distinct preference for specific sequences, since the de novo developed p53 motif based on our study is similar to those reported by genome-wide studies of cancer cells. More likely, the different chromatin landscape in normal, compared with cancer-derived cells, influences p53 binding via modulating availability of the sites. We compared the IMR90 ChIP-seq peaks to the recently published IMR90 methylome1 and demonstrated that they are enriched at hypomethylated DNA. Our study represents the first genome-wide, de novo mapping of p53 binding sites in normal human cells and reveals that p53 binding sites reside in distinct genomic landscapes in normal and cancer-derived human cells. PMID:22127205

Pot binding as a variable confounding plant phenotype: theoretical derivation and experimental observations.

PubMed

Sinclair, Thomas R; Manandhar, Anju; Shekoofa, Avat; Rosas-Anderson, Pablo; Bagherzadi, Laleh; Schoppach, Remy; Sadok, Walid; Rufty, Thomas W

2017-04-01

Theoretical derivation predicted growth retardation due to pot water limitations, i.e., pot binding. Experimental observations were consistent with these limitations. Combined, these results indicate a need for caution in high-throughput screening and phenotyping. Pot experiments are a mainstay in many plant studies, including the current emphasis on developing high-throughput, phenotyping systems. Pot studies can be vulnerable to decreased physiological activity of the plants particularly when pot volume is small, i.e., "pot binding". It is necessary to understand the conditions under which pot binding may exist to avoid the confounding influence of pot binding in interpreting experimental results. In this paper, a derivation is offered that gives well-defined conditions for the occurrence of pot binding based on restricted water availability. These results showed that not only are pot volume and plant size important variables, but the potting media is critical. Artificial potting mixtures used in many studies, including many high-throughput phenotyping systems, are particularly susceptible to the confounding influences of pot binding. Experimental studies for several crop species are presented that clearly show the existence of thresholds of plant leaf area at which various pot sizes and potting media result in the induction of pot binding even though there may be no immediate, visual plant symptoms. The derivation and experimental results showed that pot binding can readily occur in plant experiments if care is not given to have sufficiently large pots, suitable potting media, and maintenance of pot water status. Clear guidelines are provided for avoiding the confounding effects of water-limited pot binding in studying plant phenotype.

Blood safety--a focus on plasma derivatives in Mainland China.

PubMed

Zhu, Y M

2007-01-01

Plasma derivative production in Mainland China can be encapsulated by two figures: 50 years of history and 5000 tons of annually processed source plasma. Demands for albumin, immunoglobulinin and main clotting factors can barely be met, despite a relatively low average usage among China's population of 1.3 billion. The tragedy of contamination among plasma donors in Henan province in the early 1990's has left shadows on the safety of the plasma derivative industry. However, during the last ten years the Chinese government has made great strides forward. The regulation of the entire operation has been strengthened, from law and standard setting and upholding to stricter licensing regulations for plasma centers and fractionators. Public concerns in blood safety are gradually being relieved, and confidence is returning. Nevertheless, the plasma donors and hemophilia patients infected a decade ago by infected blood or plasma products represent a set of severe social and medical problems that the government and society must still deal with.

Effect of molecular parameters on the binding of phenoxyacetic acid derivatives to albumins.

PubMed

Cserháti, T; Forgács, E; Deyl, Z; Miksík, I

2001-03-25

The interaction of 12 phenoxyacetic acid derivatives with human and serum albumin as well as with egg albumin was studied by charge-transfer reversed-phase (RP) thin-layer chromatography (TLC) and the relative strength of interaction was calculated. Each phenoxyacetic acid derivative interacted with human and bovine serum albumins whereas no interaction was observed with egg albumin. Stepwise regression analysis proved that the lipophilicity of the derivatives exert a significant impact on their capacity to bind to serum albumins. This result supports the hypothesis that the binding of phenoxyacetic acid derivatives to albumins may involve hydrophobic forces occurring between the corresponding apolar substructures of these derivatives and the amino acid side chains.

Plasma Distribution in Mercury's Magnetosphere Derived from MESSENGER Magnetometer and Fast Imaging Plasma Spectrometer Observations

NASA Technical Reports Server (NTRS)

Korth, Haje; Anderson, Brian J.; Gershman, Daniel J.; Raines, Jim M.; Slavin, James A.; Zurbuchen, Thomas H.; Solomon, Sean C.; McNutt, Ralph L.

2014-01-01

We assess the statistical spatial distribution of plasma in Mercury's magnetosphere from observations of magnetic pressure deficits and plasma characteristics by the MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) spacecraft. The statistical distributions of proton flux and pressure were derived from 10months of Fast Imaging Plasma Spectrometer (FIPS) observations obtained during the orbital phase of the MESSENGER mission. The Magnetometer-derived pressure distributions compare favorably with those deduced from the FIPS observations at locations where depressions in the magnetic field associated with the presence of enhanced plasma pressures are discernible in the Magnetometer data. The magnitudes of the magnetic pressure deficit and the plasma pressure agree on average, although the two measures of plasma pressure may deviate for individual events by as much as a factor of approximately 3. The FIPS distributions provide better statistics in regions where the plasma is more tenuous and reveal an enhanced plasma population near the magnetopause flanks resulting from direct entry of magnetosheath plasma into the low-latitude boundary layer of the magnetosphere. The plasma observations also exhibit a pronounced north-south asymmetry on the nightside, with markedly lower fluxes at low altitudes in the northern hemisphere than at higher altitudes in the south on the same field line. This asymmetry is consistent with particle loss to the southern hemisphere surface during bounce motion in Mercury's offset dipole magnetic field.

Plasma protein binding of phenytoin in 100 epileptic patients.

PubMed Central

Peterson, G M; McLean, S; Aldous, S; Von Witt, R J; Millingen, K S

1982-01-01

The plasma protein binding of phenytoin was investigated in 100 epileptic patients, using equilibrium dialysis at 37 degrees C. The unbound fractions of phenytoin in plasma formed a skewed distribution, with a range of 9.7 to 24.7% and a median value of 12.3%. Most (80%) patients appeared to form one group with free phenytoin fractions from 9.7 to 14.5%, while the remainder formed a group with elevated free fractions (greater than 14.5%). Total and unbound plasma concentrations of phenytoin were strongly correlated (r=0.95, P less than 0.0001). There was a weak correlation between increasing age and the unbound phenytoin fraction (r=0.28, P less than 0.01). The results indicate that measurement of the total phenytoin concentration in plasma should usually provide a reliable index of anticonvulsant effect. However, determination of the unbound phenytoin fraction would be beneficial in the management of those patients in whom this fraction may be elevated, due to interacting drugs or biochemical abnormalities. PMID:7104186

DNA binding properties and biological evaluation of dihydropyrimidinones derivatives as potential antitumor agents.

PubMed

Wang, Gongke; Li, Xiangrong; Gou, Yaping; Chen, Yuhan; Yan, Changling; Lu, Yan

2013-10-01

The binding properties of two medicinally important dihydropyrimidinones derivatives 5-(Ethoxycarbonyl)-6-methyl-4-phenyl-3,4-dihydropyrimidin-2(1H)-one (EMPD) and 5-(Ethoxycarbonyl)-6-methyl-4-(4-chlorophenyl)-3,4-dihydropyrimidin-2(1H)-one (EMCD) with calf-thymus DNA (ctDNA) were investigated by spectroscopy, viscosity, isothermal titration calorimetry (ITC) and molecular modeling techniques. Simultaneously, their biological activities were evaluated with MTT assay method. The binding constants determined with spectroscopic titration and ITC were found to be in the same order of 10(4)M(-1). According to the results of viscosity studies, fluorescence competitive binding experiment and ITC investigations, intercalative binding was evaluated as the dominant binding modes between the two compounds and ctDNA. Furthermore, the results of molecular modeling corroborated those obtained from spectroscopic, viscosimetric and ITC investigations. Evaluation of the antitumor activities of the two derivatives against different tumor cell lines proved that they exhibited significant tumor cell inhibition rate, accordingly blocking DNA transcription and replication. The present results favor the development of potential drugs related with dihydropyrimidinones derivatives in the treatment of some diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

Chinese plasma-derived products supply under the lot release management system in 2007-2011.

PubMed

Zhang, Xuejun; Ye, Shengliang; Du, Xi; Yuan, Jing; Zhao, Chaoming; Li, Changqing

2013-11-01

In 2007, the Chinese State Food and Drug Administration (SFDA) implemented a management system for lot release of all plasma-derived products. Since then, there have been only a few systematic studies of the blood supply, which is a concern when considering the small amount of plasma collected per capita (approximately 3 L/1000 people). As a result, there may be a threat to the safety of the available blood supply. In this study, we examined the characteristics of the supply of Chinese plasma-derived products. We investigated the reports of lot-released biological products derived from all 8 national or regional regulatory authorities in China from 2007 to 2011. The market supply characteristics of Chinese plasma-derived products were analyzed by reviewing the changes in supply varieties, the batches of lot-released plasma-derived products and the actual supply. As a result, the national regulatory authorities can more accurately develop a specific understanding of the production and quality management information provided by Chinese plasma product manufacturers. The implementation of the lot release system further ensures the clinical validity of the plasma-derived products in China and improves the safety of using plasma-derived products. This work provides an assessment of the future Chinese market supply of plasma-derived products and can function as a theoretical basis for the establishment of hemovigilance. Copyright © 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives

PubMed Central

de Almeida, Sinara Mônica Vitalino; Lafayette, Elizabeth Almeida; Gomes da Silva, Lúcia Patrícia Bezerra; Amorim, Cézar Augusto da Cruz; de Oliveira, Tiago Bento; Gois Ruiz, Ana Lucia Tasca; de Carvalho, João Ernesto; de Moura, Ricardo Olímpio; Beltrão, Eduardo Isidoro Carneiro; de Lima, Maria do Carmo Alves; de Carvalho Júnior, Luiz Bezerra

2015-01-01

In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide derivatives (3a–h) were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 104 to 1.0 × 106 M−1 and quenching constants from −0.2 × 104 to 2.18 × 104 M−1 indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z)-2-(acridin-9-ylmethylene)-N-(4-chlorophenyl) hydrazinecarbothioamide (3f), while the most active compound in antiproliferative assay was (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (3a). There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties. PMID:26068233

Changes in immune and metabolic gut response in broilers fed beta-mannanase in beta-mannan containing diets

USDA-ARS?s Scientific Manuscript database

Beta-mannans, found in soy-based broiler feed, are known to cause physiological effects that are hypothesized to be related to gut inflammation. Previous studies have shown that the incorporation of beta-mannanase in the diet or as a supplement results in improvements to certain performance paramet...

Binding of canonical Wnt ligands to their receptor complexes occurs in ordered plasma membrane environments.

PubMed

Sezgin, Erdinc; Azbazdar, Yagmur; Ng, Xue W; Teh, Cathleen; Simons, Kai; Weidinger, Gilbert; Wohland, Thorsten; Eggeling, Christian; Ozhan, Gunes

2017-08-01

While the cytosolic events of Wnt/β-catenin signaling (canonical Wnt signaling) pathway have been widely studied, only little is known about the molecular mechanisms involved in Wnt binding to its receptors at the plasma membrane. Here, we reveal the influence of the immediate plasma membrane environment on the canonical Wnt-receptor interaction. While the receptors are distributed both in ordered and disordered environments, Wnt binding to its receptors selectively occurs in more ordered membrane environments which appear to cointernalize with the Wnt-receptor complex. Moreover, Wnt/β-catenin signaling is significantly reduced when the membrane order is disturbed by specific inhibitors of certain lipids that prefer to localize at the ordered environments. Similarly, a reduction in Wnt signaling activity is observed in Niemann-Pick Type C disease cells where trafficking of ordered membrane lipid components to the plasma membrane is genetically impaired. We thus conclude that ordered plasma membrane environments are essential for binding of canonical Wnts to their receptor complexes and downstream signaling activity. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Probing the nucleotide binding domain of the osmoregulator EnvZ using fluorescent nucleotide derivatives.

PubMed

Plesniak, Leigh; Horiuchi, Yuki; Sem, Daniel; Meinenger, David; Stiles, Linda; Shaffer, Jennifer; Jennings, Patricia A; Adams, Joseph A

2002-11-26

EnvZ is a histidine protein kinase important for osmoregulation in bacteria. While structural data are available for this enzyme, the nucleotide binding pocket is not well characterized. The ATP binding domain (EnvZB) was expressed, and its ability to bind nucleotide derivatives was assessed using equilbrium and stopped-flow fluorescence spectroscopy. The fluorescence emission of the trinitrophenyl derivatives, TNP-ATP and TNP-ADP, increase upon binding to EnvZB. The fluorescence enhancements were quantitatively abolished in the presence of excess ADP, indicating that the fluorescent probes occupy the nucleotide binding pocket. Both TNP-ATP and TNP-ADP bind to EnvZB with high affinity (K(d) = 2-3 microM). The TNP moiety attached to the ribose ring does not impede access of the fluorescent nucleotide into the binding pocket. The association rate constant for TNP-ADP is 7 microM(-1) s(-1), a value consistent with those for natural nucleotides and the eucaryotic protein kinases. Using competition experiments, it was found that ATP and ADP bind 30- and 150-fold more poorly, respectively, than the corresponding TNP-derivatized forms. Surprisingly, the physiological metal Mg(2+) is not required for ADP binding and only enhances ATP affinity by 3-fold. Although portions of the nucleotide pocket are disordered, the recombinant enzyme is highly stable, unfolding only at temperatures in excess of 70 degrees C. The unusually high affinity of the TNP derivatives compared to the natural nucleotides suggests that hydrophobic substitutions on the ribose ring enforce an altered binding mode that may be exploited for drug design strategies.

Binding of ACE-inhibitors to in vitro and patient-derived amyloid-β fibril models.

PubMed

Bhavaraju, Manikanthan; Phillips, Malachi; Bowman, Deborah; Aceves-Hernandez, Juan M; Hansmann, Ulrich H E

2016-01-07

Currently, no drugs exist that can prevent or reverse Alzheimer's disease, a neurodegenerative disease associated with the presence, in the brain, of plaques that are composed of β-amyloid (Aβ) peptides. Recent studies suggest that angiotensin-converting enzyme (ACE) inhibitors, a set of drugs used to treat hypertension, may inhibit amyloid formation in vitro. In the present study, we investigate through computer simulations the binding of ACE inhibitors to patient-derived Aβ fibrils and contrast it with that of ACE inhibitors binding to in vitro generated fibrils. The binding affinities of the ACE inhibitors are compared with that of Congo red, a dye that is used to identify amyloid structures and that is known to be a weak inhibitor of Aβ aggregation. We find that ACE inhibitors have a lower binding affinity to the patient-derived fibrils than to in vitro generated ones. For patient-derived fibrils, their binding affinities are even lower than that of Congo red. Our observations raise doubts on the hypothesis that these drugs inhibit fibril formation in Alzheimer patients by interacting directly with the amyloids.

Comparison of plasma pigment epithelium-derived factor (PEDF), retinol binding protein 4 (RBP-4), chitinase-3-like protein 1 (YKL-40) and brain-derived neurotrophic factor (BDNF) for the identification of insulin resistance.

PubMed

Toloza, F J K; Pérez-Matos, M C; Ricardo-Silgado, M L; Morales-Álvarez, M C; Mantilla-Rivas, J O; Pinzón-Cortés, J A; Pérez-Mayorga, M; Arévalo-García, M L; Tolosa-González, G; Mendivil, C O

2017-09-01

To evaluate and compare the association of four potential insulin resistance (IR) biomarkers (pigment-epithelium-derived factor [PEDF], retinol-binding-protein-4 [RBP-4], chitinase-3-like protein 1 [YKL-40] and brain-derived neurotrophic factor [BDNF]) with objective measures of IR. We studied 81 subjects with different metabolic profiles. All participants underwent a 5-point OGTT with calculation of multiple IR indexes. A subgroup of 21 participants additionally underwent a hyperinsulinemic-euglycemic clamp. IR was defined as belonging to the highest quartile of incremental area under the insulin curve (iAUCins), or to the lowest quartile of the insulin sensitivity index (ISI). PEDF was associated with adiposity variables. PEDF and RBP4 increased linearly across quartiles of iAUCins (for PEDF p-trend=0.029; for RBP-4 p-trend=0.053). YKL-40 and BDNF were not associated with any adiposity or IR variable. PEDF and RBP-4 levels identified individuals with IR by the iAUCins definition: A PEDF cutoff of 11.9ng/mL had 60% sensitivity and 68% specificity, while a RBP-4 cutoff of 71.6ng/mL had 70% sensitivity and 57% specificity. In multiple regression analyses simultaneously including clinical variables and the studied biomarkers, only BMI, PEDF and RBP-4 remained significant predictors of IR. Plasma PEDF and RBP4 identified IR in subjects with no prior diagnosis of diabetes. Copyright © 2017 Elsevier Inc. All rights reserved.

Multiple sup 3 H-oxytocin binding sites in rat myometrial plasma membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crankshaw, D.; Gaspar, V.; Pliska, V.

1990-01-01

The affinity spectrum method has been used to analyse binding isotherms for {sup 3}H-oxytocin to rat myometrial plasma membranes. Three populations of binding sites with dissociation constants (Kd) of 0.6-1.5 x 10(-9), 0.4-1.0 x 10(-7) and 7 x 10(-6) mol/l were identified and their existence verified by cluster analysis based on similarities between Kd, binding capacity and Hill coefficient. When experimental values were compared to theoretical curves constructed using the estimated binding parameters, good fits were obtained. Binding parameters obtained by this method were not influenced by the presence of GTP gamma S (guanosine-5'-O-3-thiotriphosphate) in the incubation medium. The bindingmore » parameters agree reasonably well with those found in uterine cells, they support the existence of a medium affinity site and may allow for an explanation of some of the discrepancies between binding and response in this system.« less

INFLAMMATORY MARKERS ASSOCIATED WITH TRAUMA AND INFECTION IN RED-TAILED HAWKS (BUTEO JAMAICENSIS) IN THE USA.

PubMed

Lee, Kelly A; Goetting, Valerie S; Tell, Lisa A

2015-10-01

Changes in inflammatory marker concentrations or activity can be used to monitor health and disease condition of domestic animals but have not been applied with the same frequency to wildlife. We measured concentrations or activity of six inflammatory markers (ceruloplasmin, haptoglobin, mannan-binding lectin-dependent complement [MBL/complement], unsaturated iron-binding capacity (UIBC) and total iron-binding capacity (TIBC), and plasma iron) in apparently healthy and sick or injured Red-tailed Hawks (Buteo jamaicensis). Haptoglobin and ceruloplasmin activities were consistently elevated in sick or injured hawks (2.1 and 2.5 times higher, respectively), and plasma iron concentrations decreased (0.46 times lower), relative to those of healthy birds. There were no differences between healthy and unhealthy hawks in TIBC and UIBC concentrations or MBL/complement activity. Therefore, haptoglobin, ceruloplasmin, and plasma iron would be useful inclusions in a panel of inflammatory markers for monitoring health in raptors.

Characterization of GTP binding and hydrolysis in plasma membranes of zucchini

NASA Technical Reports Server (NTRS)

Perdue, D. O.; Lomax, T. L.

1992-01-01

We have investigated the possibility that G-protein-like entities may be present in the plasma membrane (PM) of zucchini (Cucurbita pepo L.) hypocotyls by examining a number of criteria common to animal and yeast G-proteins. The GTP binding and hydrolysis characteristics of purified zucchini PM are similar to the characteristics of a number of known G-proteins. Our results demonstrate GTP binding to a single PM site having a Kd value between 16-31 nM. This binding has a high specificity for guanine nucleotides, and is stimulated by Mg2+, detergents, and fluoride or aluminium ions. The GTPase activity (Km = 0.49 micromole) of zucchini PM shows a sensitivity to NaF similar to that seen for other G-proteins. Localization of GTP mu 35S binding to nitrocellulose blots of proteins separated by SDS-PAGE indicates a 30-kDa protein as the predominant GTP-binding species in zucchini PM. Taken together, these data indicate that plant PM contains proteins which are biochemically similar to previously characterized G-proteins.

Characterization of GTP binding and hydrolysis in plasma membranes of zucchini.

PubMed

Perdue, D O; Lomax, T L

1992-01-01

We have investigated the possibility that G-protein-like entities may be present in the plasma membrane (PM) of zucchini (Cucurbita pepo L.) hypocotyls by examining a number of criteria common to animal and yeast G-proteins. The GTP binding and hydrolysis characteristics of purified zucchini PM are similar to the characteristics of a number of known G-proteins. Our results demonstrate GTP binding to a single PM site having a Kd value between 16-31 nM. This binding has a high specificity for guanine nucleotides, and is stimulated by Mg2+, detergents, and fluoride or aluminium ions. The GTPase activity (Km = 0.49 micromole) of zucchini PM shows a sensitivity to NaF similar to that seen for other G-proteins. Localization of GTP mu 35S binding to nitrocellulose blots of proteins separated by SDS-PAGE indicates a 30-kDa protein as the predominant GTP-binding species in zucchini PM. Taken together, these data indicate that plant PM contains proteins which are biochemically similar to previously characterized G-proteins.

SP-A binding sites on bovine alveolar macrophages.

PubMed

Plaga, S; Plattner, H; Schlepper-Schaefer, J

1998-11-25

Surfactant protein A (SP-A) binding to bovine alveolar macrophages was examined in order to characterize SP-A binding proteins on the cell surface and to isolate putative receptors from these cells that could be obtained in large amounts. Human SP-A, unlabeled or labeled with gold particles, was bound to freshly isolated macrophages and analyzed with ELISA or the transmission electron microscope. Binding of SP-A was inhibited by Ca2+ chelation, by an excess of unlabeled SP-A, or by the presence of 20 mg/ml mannan. We conclude that bovine alveolar macrophages expose binding sites for SP-A that are specific and that depend on Ca2+ and on mannose residues. For isolation of SP-A receptors with homologous SP-A as ligand we isolated SP-A from bovine lung lavage. SDS-PAGE analysis of the purified SP-A showed a protein of 32-36 kDa. Functional integrity of the protein was demonstrated. Bovine SP-A bound to Dynabeads was used to isolate SP-A binding proteins. From the fractionated and blotted proteins of the receptor preparation two proteins bound SP-A in a Ca2+-dependent manner, a 40-kDa protein showing mannose dependency and a 210-kDa protein, showing no mannose sensitivity. Copyright 1998 Academic Press.

Cloning of a heavy-metal-binding protein derived from activated-sludge microorganisms.

PubMed

Sano, Daisuke; Myojo, Ken; Omura, Tatsuo

2006-09-01

A gene of the heavy-metal-binding protein (HMBP) was newly isolated from a genetic DNA library of activated-sludge microorganisms. HMBP was produced by transformed Escherichia coli, and the copper-binding ability of HMBP was confirmed. HMBP derived from activated sludge could be available as heavy metal adsorbents in water and wastewater treatments.

DNA-Binding Interaction Studies of Microwave Assisted Synthesized Sulfonamide Substituted 8-Hydroxyquinoline Derivatives.

PubMed

Dixit, Ritu B; Patel, Tarosh S; Vanparia, Satish F; Kunjadiya, Anju P; Keharia, Harish R; Dixit, Bharat C

2011-01-01

Sulfonamide substituted 8-hydroxyquinoline derivatives were prepared using a microwave synthesizer. The interaction of sulfonamide substituted 8-hydroxyquinoline derivatives and their transition metal complexes with Plasmid (pUC 19) DNA and Calf Thymus DNA were investigated by UV spectroscopic studies and gel electrophoresis measurements. The interaction between ligand/metal complexes and DNA was carried out by increasing the concentration of DNA from 0 to 12 μl in UV spectroscopic study, while the concentration of DNA in gel electrophoresis remained constant at 10 μl. These studies supported the fact that, the complex binds to DNA by intercalation via ligand into the base pairs of DNA. The relative binding efficacy of the complexes to DNA was much higher than the binding efficacy of ligands, especially the complex of Cu-AHQMBSH had the highest binding ability to DNA. The mobility of the bands decreased as the concentration of the complex was increased, indicating that there was increase in the interaction between the metal ion and DNA. Complexes of AHQMBSH were excellent for DNA binding as compared to HQMABS.

Characterization of Rose Bengal binding to sinusoidal and bile canalicular plasma membrane from rat liver.

PubMed

Yachi, K; Sugiyama, Y; Sawada, Y; Iga, T; Ikeda, Y; Toda, G; Hanano, M

1989-01-16

The binding of Rose bengal, a model organic anion, to sinusoidal and bile canalicular membrane fractions isolated from rat liver was compared. The fluorescence change of Rose bengal after being bound to liver plasma membranes was utilized for measuring the binding. The dissociation constants (Kd = 0.1-0.12 microM) and the binding capacities (n = 11-15 nmol/mg protein) for Rose bengal are comparable between the two membrane fractions, although the n value for sinusoidal membrane is somewhat larger than that for bile canalicular membrane. The Rose bengal binding to both membrane fractions was inhibited by various organic anions at relatively low concentrations, i.e., the half-inhibition concentrations (IC50) for Indocyanine green, sulfobromophthalein, Bromophenol blue and 1-anilino-8-naphthalene sulfonate were 0.1, 100, 1.5-2.5 and 100 microM, respectively, while taurocholate did not inhibit the Rose bengal binding to either membrane fraction at these low concentration ranges. The type of inhibition of sulfobromophthalein and Indocyanine green for Rose bengal binding is different between the two membrane domains. That is, in sinusoidal and bile canalicular membrane fractions, these organic anions exhibit mixed-type and competitive-type inhibition, respectively. It was suggested that the fluorescence method using Rose bengal may provide a simple method for detecting the specific organic anion binding protein(s) in the liver plasma membrane.

Rational design of biaryl pharmacophore inserted noscapine derivatives as potent tubulin binding anticancer agents.

PubMed

Santoshi, Seneha; Manchukonda, Naresh Kumar; Suri, Charu; Sharma, Manya; Sridhar, Balasubramanian; Joseph, Silja; Lopus, Manu; Kantevari, Srinivas; Baitharu, Iswar; Naik, Pradeep Kumar

2015-03-01

We have strategically designed a series of noscapine derivatives by inserting biaryl pharmacophore (a major structural constituent of many of the microtubule-targeting natural anticancer compounds) onto the scaffold structure of noscapine. Molecular interaction of these derivatives with α,β-tubulin heterodimer was investigated by molecular docking, molecular dynamics simulation, and binding free energy calculation. The predictive binding affinity indicates that the newly designed noscapinoids bind to tubulin with a greater affinity. The predictive binding free energy (ΔG(bind, pred)) of these derivatives (ranging from -5.568 to -5.970 kcal/mol) based on linear interaction energy (LIE) method with a surface generalized Born (SGB) continuum solvation model showed improved binding affinity with tubulin compared to the lead compound, natural α-noscapine (-5.505 kcal/mol). Guided by the computational findings, these new biaryl type α-noscapine congeners were synthesized from 9-bromo-α-noscapine using optimized Suzuki reaction conditions for further experimental evaluation. The derivatives showed improved inhibition of the proliferation of human breast cancer cells (MCF-7), human cervical cancer cells (HeLa) and human lung adenocarcinoma cells (A549), compared to natural noscapine. The cell cycle analysis in MCF-7 further revealed that these compounds alter the cell cycle profile and cause mitotic arrest at G2/M phase more strongly than noscapine. Tubulin binding assay revealed higher binding affinity to tubulin, as suggested by dissociation constant (Kd) of 126 ± 5.0 µM for 5a, 107 ± 5.0 µM for 5c, 70 ± 4.0 µM for 5d, and 68 ± 6.0 µM for 5e compared to noscapine (Kd of 152 ± 1.0 µM). In fact, the experimentally determined value of ΔG(bind, expt) (calculated from the Kd value) are consistent with the predicted value of ΔG(bind, pred) calculated based on LIE-SGB. Based on these results, one of the derivative 5e of this series was used for further

The Yeast Plasma Membrane ATP Binding Cassette (ABC) Transporter Aus1

PubMed Central

Marek, Magdalena; Milles, Sigrid; Schreiber, Gabriele; Daleke, David L.; Dittmar, Gunnar; Herrmann, Andreas; Müller, Peter; Pomorski, Thomas Günther

2011-01-01

The ATP binding cassette (ABC) transporter Aus1 is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and is required for sterol uptake. These observations suggest that Aus1 promotes the translocation of sterols across membranes, but the precise transport mechanism has yet to be identified. In this study, an extraction and purification procedure was developed to characterize the Aus1 transporter. The detergent-solubilized protein was able to bind and hydrolyze ATP. Mutagenesis of the conserved lysine to methionine in the Walker A motif abolished ATP hydrolysis. Likewise, ATP hydrolysis was inhibited by classical inhibitors of ABC transporters. Upon reconstitution into proteoliposomes, the ATPase activity of Aus1 was specifically stimulated by phosphatidylserine (PS) in a stereoselective manner. We also found that Aus1-dependent sterol uptake, but not Aus1 expression and trafficking to the plasma membrane, was affected by changes in cellular PS levels. These results suggest a direct interaction between Aus1 and PS that is critical for the activity of the transporter. PMID:21521689

Public expenditure for plasma-derived and recombinant medicinal products in Italy.

PubMed

Lanzoni, Monica; Candura, Fabio; Calizzani, Gabriele; Biffoli, Claudia; Grazzini, Giuliano

2013-09-01

In Italy, the supply of plasma-derived medicinal products funded by the National Health Service can be through public healthcare facilities, accredited pharmacies or toll fractionation agreements between Regions and the manufacturer. Pharmaceutical public expenditure includes the supply related to the first two channels and costs can significantly vary because of channel-specific price reductions. This paper describes 2011 public expenditure for plasma-derived medicinal products purchased on the market, as well as the cost analysis per active substance. Analysis of the usage of plasma-derived medicinal products and of the related expenditure in public facilities has been carried out using medicinal product traceability data. The analysis related to the accredited pharmacies channel has been carried out using quantities for every medicinal package recorded by Pharmacy Associations and applying reference prices in force on March 1(st), 2012 as well as discounts for the accredited pharmaceutical expenditure imposed by law. At national and regional level, total and total per capita expenditures on plasma-derived medicinal products by market channel and funded by the National Health Service are shown. Analysis was conducted considering the active substances in three groups: substances included in toll fractionation agreements, recombinant coagulation factors, and other substances not included in toll fractionation agreements. In 2011, the national expenditure estimate for plasma-derived and recombinant medicinal product acquisition on the market was about € 535 million. The purchased volumes and mean purchased prices per unit of each substance have a significant influence on the observed regional variability of the pharmaceutical public expenditure. A strategy of regional comparison aimed at both sharing a national range of reference for purchase prices and evaluating modalities for centralised purchasing is desirable.

A major integral protein of the plant plasma membrane binds flavin.

PubMed

Lorenz, Astrid; Kaldenhoff, Ralf; Hertel, Rainer

2003-05-01

Abundant flavin binding sites have been found in membranes of plants and fungi. With flavin mononucleotide-agarose affinity columns, riboflavin-binding activity from microsomes of Cucurbita pepoL. hypocotyls was purified and identified as a specific PIP1-homologous protein of the aquaporin family. Sequences such as gi|2149955 in Phaseolus vulgaris, PIP1b of Arabidopsis thaliana, and NtAQP1 of tobacco are closely related. The identification as a riboflavin-binding protein was confirmed by binding tests with an extract of Escherichia coli cells expressing the tobacco NtAQP1 as well as leaves of transgenic tobacco plants that overexpress NtAQP1 or were inhibited in PIP1 expression by antisense constructs. When binding was assayed in the presence of dithionite, the reduced flavin formed a relatively stable association with the protein. Upon dilution under oxidizing conditions, the adduct was resolved, and free flavin reappeared with a half time of about 30 min. Such an association can also be induced photochemically, with oxidized flavin by blue light at 450 nm, in the presence of an electron donor. Several criteria, localization in the plasma membrane, high abundance, affinity to roseoflavin, and photochemistry, argue for a role of the riboflavin-binding protein PIP1 as a photoreceptor.

Study on the interaction of plasma protein binding rate between edaravone and taurine in human plasma based on HPLC analysis coupled with ultrafiltration technique.

PubMed

Tang, Dao-quan; Li, Yin-jie; Li, Zheng; Bian, Ting-ting; Chen, Kai; Zheng, Xiao-xiao; Yu, Yan-yan; Jiang, Shui-shi

2015-08-01

In this work, two high-performance liquid chromatography (HPLC) assays were developed and validated for the independent determination of edaravone and taurine using 3-methyl-1-p-tolyl-5-pyrazolone and L-glutamine as internal standards. In in vitro experiments, human plasma was separately spiked with a mixture of edaravone and taurine, edaravone or taurine alone. Plasma was precipitated with acetonitrile containing 0.1% formic acid. Ultrafiltration was employed to obtain the unbound ingredients of the two drugs. The factors that might influence the ultrafiltration effiency were elaborately optimized. Plasma supernatant and ultrafiltrate containing taurine were derivated with o-phthalaldehyde and ethanethiol in the presence of 40 mmol/L sodium borate buffer (pH 10.2) at room temperature within 1 min. Chromatographic separations were achieved on an InertSustain C18 column (250 × 4.6 mm, 5 µm). Isocratic 50 mmol/L ammonium acetate-acetonitrile and gradient 50 mmol/L sodium acetate (pH 5.3)-methanol were respectively selected as the mobile phase for the determination of edaravone and taurine. All of the validation data including linearity, extraction recovery, precision, accuracy and stability conformed to the requirements. Results showed that there were no significant alterations in the plasma protein binding rate of taurine and edaravone, implying that the proposed combination therapy was pharmacologically feasible. Copyright © 2014 John Wiley & Sons, Ltd.

The Association of Myosin IB with Actin Waves in Dictyostelium Requires Both the Plasma Membrane-Binding Site and Actin-Binding Region in the Myosin Tail

PubMed Central

Brzeska, Hanna; Pridham, Kevin; Chery, Godefroy; Titus, Margaret A.; Korn, Edward D.

2014-01-01

F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave. PMID:24747353

Effect of supplementation of prebiotic mannan-oligosaccharides and probiotic mixture on growth performance of broilers subjected to chronic heat stress

USDA-ARS?s Scientific Manuscript database

The present study was aimed at elucidating the effects of supplementing mannan-oligosaccharides (MOS) and probiotic mixture (PM) on growth performance, intestinal histology, and corticosterone concentrations in broilers kept under chronic heat stress (HS). Four hundred and fifty day-old chicks were...

Development and validation of a high-performance liquid chromatography method for the quantification of talazoparib in rat plasma: Application to plasma protein binding studies.

PubMed

Hidau, Mahendra Kumar; Kolluru, Srikanth; Palakurthi, Srinath

2018-02-01

A sensitive and selective RP-HPLC method has been developed and validated for the quantification of a highly potent poly ADP ribose polymerase inhibitor talazoparib (TZP) in rat plasma. Chromatographic separation was performed with isocratic elution method. Absorbance for TZP was measured with a UV detector (SPD-20A UV-vis) at a λ max of 227 nm. Protein precipitation was used to extract the drug from plasma samples using methanol-acetonitrile (65:35) as the precipitating solvent. The method proved to be sensitive and reproducible over a 100-2000 ng/mL linearity range with a lower limit of quantification (LLQC) of 100 ng/mL. TZP recovery was found to be >85%. Following analytical method development and validation, it was successfully employed to determine the plasma protein binding of TZP. TZP has a high level of protein binding in rat plasma (95.76 ± 0.38%) as determined by dialysis method. Copyright © 2017 John Wiley & Sons, Ltd.

Nanoplasmonic Quantification of Tumor-derived Extracellular Vesicles in Plasma Microsamples for Diagnosis and Treatment Monitoring

PubMed Central

Liang, Kai; Liu, Fei; Fan, Jia; Sun, Dali; Liu, Chang; Lyon, Christopher J.; Bernard, David W.; Li, Yan; Yokoi, Kenji; Katz, Matthew H.; Koay, Eugene J.; Zhao, Zhen; Hu, Ye

2017-01-01

Tumour-derived extracellular vesicles (EVs) are of increasing interest as a resource of diagnostic biomarkers. However, most EV assays require large samples, are time-consuming, low-throughput and costly, and thus impractical for clinical use. Here, we describe a rapid, ultrasensitive and inexpensive nanoplasmon-enhanced scattering (nPES) assay that directly quantifies tumor-derived EVs from as little as 1 μL of plasma. The assay uses the binding of antibody-conjugated gold nanospheres and nanorods to EVs captured by EV-specific antibodies on a sensor chip to produce a local plasmon effect that enhances tumour-derived EV detection sensitivity and specificity. We identified a pancreatic cancer EV biomarker, ephrin type-A receptor 2 (EphA2), and demonstrate that an nPES assay for EphA2-EVs distinguishes pancreatic cancer patients from pancreatitis patients and healthy subjects. EphA2-EVs were also informative in staging tumour progression and in detecting early responses to neoadjuvant therapy, with better performance than a conventional enzyme-linked immunosorbent assay. The nPES assay can be easily refined for clinical use, and readily adapted for diagnosis and monitoring of other conditions with disease-specific EV biomarkers. PMID:28791195

The N-Linked Outer Chain Mannans and the Dfg5p and Dcw1p Endo-α-1,6-Mannanases Are Needed for Incorporation of Candida albicans Glycoproteins into the Cell Wall

PubMed Central

Ao, Jie; Chinnici, Jennifer L.; Maddi, Abhiram

2015-01-01

A biochemical pathway for the incorporation of cell wall protein into the cell wall of Neurospora crassa was recently proposed. In this pathway, the DFG-5 and DCW-1 endo-α-1,6-mannanases function to covalently cross-link cell wall protein-associated N-linked galactomannans, which are structurally related to the yeast outer chain mannans, into the cell wall glucan-chitin matrix. In this report, we demonstrate that the mannosyltransferase enzyme Och1p, which is needed for the synthesis of the N-linked outer chain mannan, is essential for the incorporation of cell wall glycoproteins into the Candida albicans cell wall. Using endoglycosidases, we show that C. albicans cell wall proteins are cross-linked into the cell wall via their N-linked outer chain mannans. We further demonstrate that the Dfg5p and Dcw1p α-1,6-mannanases are needed for the incorporation of cell wall glycoproteins into the C. albicans cell wall. Our results support the hypothesis that the Dfg5p and Dcw1p α-1,6-mannanases incorporate cell wall glycoproteins into the C. albicans cell wall by cross-linking outer chain mannans into the cell wall glucan-chitin matrix. PMID:26048011

cAMP regulates DEP domain-mediated binding of the guanine nucleotide exchange factor Epac1 to phosphatidic acid at the plasma membrane.

PubMed

Consonni, Sarah V; Gloerich, Martijn; Spanjaard, Emma; Bos, Johannes L

2012-03-06

Epac1 is a cAMP-regulated guanine nucleotide exchange factor for the small G protein Rap. Upon cAMP binding, Epac1 undergoes a conformational change that results in its release from autoinhibition. In addition, cAMP induces the translocation of Epac1 from the cytosol to the plasma membrane. This relocalization of Epac1 is required for efficient activation of plasma membrane-located Rap and for cAMP-induced cell adhesion. This translocation requires the Dishevelled, Egl-10, Pleckstrin (DEP) domain, but the molecular entity that serves as the plasma membrane anchor and the possible mechanism of regulated binding remains elusive. Here we show that Epac1 binds directly to phosphatidic acid. Similar to the cAMP-induced Epac1 translocation, this binding is regulated by cAMP and requires the DEP domain. Furthermore, depletion of phosphatidic acid by inhibition of phospholipase D1 prevents cAMP-induced translocation of Epac1 as well as the subsequent activation of Rap at the plasma membrane. Finally, mutation of a single basic residue within a polybasic stretch of the DEP domain, which abolishes translocation, also prevents binding to phosphatidic acid. From these results we conclude that cAMP induces a conformational change in Epac1 that enables DEP domain-mediated binding to phosphatidic acid, resulting in the tethering of Epac1 at the plasma membrane and subsequent activation of Rap.

Prostatic origin of a zinc binding high molecular weight protein complex in human seminal plasma.

PubMed

Siciliano, L; De Stefano, C; Petroni, M F; Vivacqua, A; Rago, V; Carpino, A

2000-03-01

The profile of the zinc ligand high molecular weight proteins was investigated in the seminal plasma of 55 normozoospermic subjects by size exclusion high performance liquid chromatography (HPLC). The proteins were recovered from Sephadex G-75 gel filtration of seminal plasma in three zinc-containing fractions which were then submitted to HPLC analysis. The results were, that in all the samples, the protein profiles showed two peaks with apparent molecular weight of approximately 660 and approximately 250 kDa. Dialysis experiments revealed that both approximately 660 and approximately 250 kDa proteins were able to uptake zinc against gradient indicating their zinc binding capacity. The HPLC analysis of the whole seminal plasma evidenced only the approximately 660 kDa protein complex as a single well quantifying peak, furthermore a positive correlation between its peak area and the seminal zinc values (P < 0.001) was observed. This suggested a prostatic origin of the approximately 660 kDa protein complex which was then confirmed by the seminal plasma HPLC analysis of a subject with agenesis of the Wolffian ducts. Finally the study demonstrated the presence of two zinc binding proteins, approximately 660 and approximately 250 kDa respectively, in human seminal plasma and the prostatic origin of the approximately 660 kDa.

The α-galactomannan Davanat binds galectin-1 at a site different from the conventional galectin carbohydrate binding domain

PubMed Central

Miller, Michelle C; Klyosov, Anatole; Mayo, Kevin H

2009-01-01

Galectins are a sub-family of lectins, defined by their highly conserved β-sandwich structures and ability to bind to β-galactosides, like Gal β1-4 Glc (lactose). Here, we used 15N-1H HSQC and pulse field gradient (PFG) NMR spectroscopy to demonstrate that galectin-1 (gal-1) binds to the relatively large galactomannan Davanat, whose backbone is composed of β1-4-linked d-mannopyranosyl units to which single d-galactopyranosyl residues are periodically attached via α1-6 linkage (weight-average MW of 59 kDa). The Davanat binding domain covers a relatively large area on the surface of gal-1 that runs across the dimer interface primarily on that side of the protein opposite to the lactose binding site. Our data show that gal-1 binds Davanat with an apparent equilibrium dissociation constant (Kd) of 10 × 10−6 M, compared to 260 × 10−6 M for lactose, and a stiochiometry of about 3 to 6 gal-1 molecules per Davanat molecule. Mannan also interacts at the same galactomannan binding domain on gal-1, but with at least 10-fold lower avidity, supporting the role of galactose units in Davanat for relatively strong binding to gal-1. We also found that the β-galactoside binding domain remains accessible in the gal-1/Davanat complex, as lactose can still bind with no apparent loss in affinity. In addition, gal-1 binding to Davanat also modifies the supermolecular structure of the galactomannan and appears to reduce its hydrodynamic radius and disrupt inter-glycan interactions thereby reducing glycan-mediated solution viscosity. Overall, our findings contribute to understanding gal-1–carbohydrate interactions and provide insight into gal-1 function with potentially significant biological consequences. PMID:19541770

Structural and biochemical insights into the substrate-binding mechanism of a novel glycoside hydrolase family 134 β-mannanase.

PubMed

You, Xin; Qin, Zhen; Li, Yan-Xiao; Yan, Qiao-Juan; Li, Bin; Jiang, Zheng-Qiang

2018-06-01

Mannan is one of the major constituent groups of hemicellulose, which is a renewable resource from higher plants. β-Mannanases are enzymes capable of degrading lignocellulosic biomass. Here, an endo-β-mannanase from Rhizopus microsporus (RmMan134A) was cloned and expressed. The recombinant RmMan134A showed maximal activity at pH 5.0 and 50 °C, and exhibited high specific activity towards locust bean gum (2337 U/mg). To gain insight into the substrate-binding mechanism of RmMan134A, four complex structures (RmMan134A-M3, RmMan134A-M4, RmMan134A-M5 and RmMan134A-M6) were further solved. These structures showed that there were at least seven subsites (-3 to +4) in the catalytic groove of RmMan134A. Mannose in the -1 subsite hydrogen bonded with His113 and Tyr131, revealing a unique conformation. Lys48 and Val159 formed steric hindrance, which impedes to bond with galactose branches. In addition, the various binding modes of RmMan134A-M5 indicated that subsites -2 to +2 are indispensable during the hydrolytic process. The structure of RmMan134A-M4 showed that mannotetrose only binds at subsites +1 to +4, and RmMan134A could therefore not hydrolyze mannan oligosaccharides with degree of polymerization ≤4. Through rational design, the specific activity and optimal conditions of RmMan134A were significantly improved. The purpose of this paper is to investigate the structure and function of fungal GH family 134 β-1,4-mannanases, and substrate-binding mechanism of GH family 134 members. Copyright © 2018 Elsevier B.V. All rights reserved.

Genome wide association identifies common variants at the SERPINA6/SERPINA1 locus influencing plasma cortisol and corticosteroid binding globulin.

PubMed

Bolton, Jennifer L; Hayward, Caroline; Direk, Nese; Lewis, John G; Hammond, Geoffrey L; Hill, Lesley A; Anderson, Anna; Huffman, Jennifer; Wilson, James F; Campbell, Harry; Rudan, Igor; Wright, Alan; Hastie, Nicholas; Wild, Sarah H; Velders, Fleur P; Hofman, Albert; Uitterlinden, Andre G; Lahti, Jari; Räikkönen, Katri; Kajantie, Eero; Widen, Elisabeth; Palotie, Aarno; Eriksson, Johan G; Kaakinen, Marika; Järvelin, Marjo-Riitta; Timpson, Nicholas J; Davey Smith, George; Ring, Susan M; Evans, David M; St Pourcain, Beate; Tanaka, Toshiko; Milaneschi, Yuri; Bandinelli, Stefania; Ferrucci, Luigi; van der Harst, Pim; Rosmalen, Judith G M; Bakker, Stephen J L; Verweij, Niek; Dullaart, Robin P F; Mahajan, Anubha; Lindgren, Cecilia M; Morris, Andrew; Lind, Lars; Ingelsson, Erik; Anderson, Laura N; Pennell, Craig E; Lye, Stephen J; Matthews, Stephen G; Eriksson, Joel; Mellstrom, Dan; Ohlsson, Claes; Price, Jackie F; Strachan, Mark W J; Reynolds, Rebecca M; Tiemeier, Henning; Walker, Brian R

2014-07-01

Variation in plasma levels of cortisol, an essential hormone in the stress response, is associated in population-based studies with cardio-metabolic, inflammatory and neuro-cognitive traits and diseases. Heritability of plasma cortisol is estimated at 30-60% but no common genetic contribution has been identified. The CORtisol NETwork (CORNET) consortium undertook genome wide association meta-analysis for plasma cortisol in 12,597 Caucasian participants, replicated in 2,795 participants. The results indicate that <1% of variance in plasma cortisol is accounted for by genetic variation in a single region of chromosome 14. This locus spans SERPINA6, encoding corticosteroid binding globulin (CBG, the major cortisol-binding protein in plasma), and SERPINA1, encoding α1-antitrypsin (which inhibits cleavage of the reactive centre loop that releases cortisol from CBG). Three partially independent signals were identified within the region, represented by common SNPs; detailed biochemical investigation in a nested sub-cohort showed all these SNPs were associated with variation in total cortisol binding activity in plasma, but some variants influenced total CBG concentrations while the top hit (rs12589136) influenced the immunoreactivity of the reactive centre loop of CBG. Exome chip and 1000 Genomes imputation analysis of this locus in the CROATIA-Korcula cohort identified missense mutations in SERPINA6 and SERPINA1 that did not account for the effects of common variants. These findings reveal a novel common genetic source of variation in binding of cortisol by CBG, and reinforce the key role of CBG in determining plasma cortisol levels. In turn this genetic variation may contribute to cortisol-associated degenerative diseases.

SPM analysis of parametric (R)-[11C]PK11195 binding images: plasma input versus reference tissue parametric methods.

PubMed

Schuitemaker, Alie; van Berckel, Bart N M; Kropholler, Marc A; Veltman, Dick J; Scheltens, Philip; Jonker, Cees; Lammertsma, Adriaan A; Boellaard, Ronald

2007-05-01

(R)-[11C]PK11195 has been used for quantifying cerebral microglial activation in vivo. In previous studies, both plasma input and reference tissue methods have been used, usually in combination with a region of interest (ROI) approach. Definition of ROIs, however, can be labourious and prone to interobserver variation. In addition, results are only obtained for predefined areas and (unexpected) signals in undefined areas may be missed. On the other hand, standard pharmacokinetic models are too sensitive to noise to calculate (R)-[11C]PK11195 binding on a voxel-by-voxel basis. Linearised versions of both plasma input and reference tissue models have been described, and these are more suitable for parametric imaging. The purpose of this study was to compare the performance of these plasma input and reference tissue parametric methods on the outcome of statistical parametric mapping (SPM) analysis of (R)-[11C]PK11195 binding. Dynamic (R)-[11C]PK11195 PET scans with arterial blood sampling were performed in 7 younger and 11 elderly healthy subjects. Parametric images of volume of distribution (Vd) and binding potential (BP) were generated using linearised versions of plasma input (Logan) and reference tissue (Reference Parametric Mapping) models. Images were compared at the group level using SPM with a two-sample t-test per voxel, both with and without proportional scaling. Parametric BP images without scaling provided the most sensitive framework for determining differences in (R)-[11C]PK11195 binding between younger and elderly subjects. Vd images could only demonstrate differences in (R)-[11C]PK11195 binding when analysed with proportional scaling due to intersubject variation in K1/k2 (blood-brain barrier transport and non-specific binding).

Binding of Thyrotropin-Releasing Hormone to Plasma Membranes of Bovine Anterior Pituitary Gland

PubMed Central

Labrie, Fernand; Barden, Nicholas; Poirier, Guy; De Lean, Andre

1972-01-01

An assay for the binding of [3H]thyrotropin-releasing hormone ([3H]TRH) is described. Plasma membranes isolated from bovine anterior pituitary gland bind about 600 femtomoles of this hormone per mg of protein, as compared to 15 femtomoles per mg of protein in the total adenohypophyseal homogenate (40-fold purification). The equilibrium constant of membrane receptor-[3H]TRH binding at 0°C is 4.3 × 107 L·M-1, or a half-maximal binding of this hormone at 23 nM. The binding is time-dependent; addition of unlabeled hormone induces dissociation of the receptor-[3H]TRH complex with a half-life of 14 min. The binding of TRH is not altered by 10 μM melanocyte-stimulating hormone-release inhibiting hormone, lysine-vasopressin, adrenocorticotropin, growth hormone, prolactin, luteinizing hormone, insulin, glucagon, L-thyroxine, or L-triiodothyronine. K+ and Mg++ increase formation of the receptor-TRH complex at optimal concentrations of 5-25 mM and 0.5-2.5 mM, respectively, with inhibition at higher concentrations. Ca++ inhibits binding of TRH at all concentrations tested. PMID:4621548

Plasma brain-derived neurotrophic factor in women after bariatric surgery: a pilot study.

PubMed

Merhi, Zaher O; Minkoff, Howard; Lambert-Messerlian, Geralyn M; Macura, Jerzy; Feldman, Joseph; Seifer, David B

2009-04-01

Eighteen morbidly obese women had plasma brain-derived neurotrophic factor (BDNF) measured before bariatric surgery and 3 months postoperatively. We analyzed plasma BDNF levels in all the participants then subdivided according to menopausal status and type of surgery. Brain-derived neurotrophic factor decreased significantly in all the participants and in the premenopausal group when looked at in isolation.

Isolation and characterization of a new zinc-binding protein from albacore tuna plasma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dyke, B.; Hegenauer, J.; Saltman, P.

1987-06-02

The protein responsible for sequestering high levels of zinc in the plasma of the albacore tuna (Thunnus alalunga) has been isolated by sequential chromatography. The glycoprotein has a molecular weight of 66,000. Approximately 8.2% of its amino acid residues are histidines. Equilibrium dialysis experiments show it to bind 3 mol of zinc/mol of protein. The stoichiometric constant for the association of zinc with a binding site containing three histidines was determined to be 10/sup 9.4/. This protein is different from albumin and represents a previously uncharacterized zinc transport protein.

Changes in the Expression and Distribution of Claudins, Increased Epithelial Apoptosis, and a Mannan-Binding Lectin-Associated Immune Response Lead to Barrier Dysfunction in Dextran Sodium Sulfate-Induced Rat Colitis

PubMed Central

Yuan, Bosi; Zhou, Shuping; Lu, Youke; Liu, Jiong; Jin, Xinxin; Wan, Haijun; Wang, Fangyu

2015-01-01

Background/Aims This animal study aimed to define the underlying cellular mechanisms of intestinal barrier dysfunction. Methods Rats were fed 4% with dextran sodium sulfate (DSS) to induce experimental colitis. We analyzed the sugars in 24-hour urine output by high pressure liquid chromatography. The expression of claudins, mannan-binding lectin (MBL), and MBL-associated serine proteases 2 (MASP-2) were detected in the colonic mucosa by immunohistochemistry; and apoptotic cells in the colonic epithelium were detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method assay. Results The lactulose and sucralose excretion levels in the urine of rats with DSS-induced colitis were significantly higher than those in the control rats. Mannitol excretion was lower and lactulose/mannitol ratios and sucralose/mannitol ratios were significantly increased compared with those in the control group (p<0.05). Compared with the controls, the expression of sealing claudins (claudin 3, claudin 5, and claudin 8) was significantly decreased, but that of claudin 1 was increased. The expression of pore-forming claudin 2 was upregulated and claudin 7 was downregulated in DSS-induced colitis. The epithelial apoptotic ratio was 2.8%±1.2% in controls and was significantly increased to 7.2%±1.2% in DSS-induced colitis. The expression of MBL and MASP-2 in the intestinal mucosa showed intense staining in controls, whereas there was weak staining in the rats with colitis. Conclusions There was increased intestinal permeability in DSS-induced colitis. Changes in the expression and distribution of claudins, increased epithelial apoptosis, and the MASP-2-induced immune response impaired the intestinal epithelium and contributed to high intestinal permeability. PMID:25717051

Changes in the Expression and Distribution of Claudins, Increased Epithelial Apoptosis, and a Mannan-Binding Lectin-Associated Immune Response Lead to Barrier Dysfunction in Dextran Sodium Sulfate-Induced Rat Colitis.

PubMed

Yuan, Bosi; Zhou, Shuping; Lu, Youke; Liu, Jiong; Jin, Xinxin; Wan, Haijun; Wang, Fangyu

2015-11-23

This animal study aimed to define the underlying cellular mechanisms of intestinal barrier dysfunction. Rats were fed 4% with dextran sodium sulfate (DSS) to induce experimental colitis. We analyzed the sugars in 24-hour urine output by high pressure liquid chromatography. The expression of claudins, mannan-binding lectin (MBL), and MBL-associated serine proteases 2 (MASP-2) were detected in the colonic mucosa by immunohistochemistry; and apoptotic cells in the colonic epithelium were detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method assay. The lactulose and sucralose excretion levels in the urine of rats with DSS-induced colitis were significantly higher than those in the control rats. Mannitol excretion was lower and lactulose/mannitol ratios and sucralose/mannitol ratios were significantly increased compared with those in the control group (p<0.05). Compared with the controls, the expression of sealing claudins (claudin 3, claudin 5, and claudin 8) was significantly decreased, but that of claudin 1 was increased. The expression of pore-forming claudin 2 was upregulated and claudin 7 was downregulated in DSS-induced colitis. The epithelial apoptotic ratio was 2.8%±1.2% in controls and was significantly increased to 7.2%±1.2% in DSS-induced colitis. The expression of MBL and MASP-2 in the intestinal mucosa showed intense staining in controls, whereas there was weak staining in the rats with colitis. There was increased intestinal permeability in DSS-induced colitis. Changes in the expression and distribution of claudins, increased epithelial apoptosis, and the MASP-2-induced immune response impaired the intestinal epithelium and contributed to high intestinal permeability.

Comparative Toxicokinetics and Plasma Protein Binding of Ochratoxin A in Four Avian Species.

PubMed

Devreese, Mathias; Croubels, Siska; De Baere, Siegrid; Gehring, Ronette; Antonissen, Gunther

2018-03-07

Ochratoxin A (OTA, 0.25 mg/kg body weight) was absorbed rapidly ( T max = 0.31-1.88 h) in all avian species (broiler chickens, laying hens, turkeys, and Muscovy ducks) but more slowly in broiler chickens ( T max = 1.43-4.63 h). The absolute oral bioavailability was complete in these bird species (88.0-109.6%). Ducks have a significantly higher volume of distribution ( V d ) and turkeys a lower V d compared to chickens and layers (broiler chickens, 0.27 ± 0.12 L/kg; layers, 0.23 ± 0.08 L/kg; turkeys, 0.18 ± 0.04 L/kg; ducks, 0.76 ± 0.44 L/kg). This difference in V d can be attributed to the species-dependent differences in plasma protein binding of OTA, namely ranging between 82.2 and 88.9% in ducks and between 96.5 and 98.8% in turkeys. No significant gender differences were found in toxicokinetics or plasma protein binding.

Prostate Secretory Protein of 94 Amino Acids (PSP94) Binds to Prostatic Acid Phosphatase (PAP) in Human Seminal Plasma

PubMed Central

Anklesaria, Jenifer H.; Jagtap, Dhanashree D.; Pathak, Bhakti R.; Kadam, Kaushiki M.; Joseph, Shaini; Mahale, Smita D.

2013-01-01

Prostate Secretory Protein of 94 amino acids (PSP94) is one of the major proteins present in the human seminal plasma. Though several functions have been predicted for this protein, its exact role either in sperm function or in prostate pathophysiology has not been clearly defined. Attempts to understand the mechanism of action of PSP94 has led to the search for its probable binding partners. This has resulted in the identification of PSP94 binding proteins in plasma and seminal plasma from human. During the chromatographic separation step of proteins from human seminal plasma by reversed phase HPLC, we had observed that in addition to the main fraction of PSP94, other fractions containing higher molecular weight proteins also showed the presence of detectable amounts of PSP94. This prompted us to hypothesize that PSP94 could be present in the seminal plasma complexed with other protein/s of higher molecular weight. One such fraction containing a major protein of ∼47 kDa, on characterization by mass spectrometric analysis, was identified to be Prostatic Acid Phosphatase (PAP). The ability of PAP present in this fraction to bind to PSP94 was demonstrated by affinity chromatography. Co-immunoprecipitation experiments confirmed the presence of PSP94-PAP complex both in the fraction studied and in the fresh seminal plasma. In silico molecular modeling of the PSP94-PAP complex suggests that β-strands 1 and 6 of PSP94 appear to interact with domain 2 of PAP, while β-strands 7 and 10 with domain 1 of PAP. This is the first report which suggests that PSP94 can bind to PAP and the PAP-bound PSP94 is present in human seminal plasma. PMID:23469287

Food proteins and maturation of small intestinal microvillus membranes (MVM). II. Binding of gliadin hydrolysate fractions and of the gliadin peptide B3142.

PubMed

Stern, M; Gellermann, B; Belitz, H D; Wieser, H

1988-01-01

To investigate in vitro interactions between gliadin peptide fractions that have been shown to be toxic to celiac small intestinal mucosa in humans and small intestinal microvillus membranes (MVM) from rats during postnatal maturation, MVM were prepared from newborn, 18-day-old preweanling, and adult rats. Partially hydrolyzed gliadin peptide fractions B1-B4, and the pure gliadin peptide B3142 were radioiodinated and used for binding assays. Miniature ultracentrifugation was used for separation of unbound material. Binding of gliadin fractions to MVM was weak and nonspecific in terms of lacking saturation and inhibition. There was no inhibition of binding by mannan. Enzyme pretreatment of MVM (trypsin, neuraminidase, phospholipase C) did not result in any significant change of binding. Compared with peptides prepared from bovine serum albumin as a control, there was no significant difference in binding of gliadin peptide fractions to MVM. Thus, a lectin-like effect of gliadin peptides toward MVM, or the existence of a specific intestinal surface receptor for gliadin peptides appeared improbable. There were, however, consistent maturational changes in MVM binding in that newborn MVM bound more B1-B4 and B3142 compared with adult controls (p less than 0.001). Nonspecific binding of gliadin fractions to MVM might be related to the initiation of nonspecific in vitro effects of gliadin, particularly toward the immature small intestine. The MVM binding model in the rat clearly does not provide a system for studying celiac disease pathogenesis, but it might help clarify basic processes in the interaction between food-derived substances and elements of the gastrointestinal mucosal barrier.

Genome Wide Association Identifies Common Variants at the SERPINA6/SERPINA1 Locus Influencing Plasma Cortisol and Corticosteroid Binding Globulin

PubMed Central

Direk, Nese; Lewis, John G.; Hammond, Geoffrey L.; Hill, Lesley A.; Anderson, Anna; Huffman, Jennifer; Wilson, James F.; Campbell, Harry; Rudan, Igor; Wright, Alan; Hastie, Nicholas; Wild, Sarah H.; Velders, Fleur P.; Hofman, Albert; Uitterlinden, Andre G.; Lahti, Jari; Räikkönen, Katri; Kajantie, Eero; Widen, Elisabeth; Palotie, Aarno; Eriksson, Johan G.; Kaakinen, Marika; Järvelin, Marjo-Riitta; Timpson, Nicholas J.; Davey Smith, George; Ring, Susan M.; Evans, David M.; St Pourcain, Beate; Tanaka, Toshiko; Milaneschi, Yuri; Bandinelli, Stefania; Ferrucci, Luigi; van der Harst, Pim; Rosmalen, Judith G. M.; Bakker, Stephen J. L.; Verweij, Niek; Dullaart, Robin P. F.; Mahajan, Anubha; Lindgren, Cecilia M.; Morris, Andrew; Lind, Lars; Ingelsson, Erik; Anderson, Laura N.; Pennell, Craig E.; Lye, Stephen J.; Matthews, Stephen G.; Eriksson, Joel; Mellstrom, Dan; Ohlsson, Claes; Price, Jackie F.; Strachan, Mark W. J.; Reynolds, Rebecca M.; Tiemeier, Henning; Walker, Brian R.

2014-01-01

Variation in plasma levels of cortisol, an essential hormone in the stress response, is associated in population-based studies with cardio-metabolic, inflammatory and neuro-cognitive traits and diseases. Heritability of plasma cortisol is estimated at 30–60% but no common genetic contribution has been identified. The CORtisol NETwork (CORNET) consortium undertook genome wide association meta-analysis for plasma cortisol in 12,597 Caucasian participants, replicated in 2,795 participants. The results indicate that <1% of variance in plasma cortisol is accounted for by genetic variation in a single region of chromosome 14. This locus spans SERPINA6, encoding corticosteroid binding globulin (CBG, the major cortisol-binding protein in plasma), and SERPINA1, encoding α1-antitrypsin (which inhibits cleavage of the reactive centre loop that releases cortisol from CBG). Three partially independent signals were identified within the region, represented by common SNPs; detailed biochemical investigation in a nested sub-cohort showed all these SNPs were associated with variation in total cortisol binding activity in plasma, but some variants influenced total CBG concentrations while the top hit (rs12589136) influenced the immunoreactivity of the reactive centre loop of CBG. Exome chip and 1000 Genomes imputation analysis of this locus in the CROATIA-Korcula cohort identified missense mutations in SERPINA6 and SERPINA1 that did not account for the effects of common variants. These findings reveal a novel common genetic source of variation in binding of cortisol by CBG, and reinforce the key role of CBG in determining plasma cortisol levels. In turn this genetic variation may contribute to cortisol-associated degenerative diseases. PMID:25010111

Synthesis, characterization and serum albumin binding studies of vitamin K3 derivatives.

PubMed

Suganthi, Murugesan; Elango, Kuppanagounder P

2017-01-01

Synthesis, characterization and bovine serum albumin (BSA) binding properties of three derivatives of vitamin K3 have been described. Results of UV-Vis and fluorescence spectra indicate complexation between BSA and the ligands with conformational changes in protein, which is strongly supported by synchronous and three dimensional fluorescence studies. Addition of the ligands quenches the fluorescence of BSA which is accompanied by reduction in quantum yield (Ф) from 0.1010 to 0.0775-0.0986 range. Thermodynamic investigations reveal that hydrophobic interaction is the major binding force in the spontaneous binding of these ligands with BSA. The binding constants obtained depend on the substituent present in the quinone ring, which correlates linearly with the Taft's field substituent constant (σ F ). The results show that compound with strong electron withdrawing nitro-group forms relatively stronger complex with BSA than amino and thioglycolate substituted ones. Circular dichroism studies show that the α-helical content of the protein, upon complexation with the ligands, decreases in the case of amino and nitro substituted vitamin K3 while increases in thioglycolate substituted compound. Molecular docking studies indicated that the vitamin K3 derivatives are surrounded by hydrophobic residues of the BSA molecule, which is in good agreement with the results of fluorescence spectral and thermodynamic studies. Copyright © 2016 Elsevier B.V. All rights reserved.

Probing the Allosteric Modulator Binding Site of GluR2 with Thiazide Derivatives

PubMed Central

Ptak, Christopher P.; Ahmed, Ahmed H.; Oswald, Robert E.

2009-01-01

Ionotropic glutamate receptors mediate the majority of vertebrate excitatory synaptic transmission and are therapeutic targets for cognitive enhancement and treatment of schizophrenia. The binding domains of these tetrameric receptors consist of two dimers, and the dissociation of the dimer interface of the ligand-binding domain leads to desensitization in the continued presence of agonist. Positive allosteric modulators act by strengthening the dimer interface and reducing desensitization, thereby increasing steady-state activation. Removing the desensitized state for simplified analysis of receptor activation is commonly achieved using cyclothiazide (CTZ), the most potent modulator of the benzothiadiazide class, with the flip form of the AMPA receptor subtype. IDRA-21, the first benzothiadiazide to have an effect in behavioral tests, is an important lead compound in clinical trials for cognitive enhancement as it can cross the blood-brain barrier. Intermediate structures between CTZ and IDRA-21 show reduced potency suggesting that these two compounds have different contact points associated with binding. To understand how benzothiadiazides bind to the pocket bridging the dimer interface, we generated a series of crystal structures of the GluR2 ligand-binding domain complexed with benzothiadiazide derivatives (IDRA-21, hydroflumethiazide, hydrochlorothiazide, chlorothiazide, trichlormethiazide, and althiazide) for comparison with an existing structure for cyclothiazide. The structures detail how changes in the substituents in the 3- and 7-positions of the hydrobenzothiadiazide ring shift the orientation of the drug in the binding site and, in some cases, change the stoichiometry of binding. All derivatives maintain a hydrogen bond with the Ser754 hydroxyl, affirming the partial selectivity of the benzothiadiazides for the flip form of AMPA receptors. PMID:19673491

Rational design of biaryl pharmacophore inserted noscapine derivatives as potent tubulin binding anticancer agents

NASA Astrophysics Data System (ADS)

Santoshi, Seneha; Manchukonda, Naresh Kumar; Suri, Charu; Sharma, Manya; Sridhar, Balasubramanian; Joseph, Silja; Lopus, Manu; Kantevari, Srinivas; Baitharu, Iswar; Naik, Pradeep Kumar

2015-03-01

We have strategically designed a series of noscapine derivatives by inserting biaryl pharmacophore (a major structural constituent of many of the microtubule-targeting natural anticancer compounds) onto the scaffold structure of noscapine. Molecular interaction of these derivatives with α,β-tubulin heterodimer was investigated by molecular docking, molecular dynamics simulation, and binding free energy calculation. The predictive binding affinity indicates that the newly designed noscapinoids bind to tubulin with a greater affinity. The predictive binding free energy (ΔGbind, pred) of these derivatives (ranging from -5.568 to -5.970 kcal/mol) based on linear interaction energy (LIE) method with a surface generalized Born (SGB) continuum solvation model showed improved binding affinity with tubulin compared to the lead compound, natural α-noscapine (-5.505 kcal/mol). Guided by the computational findings, these new biaryl type α-noscapine congeners were synthesized from 9-bromo-α-noscapine using optimized Suzuki reaction conditions for further experimental evaluation. The derivatives showed improved inhibition of the proliferation of human breast cancer cells (MCF-7), human cervical cancer cells (HeLa) and human lung adenocarcinoma cells (A549), compared to natural noscapine. The cell cycle analysis in MCF-7 further revealed that these compounds alter the cell cycle profile and cause mitotic arrest at G2/M phase more strongly than noscapine. Tubulin binding assay revealed higher binding affinity to tubulin, as suggested by dissociation constant (Kd) of 126 ± 5.0 µM for 5a, 107 ± 5.0 µM for 5c, 70 ± 4.0 µM for 5d, and 68 ± 6.0 µM for 5e compared to noscapine (Kd of 152 ± 1.0 µM). In fact, the experimentally determined value of ΔGbind, expt (calculated from the Kd value) are consistent with the predicted value of ΔGbind, pred calculated based on LIE-SGB. Based on these results, one of the derivative 5e of this series was used for further toxicological

Binding of plasma proteins to titanium dioxide nanotubes with different diameters

PubMed Central

Kulkarni, Mukta; Flašker, Ajda; Lokar, Maruša; Mrak-Poljšak, Katjuša; Mazare, Anca; Artenjak, Andrej; Čučnik, Saša; Kralj, Slavko; Velikonja, Aljaž; Schmuki, Patrik; Kralj-Iglič, Veronika; Sodin-Semrl, Snezna; Iglič, Aleš

2015-01-01

Titanium and titanium alloys are considered to be one of the most applicable materials in medical devices because of their suitable properties, most importantly high corrosion resistance and the specific combination of strength with biocompatibility. In order to improve the biocompatibility of titanium surfaces, the current report initially focuses on specifying the topography of titanium dioxide (TiO2) nanotubes (NTs) by electrochemical anodization. The zeta potential (ζ-potential) of NTs showed a negative value and confirmed the agreement between the measured and theoretically predicted dependence of ζ-potential on salt concentration, whereby the absolute value of ζ-potential diminished with increasing salt concentrations. We investigated binding of various plasma proteins with different sizes and charges using the bicinchoninic acid assay and immunofluorescence microscopy. Results showed effective and comparatively higher protein binding to NTs with 100 nm diameters (compared to 50 or 15 nm). We also showed a dose-dependent effect of serum amyloid A protein binding to NTs. These results and theoretical calculations of total available surface area for binding of proteins indicate that the largest surface area (also considering the NT lengths) is available for 100 nm NTs, with decreasing surface area for 50 and 15 nm NTs. These current investigations will have an impact on increasing the binding ability of biomedical devices in the body leading to increased durability of biomedical devices. PMID:25733829

Prediction of the binding mode of N2-phenylguanine derivative inhibitors to herpes simplex virus type 1 thymidine kinase

NASA Astrophysics Data System (ADS)

Gaudio, Anderson Coser; Takahata, Yuji; Richards, William Graham

1998-01-01

The probable binding mode of the herpes simplex virus thymidine kinase (HSV1 TK) N2-[substituted]-phenylguanine inhibitors is proposed. A computational experiment was designed to check some qualitative binding parameters and to calculate the interaction binding energies of alternative binding modes of N2-phenylguanines. The known binding modes of the HSV1 TK natural substrate deoxythymidine and one of its competitive inhibitors ganciclovir were used as templates. Both the qualitative and quantitative parts of the computational experiment indicated that the N2-phenylguanine derivatives bind to the HSV1 TK active site in the deoxythymidine-like binding mode. An experimental observation that N2-phenylguanosine derivatives are not phosphorylated during the interaction with the HSV1 TK gives support to the proposed binding mode.

Informing the Human Plasma Protein Binding of ...

EPA Pesticide Factsheets

The free fraction of a xenobiotic in plasma (Fub) is an important determinant of chemical adsorption, distribution, metabolism, elimination, and toxicity, yet experimental plasma protein binding data is scarce for environmentally relevant chemicals. The presented work explores the merit of utilizing available pharmaceutical data to predict Fub for environmentally relevant chemicals via machine learning techniques. Quantitative structure-activity relationship (QSAR) models were constructed with k nearest neighbors (kNN), support vector machines (SVM), and random forest (RF) machine learning algorithms from a training set of 1045 pharmaceuticals. The models were then evaluated with independent test sets of pharmaceuticals (200 compounds) and environmentally relevant ToxCast chemicals (406 total, in two groups of 238 and 168 compounds). The selection of a minimal feature set of 10-15 2D molecular descriptors allowed for both informative feature interpretation and practical applicability domain assessment via a bounded box of descriptor ranges and principal component analysis. The diverse pharmaceutical and environmental chemical sets exhibit similarities in terms of chemical space (99-82% overlap), as well as comparable bias and variance in constructed learning curves. All the models exhibit significant predictability with mean absolute errors (MAE) in the range of 0.10-0.18 Fub. The models performed best for highly bound chemicals (MAE 0.07-0.12), neutrals (MAE 0

Inactivation of Zika virus by solvent/detergent treatment of human plasma and other plasma-derived products and pasteurization of human serum albumin.

PubMed

Kühnel, Denis; Müller, Sebastian; Pichotta, Alexander; Radomski, Kai Uwe; Volk, Andreas; Schmidt, Torben

2017-03-01

In 2016 the World Health Organization declared the mosquito-borne Zika virus (ZIKV) a "public health emergency of international concern." ZIKV is a blood-borne pathogen, which therefore causes concerns regarding the safety of human plasma-derived products due to potential contamination of the blood supply. This study investigated the effectiveness of viral inactivation steps used during the routine manufacturing of various plasma-derived products to reduce ZIKV infectivity. Human plasma and intermediates from the production of various plasma-derived products were spiked with ZIKV and subjected to virus inactivation using the identical techniques (either solvent/detergent [S/D] treatment or pasteurization) and conditions used for the actual production of the respective products. Samples were taken and the viral loads measured before and after inactivation. After S/D treatment of spiked intermediates of the plasma-derived products Octaplas(LG), Octagam, and Octanate, the viral loads were below the limit of detection in all cases. The mean log reduction factor (LRF) was at least 6.78 log for Octaplas(LG), at least 7.00 log for Octagam, and at least 6.18 log for Octanate after 60, 240, and 480 minutes of S/D treatment, respectively. For 25% human serum albumin (HSA), the mean LRF for ZIKV was at least 7.48 log after pasteurization at 60°C for 120 minutes. These results demonstrate that the commonly used virus inactivation processes utilized during the production of human plasma and plasma-derived products, namely, S/D treatment or pasteurization, are effective for inactivation of ZIKV. © 2016 The Authors Transfusion published by Wiley Periodicals, Inc. on behalf of AABB.

Interaction of gold and silver nanoparticles with human plasma: Analysis of protein corona reveals specific binding patterns.

PubMed

Lai, Wenjia; Wang, Qingsong; Li, Lumeng; Hu, Zhiyuan; Chen, Jiankui; Fang, Qiaojun

2017-04-01

Determining how nanomaterials interact with plasma will assist in understanding their effects on the biological system. This work presents a systematic study of the protein corona formed from human plasma on 20nm silver and gold nanoparticles with three different surface modifications, including positive and negative surface charges. The results show that all nanoparticles, even those with positive surface modifications, acquire negative charges after interacting with plasma. Approximately 300 proteins are identified on the coronas, while 99 are commonly found on each nanomaterial. The 20 most abundant proteins account for over 80% of the total proteins abundance. Remarkably, the surface charge and core of the nanoparticles, as well as the isoelectric point of the plasma proteins, are found to play significant roles in determining the nanoparticle coronas. Albumin and globulins are present at levels of less than 2% on these nanoparticle coronas. Fibrinogen, which presents in the plasma but not in the serum, preferably binds to negatively charged gold nanoparticles. These observations demonstrate the specific plasma protein binding pattern of silver and gold nanoparticles, as well as the importance of the surface charge and core in determining the protein corona compositions. The potential downstream biological impacts of the corona proteins were also investigated. Copyright © 2017 Elsevier B.V. All rights reserved.

Estimation of the binding ability of main transport proteins of blood plasma with liver cirrhosis by the fluorescent probe method

NASA Astrophysics Data System (ADS)

Korolenko, E. A.; Korolik, E. V.; Korolik, A. K.; Kirkovskii, V. V.

2007-07-01

We present results from an investigation of the binding ability of the main transport proteins (albumin, lipoproteins, and α-1-acid glycoprotein) of blood plasma from patients at different stages of liver cirrhosis by the fluorescent probe method. We used the hydrophobic fluorescent probes anionic 8-anilinonaphthalene-1-sulfonate, which interacts in blood plasma mainly with albumin; cationic Quinaldine red, which interacts with α-1-acid glycoprotein; and neutral Nile red, which redistributes between lipoproteins and albumin in whole blood plasma. We show that the binding ability of albumin and α-1-acid glycoprotein to negatively charged and positively charged hydrophobic metabolites, respectively, increases in the compensation stage of liver cirrhosis. As the pathology process deepens and transitions into the decompensation stage, the transport abilities of albumin and α-1-acid glycoprotein decrease whereas the binding ability of lipoproteins remains high.

Effects of MASP-1 of the Complement System on Activation of Coagulation Factors and Plasma Clot Formation

PubMed Central

Hess, Katharina; Ajjan, Ramzi; Phoenix, Fladia; Dobó, József; Gál, Péter; Schroeder, Verena

2012-01-01

Background Numerous interactions between the coagulation and complement systems have been shown. Recently, links between coagulation and mannan-binding lectin-associated serine protease-1 (MASP-1) of the complement lectin pathway have been proposed. Our aim was to investigate MASP-1 activation of factor XIII (FXIII), fibrinogen, prothrombin, and thrombin-activatable fibrinolysis inhibitor (TAFI) in plasma-based systems, and to analyse effects of MASP-1 on plasma clot formation, structure and lysis. Methodology/Principal Findings We used a FXIII incorporation assay and specific assays to measure the activation products prothrombin fragment F1+2, fibrinopeptide A (FPA), and activated TAFI (TAFIa). Clot formation and lysis were assessed by turbidimetric assay. Clot structure was studied by scanning electron microscopy. MASP-1 activated FXIII and, contrary to thrombin, induced FXIII activity faster in the Val34 than the Leu34 variant. MASP-1-dependent generation of F1+2, FPA and TAFIa showed a dose-dependent response in normal citrated plasma (NCP), albeit MASP-1 was much less efficient than FXa or thrombin. MASP-1 activation of prothrombin and TAFI cleavage were confirmed in purified systems. No FPA generation was observed in prothrombin-depleted plasma. MASP-1 induced clot formation in NCP, affected clot structure, and prolonged clot lysis. Conclusions/Significance We show that MASP-1 interacts with plasma clot formation on different levels and influences fibrin structure. Although MASP-1-induced fibrin formation is thrombin-dependent, MASP-1 directly activates prothrombin, FXIII and TAFI. We suggest that MASP-1, in concerted action with other complement and coagulation proteins, may play a role in fibrin clot formation. PMID:22536427

Components in Plasma-Derived Factor VIII, But Not in Recombinant Factor VIII Downregulate Anti-Inflammatory Surface Marker CD163 in Human Macrophages through Release of CXCL4 (Platelet Factor 4)

PubMed Central

Bertling, Anne; Brodde, Martin F.; Visser, Mayken; Treffon, Janina; Fennen, Michelle; Fender, Anke C.; Kelsch, Reinhard; Kehrel, Beate E.

2017-01-01

Background Hemarthrosis, or bleeding into the joints, is a hallmark of hemophilia. Heme triggers oxidative stress, inflammation, and destruction of cartilage and bone. The haptoglobin-CD163-heme oxygenase-1 (HO-1) pathway circumvents heme toxicity through enzymatic degradation of heme and transcription of antioxidant genes. Plasma-derived factor concentrates contain many proteins that might impact on cellular pathways in joints, blood, and vessels. Methods Activation of platelets from healthy volunteers was assessed by flow cytometry analysis of fibrinogen binding and CD62P expression. Platelet CXCL4 release was measured by ELISA. Human peripheral blood mononuclear cells were exposed to CXCL4 or platelet supernatants (untreated or pre-stimulated with factor VIII (FVIII) products) during their differentiation to macrophages and analyzed for CD163 expression. Some macrophage cultures were additionally incubated with autologous hemoglobin for 18 h for analysis of HO-1 expression. Results Platelet CXCL4 release was increased by all 8 tested plasma-derived FVIII products but not the 3 recombinant products. Macrophages exposed to supernatant from platelets treated with some plasma-derived FVIII products downregulated CD163 surface expression and failed to upregulate the athero- and joint protective enzyme HO-1 in response to hemoglobin. Conclusion Plasma-derived FVIII products might promote bleeding-induced joint injury via generation of macrophages that are unable to counteract redox stress. PMID:29070980

Components in Plasma-Derived Factor VIII, But Not in Recombinant Factor VIII Downregulate Anti-Inflammatory Surface Marker CD163 in Human Macrophages through Release of CXCL4 (Platelet Factor 4).

PubMed

Bertling, Anne; Brodde, Martin F; Visser, Mayken; Treffon, Janina; Fennen, Michelle; Fender, Anke C; Kelsch, Reinhard; Kehrel, Beate E

2017-09-01

Hemarthrosis, or bleeding into the joints, is a hallmark of hemophilia. Heme triggers oxidative stress, inflammation, and destruction of cartilage and bone. The haptoglobin-CD163-heme oxygenase-1 (HO-1) pathway circumvents heme toxicity through enzymatic degradation of heme and transcription of antioxidant genes. Plasma-derived factor concentrates contain many proteins that might impact on cellular pathways in joints, blood, and vessels. Activation of platelets from healthy volunteers was assessed by flow cytometry analysis of fibrinogen binding and CD62P expression. Platelet CXCL4 release was measured by ELISA. Human peripheral blood mononuclear cells were exposed to CXCL4 or platelet supernatants (untreated or pre-stimulated with factor VIII (FVIII) products) during their differentiation to macrophages and analyzed for CD163 expression. Some macrophage cultures were additionally incubated with autologous hemoglobin for 18 h for analysis of HO-1 expression. Platelet CXCL4 release was increased by all 8 tested plasma-derived FVIII products but not the 3 recombinant products. Macrophages exposed to supernatant from platelets treated with some plasma-derived FVIII products downregulated CD163 surface expression and failed to upregulate the athero- and joint protective enzyme HO-1 in response to hemoglobin. Plasma-derived FVIII products might promote bleeding-induced joint injury via generation of macrophages that are unable to counteract redox stress.

Characterization of auxin-binding proteins from zucchini plasma membrane

NASA Technical Reports Server (NTRS)

Hicks, G. R.; Rice, M. S.; Lomax, T. L.

1993-01-01

We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may

Characterization of auxin-binding proteins from zucchini plasma membrane.

PubMed

Hicks, G R; Rice, M S; Lomax, T L

1993-01-01

We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may

Determination of nuvenzepine in human plasma by a sensitive sup 3 Hpirenzepine radioreceptor binding assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Caselli, G.; Ferrari, M.P.; Tonon, G.

1991-02-01

A sensitive method for the quantitation of small amounts of nuvenzepine, a new M1-selective antimuscarinic drug, in plasma is described. The analytical method involves the use of a radioreceptor binding assay based on {sup 3}Hpirenzepine displacement in rat cerebral cortex homogenates; no previous extraction is required. The method is reliable, with an interassay CV ranging from 5 to 10%, and allows the analysis of greater than 100 samples/experiment. The limit of detection is {approximately} 0.1 ng/assay. Using this method we have determined the plasma levels of nuvenzepine in eight healthy volunteers treated PO with 15 or 25 mg of nuvenzepine.HCl.more » The pharmacokinetic parameters obtained were (for 15 and 25 mg): Cmax, 64 and 131 ng/mL; AUC0-infinity, 851 and 1379 ng.h/mL; t1/2, 8.6 and 7.2 h. These values are in good agreement with those obtained using an HPLC method. Therefore, this radioreceptor binding assay proved to be simple, rapid, and specific for the determination of low levels of nuvenzepine in human plasma.« less

Actin-Binding Protein Requirement for Cortical Stability and Efficient Locomotion

NASA Astrophysics Data System (ADS)

Cunningham, C. Casey; Gorlin, Jed B.; Kwiatkowski, David J.; Hartwig, John H.; Janmey, Paul A.; Randolph Byers, H.; Stossel, Thomas P.

1992-01-01

Three unrelated tumor cell lines derived from human malignant melanomas lack actin-binding protein (ABP), which cross-links actin filaments in vitro and connects these filaments to plasma membrane glycoproteins. The ABP-deficient cells have impaired locomotion and display circumferential blebbing of the plasma membrane. Expression of ABP in one of the lines after transfection restored translocational motility and reduced membrane blebbing. These findings establish that ABP functions to stabilize cortical actin in vivo and is required for efficient cell locomotion.

Evaluation of metabolism, plasma protein binding and other biological parameters after administration of (-)-[(18)F]Flubatine in humans.

PubMed

Patt, Marianne; Becker, Georg A; Grossmann, Udo; Habermann, Bernd; Schildan, Andreas; Wilke, Stephan; Deuther-Conrad, Winnie; Graef, Susanne; Fischer, Steffen; Smits, René; Hoepping, Alexander; Wagenknecht, Gudrun; Steinbach, Jörg; Gertz, Hermann-Josef; Hesse, Swen; Schönknecht, Peter; Brust, Peter; Sabri, Osama

2014-07-01

(-)-[(18)F]Flubatine is a PET tracer with high affinity and selectivity for the nicotinic acetylcholine α4β2 receptor subtype. A clinical trial assessing the availability of this subtype of nAChRs was performed. From a total participant number of 21 Alzheimer's disease (AD) patients and 20 healthy controls (HCs), the following parameters were determined: plasma protein binding, metabolism and activity distribution between plasma and whole blood. Plasma protein binding and fraction of unchanged parent compound were assessed by ultracentrifugation and HPLC, respectively. The distribution of radioactivity (parent compound+metabolites) between plasma and whole blood was determined ex vivo at different time-points after injection by gamma counting after separation of whole blood by centrifugation into the cellular and non-cellular components. In additional experiments in vitro, tracer distribution between these blood components was assessed for up to 90min. A fraction of 15%±2% of (-)-[(18)F]Flubatine was found to be bound to plasma proteins. Metabolic degradation of (-)-[(18)F]Flubatine was very low, resulting in almost 90% unchanged parent compound at 90min p.i. with no significant difference between AD and HC. The radioactivity distribution between plasma and whole blood changed in vivo only slightly over time from 0.82±0.03 at 3min p.i. to 0.87±0.03 at 270min p.i. indicating the contribution of only a small amount of metabolites. In vitro studies revealed that (-)-[(18)F]Flubatine was instantaneously distributed between cellular and non-cellular blood parts. (-)-[(18)F]Flubatine exhibits very favourable characteristics for a PET radiotracer such as slow metabolic degradation and moderate plasma protein binding. Equilibrium of radioactivity distribution between plasma and whole blood is reached instantaneously and remains almost constant over time allowing both convenient sample handling and facilitated fractional blood volume contribution assessment. Copyright

Binding Mode Analyses and Pharmacophore Model Development for Stilbene Derivatives as a Novel and Competitive Class of α-Glucosidase Inhibitors

PubMed Central

Kim, Jun Young; Arooj, Mahreen; Kim, Siu; Hwang, Swan; Kim, Byeong-Woo; Park, Ki Hun; Lee, Keun Woo

2014-01-01

Stilbene urea derivatives as a novel and competitive class of non-glycosidic α-glucosidase inhibitors are effective for the treatment of type II diabetes and obesity. The main purposes of our molecular modeling study are to explore the most suitable binding poses of stilbene derivatives with analyzing the binding affinity differences and finally to develop a pharmacophore model which would represents critical features responsible for α-glucosidase inhibitory activity. Three-dimensional structure of S. cerevisiae α-glucosidase was built by homology modeling method and the structure was used for the molecular docking study to find out the initial binding mode of compound 12, which is the most highly active one. The initial structure was subjected to molecular dynamics (MD) simulations for protein structure adjustment at compound 12-bound state. Based on the adjusted conformation, the more reasonable binding modes of the stilbene urea derivatives were obtained from molecular docking and MD simulations. The binding mode of the derivatives was validated by correlation analysis between experimental Ki value and interaction energy. Our results revealed that the binding modes of the potent inhibitors were engaged with important hydrogen bond, hydrophobic, and π-interactions. With the validated compound 12-bound structure obtained from combining approach of docking and MD simulation, a proper four featured pharmacophore model was generated. It was also validated by comparison of fit values with the Ki values. Thus, these results will be helpful for understanding the relationship between binding mode and bioactivity and for designing better inhibitors from stilbene derivatives. PMID:24465730

Inclusion of live yeast and mannan-oligosaccharides in high grain-based diets for sheep: Ruminal parameters, inflammatory response and rumen morphology

PubMed Central

Jacovaci, Fernando Alberto; Cabreira Jobim, Clóves; Bolson, Dheyme Cristina; Pratti Daniel, João Luiz

2018-01-01

The objective of this study was to evaluate the effects of dietary supplementation with live yeast (Saccharomyces cerevisiae), mannan-oligosaccharides and the combination of these additives on the inflammatory response, ruminal parameters and rumen morphology of sheep fed a high grain-based diet. Thirty-Two Dorper x Santa Ines crossbred lambs with an average weight of 24±2 kg were distributed in a completely randomized design. The animals were housed in individual stalls and fed ad libitum. Diet treatments were: Control (without additive); LY (2 g/kg DM of live yeast, Saccharomyces cerevisiae), MOS (2 g/kg DM of mannan-oligosaccharides) and LY+MOS (2 g/kg DM of LY + 2 g/kg DM of MOS). The experiment lasted 42 days. The supplementation with MOS alone and the additives combination resulted in increased ruminal pH (P<0.01), while the total concentrations of short chain fatty acids (SCFA) in the rumen were higher (P<0.05) only in the diets with LY and MOS. Ammonia (NH3) concentration in the rumen decreased (P<0.04) with the additives usage. Diets with LY, MOS and with additives combination reduced (P<0.01) the levels of lipopolysaccharides (LPS) in the plasma with values of 0.46; 0.44 and 0.04 EU/mL, respectively when compared to the control (0.93 EU/mL). MOS and LY+MOS treatments had reduced stratum corneum thickness (P<0.01) in comparison to the control treatment. The total thickness of ruminal epithelium was lower with the addition of MOS in the diet (P<0.03) than with LY additive. The incidence and severity of hepatic abscesses in animals whose diet was supplemented with LY and LY+MOS was lower (P<0.05) than in animals fed the control diet. The use of LY, MOS and LY+MOS in the high-concentrate diets for sheep reduced NH3 concentrations and LPS translocation into the bloodstream. Diets containing MOS and LY+MOS enhanced the health of the ruminal epithelium by reducing the thickness of the stratum corneum, and diets containing LY and LY+MOS decreased the incidence

Novel acyl-CoA: cholesterol acyltransferase inhibitor: indoline-based sulfamide derivatives with low lipophilicity and protein binding ratio.

PubMed

Takahashi, Kenji; Ohta, Masaru; Shoji, Yoshimichi; Kasai, Masayasu; Kunishiro, Kazuyoshi; Miike, Tomohiro; Kanda, Mamoru; Shirahase, Hiroaki

2010-08-01

To find a novel acyl-CoA: cholesterol acyltransferase inhibitor, a series of sulfamide derivatives were synthesized and evaluated. Compound 1d, in which carboxymethyl moiety at the 5-position of Pactimibe was replaced by a sulfamoylamino group, showed 150-fold more potent anti-foam cell formation activity (IC(50): 0.02 microM), 1.6-fold higher log D(7.0) (4.63), and a slightly lower protein binding ratio (93.2%) than Pactimibe. Compound 1i, in which the octyl chain at the 1-position in 1d was replaced by an ethoxyethyl, showed markedly low log D(7.0) (1.73) and maintained 3-fold higher anti-foam cell formation activity (IC(50): 1.0 microM), than Pactimibe. The plasma protein binding ratio (PBR) of 1i was much lower than that of Pactimibe (62.5% vs. 98.1%), and its partition ratio to the rabbit atherosclerotic aorta after oral administration was higher than that of Pactimibe. Compound 1i at 10 microM markedly inhibited cholesterol esterification in atherosclerotic rabbit aortas even when incubated with serum, while Pactimibe had little effect probably due to its high PBR. In conclusion, compound 1i is expected to more efficiently inhibit the progression of atherosclerosis than Pactimibe.

Partially ionized hydrogen plasma in strong magnetic fields.

PubMed

Potekhin, A Y; Chabrier, G; Shibanov, Y A

1999-08-01

We study the thermodynamic properties of a partially ionized hydrogen plasma in strong magnetic fields, B approximately 10(12)-10(13) G, typical of neutron stars. The properties of the plasma depend significantly on the quantum-mechanical sizes and binding energies of the atoms, which are strongly modified by thermal motion across the field. We use new fitting formulas for the atomic binding energies and sizes, based on accurate numerical calculations and valid for any state of motion of the atom. In particular, we take into account decentered atomic states, neglected in previous studies of thermodynamics of magnetized plasmas. We also employ analytic fits for the thermodynamic functions of nonideal fully ionized electron-ion Coulomb plasmas. This enables us to construct an analytic model of the free energy. An ionization equilibrium equation is derived, taking into account the strong magnetic field effects and the nonideality effects. This equation is solved by an iteration technique. Ionization degrees, occupancies, and the equation of state are calculated.

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

PubMed Central

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

Cinnamon extract regulates plasma levels of adipose-derived factors and expression of multiple genes related to carbohydrate metabolism and lipogenesis in adipose tissue of fructose-fed rats.

PubMed

Qin, B; Polansky, M M; Anderson, R A

2010-03-01

We reported earlier that dietary cinnamon extract (CE) improves systemic insulin sensitivity and dyslipidemia by enhancing insulin signaling. In the present study, we have examined the effects of CE on several biomarkers including plasma levels of adipose-derived adipokines, and the potential molecular mechanisms of CE in epididymal adipose tissue (EAT). In Wistar rats fed a high-fructose diet (HFD) to induce insulin resistance, supplementation with a CE (Cinnulin PF, 50 mg/kg daily) for 8 weeks reduced blood glucose, plasma insulin, triglycerides, total cholesterol, chylomicron-apoB48, VLDL-apoB100, and soluble CD36. CE also inhibited plasma retinol binding protein 4 (RBP4) and fatty acid binding protein 4 (FABP4) levels. CE-induced increases in plasma adiponectin were not significant. CE did not affect food intake, bodyweight, and EAT weight. In EAT, there were increases in the insulin receptor ( IR) and IR substrate 2 ( IRS2) mRNA, but CE-induced increases in mRNA expression of IRS1, phosphoinositide-3-kinase, AKT1, glucose transporters 1 and 4 , and glycogen synthase 1 expression and decreased trends in mRNA expression of glycogen synthase kinase 3beta were not statistically significant. CE also enhanced the mRNA levels of ADIPOQ, and inhibited sterol regulatory element binding protein-1c mRNA levels. mRNA and protein levels of fatty acid synthase and FABP4 were inhibited by CE and RBP4, and CD36 protein levels were also decreased by CE. These results suggest that CE effectively ameliorates circulating levels of adipokines partially mediated via regulation of the expression of multiple genes involved in insulin sensitivity and lipogenesis in the EAT.

In-silico identification of the binding mode of synthesized adamantyl derivatives inside cholinesterase enzymes

PubMed Central

Al-Aboudi, Amal; Al-Qawasmeh, Raed A; Shahwan, Alaa; Mahmood, Uzma; Khalid, Asaad; Ul-Haq, Zaheer

2015-01-01

Aim: To investigate the binding mode of synthesized adamantly derivatives inside of cholinesterase enzymes using molecular docking simulations. Methods: A series of hybrid compounds containing adamantane and hydrazide moieties was designed and synthesized. Their inhibitory activities against acetylcholinesterase (AChE) and (butyrylcholinesterase) BChE were assessed in vitro. The binding mode of the compounds inside cholinesterase enzymes was investigated using Surflex-Dock package of Sybyl7.3 software. Results: A total of 26 adamantyl derivatives were synthesized. Among them, adamantane-1-carboxylic acid hydrazide had an almost equal inhibitory activity towards both enzymes, whereas 10 other compounds exhibited moderate inhibitory activity against BChE. The molecular docking studies demonstrated that hydrophobic interactions between the compounds and their surrounding residues in the active site played predominant roles, while hydrophilic interactions were also found. When the compounds were docked inside each enzyme, they exhibited stronger interactions with BChE over AChE, possibly due to the larger active site of BChE. The binding affinities of the compounds for BChE and AChE estimated were in agreement with the experimental data. Conclusion: The new adamantly derivatives selectively inhibit BChE with respect to AChE, thus making them good candidates for testing the hypothesis that BChE inhibitors would be more efficient and better tolerated than AChE inhibitors in the treatment of Alzheimer's disease. PMID:25937631

Neuronal pentraxin 1: A synaptic-derived plasma biomarker in Alzheimer's disease.

PubMed

Ma, Qiu-Lan; Teng, Edmond; Zuo, Xiaohong; Jones, Mychica; Teter, Bruce; Zhao, Evan Y; Zhu, Cansheng; Bilousova, Tina; Gylys, Karen H; Apostolova, Liana G; LaDu, Mary Jo; Hossain, Mir Ahamed; Frautschy, Sally A; Cole, Gregory M

2018-06-01

Synaptic neurodegeneration is thought to be an early event initiated by soluble β-amyloid (Aβ) aggregates that closely correlates with cognitive decline in Alzheimer disease (AD). Apolipoprotein ε4 (APOE4) is the most common genetic risk factor for both familial AD (FAD) and sporadic AD; it accelerates Aβ aggregation and selectively impairs glutamate receptor function and synaptic plasticity. However, its molecular mechanisms remain elusive and these synaptic deficits are difficult to monitor. AD- and APOE4-dependent plasma biomarkers have been proposed, but synapse-related plasma biomarkers are lacking. We evaluated neuronal pentraxin 1 (NP1), a potential CNS-derived plasma biomarker of excitatory synaptic pathology. NP1 is preferentially expressed in brain and involved in glutamate receptor internalization. NP1 is secreted presynaptically induced by Aβ oligomers, and implicated in excitatory synaptic and mitochondrial deficits. Levels of NP1 and its fragments were increased in a correlated fashion in both brain and plasma of 7-8 month-old E4FAD mice relative to E3FAD mice. NP1 was also found in exosome preparations and reduced by dietary DHA supplementation. Plasma NP1 was higher in E4FAD+ (APOE4 +/+ /FAD +/- ) relative to E4FAD- (non-carrier; APOE4 +/+ /FAD -/- ) mice, suggesting NP1 is modulated by Aβ expression. Finally, relative to normal elderly, plasma NP1 was also elevated in patients with mild cognitive impairment (MCI) and elevated further in the subset who progressed to early-stage AD. In those patients, there was a trend towards increased NP1 levels in APOE4 carriers relative to non-carriers. These findings indicate that NP1 may represent a potential synapse-derived plasma biomarker relevant to early alterations in excitatory synapses in MCI and early-stage AD. Copyright © 2018. Published by Elsevier Inc.

Ontogeny of growth hormone (GH) binding in the domestic turkey: evidence of sexual dimorphism and developmental changes in relationship to plasma GH.

PubMed

Vasilatos-Younken, R; Gray, K S; Bacon, W L; Nestor, K E; Long, D W; Rosenberger, J L

1990-07-01

The post-hatch ontogeny of hepatic GH binding and its relationship to GH plasma profile characteristics in male and female turkeys of slow- (RBC-2) and fast-growing (F; selected from RBC-2) genetic lines were determined. Specific binding of 125I-labelled recombinant chicken GH to crude hepatic membrane preparations (100,000 g pellet) was determined at 2, 4, 8, 14 and 24 weeks of age for both total (occupied plus free; 4 mol MgCl2/l pretreatment) and free (without MgCl2 pretreatment) binding sites. Characteristics of the plasma GH profile were measured at each age by serial blood sampling through indwelling jugular vein catheters. When specific binding to either free or total sites was expressed on a whole organ basis (i.e. hepatic GH-binding capacity/bird), binding increased dramatically (P less than 0.0001) with increasing age over both lines and sexes. Total binding capacity (free plus occupied sites) per bird was greater for females than for males at 24 weeks of age (P less than 0.04), as birds reached sexual maturity, but did not differ between fast- and slow-growing lines at any age. Available binding capacity (free sites) per bird was greater for the faster growing F than RBC-2 line at the older ages when body size was most divergent (14 and 24 weeks of age; P less than 0.01, P less than 0.06 respectively), but did not differ between sexes. Correlation analysis at individual ages revealed a progressive change in the nature of the relationship between hepatic GH binding, plasma GH and somatic growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes.

PubMed

Csermely, P; Szamel, M; Resch, K; Somogyi, J

1988-05-15

In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested (Parker, P.J., Coussens, L., Totty, N., Rhee, L., Young, S., Chen, E., Stabel, S., Waterfield, M.D., and Ullrich, A. (1986) Science 233, 853-859). In the present report, we demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes, and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn2+, while Fe2+ and Mn2+ are only partially counteractive. Our results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca2+, phorbol ester, or antigen.

SYSTEMATICS OF THE GENERA SACCHAROMYCES, SCHIZOSACCHAROMYCES, ENDOMYCOPSIS, KLUYVEROMYCES, SCHWANNIOMYCES AND BRETTANOMYCES: PROTON MAGNETIC RESONANCE SPECTRA OF THE MANNANS AND MANNOSE-CONTAINING POLYSACCHARIDES AS AN AID IN CLASSIFICATION,

DTIC Science & Technology

Endomycopsis, Kluyveromyces, Brettanomyces , Nematospora and Schwanniomyces and of some apparently related species of Torulopsis were determined, grouped...mannans produced by Saccharomyces, Kluyveromyces, Nematospora, Brettanomyces and Torulopsis were placed in 10 groups. The galactomannans formed by the

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE.

PubMed

Pidatala, Venkataramana R; Mahboubi, Amir; Mortimer, Jenny C

2017-10-16

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts of purified material. Here, we describe a simple method for gaining detailed polysaccharide structural information, including resolution of structural isomers. For polysaccharide analysis by gel electrophoresis (PACE), plant cell wall material is hydrolyzed with glycosyl hydrolases specific to the polysaccharide of interest (e.g., mannanases for mannan). Large format polyacrylamide gels are then used to separate the released oligosaccharides, which have been fluorescently labeled. Gels can be visualized with a modified gel imaging system (see Table of Materials). The resulting oligosaccharide fingerprint can either be compared qualitatively or, with replication, quantitatively. Linkage and branching information can be established using additional glycosyl hydrolases (e.g., mannosidases and galactosidases). Whilst this protocol describes a method for analyzing glucomannan structure, it can be applied to any polysaccharide for which characterized glycosyl hydrolases exist. Alternatively, it can be used to characterize novel glycosyl hydrolases using defined polysaccharide substrates.

Comparative molecular field analysis of the binding of the stereoisomers of fenoterol and fenoterol derivatives to the beta2 adrenergic receptor.

PubMed

Jozwiak, Krzysztof; Khalid, Chakir; Tanga, Mary J; Berzetei-Gurske, Ilona; Jimenez, Lucita; Kozocas, Joseph A; Woo, Anthony; Zhu, Weizhong; Xiao, Rui-Ping; Abernethy, Darrell R; Wainer, Irving W

2007-06-14

Stereoisomers of fenoterol and six fenoterol derivatives have been synthesized and their binding affinities for the beta2 adrenergic receptor (Kibeta2-AR), the subtype selectivity relative to the beta1-AR (Kibeta1-AR/Kibeta2-AR) and their functional activities were determined. Of the 26 compounds synthesized in the study, submicromolar binding affinities were observed for (R,R)-fenoterol, the (R,R)-isomer of the p-methoxy, and (R,R)- and (R,S)-isomers of 1-naphthyl derivatives and all of these compounds were active at submicromolar concentrations in cardiomyocyte contractility tests. The Kibeta1-AR/Kibeta2-AR ratios were >40 for (R,R)-fenoterol and the (R,R)-p-methoxy and (R,S)-1-naphthyl derivatives and 14 for the (R,R)-1-napthyl derivative. The binding data was analyzed using comparative molecular field analysis (CoMFA), and the resulting model indicated that the fenoterol derivatives interacted with two separate binding sites and one steric restricted site on the pseudo-receptor and that the chirality of the second stereogenic center affected Kibeta2 and subtype selectivity.

Cyanide binding to human plasma heme-hemopexin: A comparative study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ascenzi, Paolo, E-mail: ascenzi@uniroma3.it; Istituto Nazionale di Biostrutture e Biosistemi, Roma; Leboffe, Loris

Highlights: Black-Right-Pointing-Pointer Cyanide binding to ferric HHPX-heme-Fe. Black-Right-Pointing-Pointer Cyanide binding to ferrous HHPX-heme-Fe. Black-Right-Pointing-Pointer Dithionite-mediated reduction of ferric HHPX-heme-Fe-cyanide. Black-Right-Pointing-Pointer Cyanide binding to HHPX-heme-Fe is limited by ligand deprotonation. Black-Right-Pointing-Pointer Cyanide dissociation from HHPX-heme-Fe-cyanide is limited by ligand protonation. -- Abstract: Hemopexin (HPX) displays a pivotal role in heme scavenging and delivery to the liver. In turn, heme-Fe-hemopexin (HPX-heme-Fe) displays heme-based spectroscopic and reactivity properties. Here, kinetics and thermodynamics of cyanide binding to ferric and ferrous hexa-coordinate human plasma HPX-heme-Fe (HHPX-heme-Fe(III) and HHPX-heme-Fe(II), respectively), and for the dithionite-mediated reduction of the HHPX-heme-Fe(III)-cyanide complex, at pH 7.4 and 20.0 Degree-Sign C,more » are reported. Values of thermodynamic and kinetic parameters for cyanide binding to HHPX-heme-Fe(III) and HHPX-heme-Fe(II) are K = (4.1 {+-} 0.4) Multiplication-Sign 10{sup -6} M, k{sub on} = (6.9 {+-} 0.5) Multiplication-Sign 10{sup 1} M{sup -1} s{sup -1}, and k{sub off} = 2.8 Multiplication-Sign 10{sup -4} s{sup -1}; and H = (6 {+-} 1) Multiplication-Sign 10{sup -1} M, h{sub on} = 1.2 Multiplication-Sign 10{sup -1} M{sup -1} s{sup -1}, and h{sub off} = (7.1 {+-} 0.8) Multiplication-Sign 10{sup -2} s{sup -1}, respectively. The value of the rate constant for the dithionite-mediated reduction of the HHPX-heme-Fe(III)-cyanide complex is l = 8.9 {+-} 0.8 M{sup -1/2} s{sup -1}. HHPX-heme-Fe reactivity is modulated by proton acceptor/donor amino acid residue(s) (e.g., His236) assisting the deprotonation and protonation of the incoming and outgoing ligand, respectively.« less

Plasma proteins of rainbow trout (Oncorhynchus mykiss) isolated by binding to lipopolysaccharide from Aeromonas salmonicida.

PubMed

Hoover, G J; el-Mowafi, A; Simko, E; Kocal, T E; Ferguson, H W; Hayes, M A

1998-07-01

In an attempt to find plasma proteins that might be involved in the constitutive resistance of rainbow trout to furunculosis, a disease caused by Aeromonas salmonicida (AS), we purified serum and plasma proteins based on their calcium- and carbohydrate-dependent affinity for A. salmonicida lipopolysaccharide (LPS) coupled to an epoxy-activated synthetic matrix (Toyopearl AF Epoxy 650M). A multimeric family of high molecular weight (96 to 200-kDa) LPS-binding proteins exhibiting both calcium and mannose dependent binding was isolated. Upon reduction the multimers collapsed to subunits of approximately 16-kDa as estimated by 1D-PAGE and exhibited pI values of 5.30 and 5.75 as estimated from 2D-PAGE. Their N-terminal sequences were related to rainbow trout ladderlectin (RT-LL), a Sepharose-binding protein. Polyclonal antibodies to the LPS-purified 16-kDa subunits recognized both the reduced 16-kDa subunits and the non-reduced multimeric forms. A calcium- and N-acetylglucosamine (GlcNAc)-dependent LPS-binding multimeric protein (approximately 207-kDa) composed of 34.5-kDa subunits was purified and found to be identical to trout serum amyloid P (SAP) by N-terminal sequence (DLQDLSGKVFV). A protein of 24-kDa, in reduced and non-reduced conditions, was isolated and had N-terminal sequence identity with a known C-reactive protein (CRP) homologue, C-polysaccharide-binding protein 2 (TCBP2) of rainbow trout. A novel calcium-dependent LPS-binding protein was purified and termed rainbow trout lectin 37 (RT-L37). This protein, composed of dimers, tetramers and pentamers of 37 kDa subunits (pI 5.50-6.10) with N-terminal sequence (IQE(D/N)GHAEAPGATTVLNEILR) showed no close homology to proteins known or predicted from cDNA sequences. These findings demonstrate that rainbow trout have several blood proteins with lectin properties for the LPS of A. salmonicida; the biological functions of these proteins in resistance to furunculosis are still unknown.

Quartz crystal microbalance for the cardiac markers/antibodies binding kinetic measurements in the plasma samples

NASA Astrophysics Data System (ADS)

Agafonova, L. E.; Shumyantseva, V. V.; Archakov, A. I.

2014-06-01

The quartz crystal microbalance (QCM) was exploited for cardiac markers detection and kinetic studies of immunochemical reaction of cardiac troponin I (cTnI) and human heart fatty acid binding protein (H-FABP) with the corresponding monoclonal antibodies in undiluted plasma (serum) and standard solutions. The QCM technique allowed to dynamically monitor the kinetic differences in specific interactions and nonspecific sorption, without multiple labeling procedures and separation steps. The affinity binding process was characterized by the association (ka) and the dissociation (kd) kinetic constants and the equilibrium association (K) constant, all of which were obtained from experimental data.

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

PubMed Central

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

Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

PubMed

Oshiro, Satoshi; Honda, Shinya

2014-04-18

Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

Development of a lectin binding assay to differentiate between recombinant and endogenous proteins in pharmacokinetic studies of protein-biopharmaceuticals.

PubMed

Weber, Alfred; Minibeck, Eva; Scheiflinger, Friedrich; Turecek, Peter L

2015-04-10

Human glycoproteins, expressed in hamster cell lines, show similar glycosylation patterns to naturally occurring human molecules except for a minute difference in the linkage of terminal sialic acid: both cell types lack α2,6-galactosyl-sialyltransferase, abundantly expressed in human hepatocytes and responsible for the α2,6-sialylation of circulating glycoproteins. This minute difference, which is currently not known to have any physiological relevance, was the basis for the selective measurement of recombinant glycoproteins in the presence of their endogenous counterparts. The assay is based on using the lectin Sambucus nigra agglutinin (SNA), selectively binding to α2,6-sialylated N-glycans. Using von Willebrand factor (VWF), factor IX (FIX), and factor VIIa (FVIIa), it was demonstrated that (i) the plasma-derived proteins, but not the corresponding recombinant proteins, specifically bind to SNA and (ii) this binding can be used to deplete the plasma-derived proteins. The feasibility of this approach was confirmed in spike-recovery studies for all three recombinant coagulation proteins in human plasma and for recombinant VWF (rVWF) in macaque plasma. Analysis of plasma samples from macaques after administration of recombinant and a plasma-derived VWF demonstrated the suitability and robustness of this approach. Data showed that rVWF could be selectively measured without changing the ELISAs and furthermore revealed the limitations of baseline adjustment using a single measurement of the predose concentration only. The SNA gel-based depletion procedure can easily be integrated in existing procedures as a specific sample pre-treatment step. While ELISA-based methods were used to measure the recombinant coagulation proteins in the supernatants obtained by depletion, this procedure is applicable for all biochemical analyses. Copyright © 2015 Elsevier B.V. All rights reserved.

Reduced fasting plasma levels of diazepam-binding inhibitor in adolescents with anorexia nervosa.

PubMed

Conti, Elisa; Tremolizzo, Lucio; Bomba, Monica; Uccellini, Orlando; Rossi, Maria Sara; Raggi, Maria Elisabetta; Neri, Francesca; Ferrarese, Carlo; Nacinovich, Renata

2013-09-01

Altered expression and/or function, both peripherally and centrally, of various neuropeptides is involved in the neurophysiology of anorexia nervosa (AN). Diazepam-binding inhibitor (DBI) is an interesting peptide for understanding this crosstalk. The aim of this work was to assess fasting plasma levels of DBI and leptin in patients with AN. Twenty-four AN adolescents were recruited together with 10 age-comparable healthy controls. Neuropeptide determinations were performed on plasma samples by enzyme-linked immunosorbent assays. Patients with AN were further characterized for the presence of a depressive state or anxiety by using, respectively, the Children's Depression Inventory or the State-Trait Anxiety Inventory form Y. Levels of both plasma DBI and leptin were reduced in patients with AN (∼40 and ∼70%, respectively). DBI levels displayed a tendency to increase in the presence of a depressive state, although not with anxiety, whereas leptin levels correlated exclusively with body mass index. These data further extend our knowledge of neuropeptide dysfunction in AN, and plasma DBI may represent a marker for this disease, in particular considering its correlation with comorbid mood disorders. Copyright © 2013 Wiley Periodicals, Inc.

Synthesis and evaluation of 7-substituted-5,6-dihydrobenzo[c]acridine derivatives as new c-KIT promoter G-quadruplex binding ligands.

PubMed

Guo, Qian-Liang; Su, Hua-Fei; Wang, Ning; Liao, Sheng-Rong; Lu, Yu-Ting; Ou, Tian-Miao; Tan, Jia-Heng; Li, Ding; Huang, Zhi-Shu

2017-04-21

It has been shown that treatment of cancer cells with c-KIT G-quadruplex binding ligands can reduce their c-KIT expression levels thus inhibiting cell proliferation and inducing cell apoptosis. Herein, a series of new 7-substituted-5,6-dihydrobenzo[c]acridine derivatives were designed and synthesized. Subsequent biophysical evaluation demonstrated that the derivatives could effectively bind to and stabilize c-KIT G-quadruplex with good selectivity against duplex DNA. It was found that 12-N-methylated derivatives with a positive charge introduced at 12-position of 5,6-dihydrobenzo[c]acridine ring had similar binding affinity but lower stabilizing ability to c-KIT G-quadruplex DNA, compared with those of nonmethylated derivatives. Further molecular modeling studies showed possible binding modes of G-quadruplex with the ligands. RT-PCR assay and Western blot showed that compound 2b suppressed transcription and translation of c-KIT gene in K562 cells, which was consistent with the property of an effective G-quadruplex binding ligand targeting c-KIT oncogene promoter. Further biological evaluation showed that compound 2b could induce apoptosis through activation of the caspase-3 cascade pathway. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.

PubMed

Nishimura, R; Li, W; Kashishian, A; Mondino, A; Zhou, M; Cooper, J; Schlessinger, J

1993-11-01

Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth factors stimulate the phosphorylation of Nck and its association with autophosphorylated growth factor receptors. A panel of platelet-derived growth factor (PDGF) receptor mutations at tyrosine residues has been used to identify the Nck binding site. Here we show that mutation at Tyr-751 of the PDGF beta-receptor eliminates Nck binding both in vitro and in living cells. Moreover, the Y751F PDGF receptor mutant failed to mediate PDGF-stimulated phosphorylation of Nck in intact cells. A phosphorylated Tyr-751 is also required for binding of phosphatidylinositol-3 kinase to the PDGF receptor. Hence, the SH2 domains of p85 and Nck share a binding site in the PDGF receptor. Competition experiments with different phosphopeptides derived from the PDGF receptor suggest that binding of Nck and p85 is influenced by different residues around Tyr-751. Thus, a single tyrosine autophosphorylation site is able to link the PDGF receptor to two distinct SH2 domain-containing signaling molecules.

Two factors of the lectin pathway of complement, l-ficolin and mannan-binding lectin, and their associations with prematurity, low birthweight and infections in a large cohort of Polish neonates.

PubMed

Swierzko, Anna St; Atkinson, Anne P M; Cedzynski, Maciej; Macdonald, Shirley L; Szala, Agnieszka; Domzalska-Popadiuk, Iwona; Borkowska-Klos, Monika; Jopek, Aleksandra; Szczapa, Jerzy; Matsushita, Misao; Szemraj, Janusz; Turner, Marc L; Kilpatrick, David C

2009-02-01

Ficolins and one collectin, mannan-binding lectin (MBL), are the only factors known to activate the lectin pathway (LP) of complement. There is considerable circumstantial evidence that MBL insufficiency can increase susceptibility to various infections and influence the course of several non-infectious diseases complicated by infections. Much less information is available concerning l-ficolin. We report the results of a prospective study to investigate any association between either MBL deficiency or l-ficolin deficiency with prematurity, low birthweight or perinatal infections in a large cohort of Polish neonates, representing an ethnically homogenous population (n=1832). Cord blood samples were analysed to determine mbl-2 gene variants, MBL concentrations and MBL-MASP-2 complex activities (MBL-dependent lectin pathway activity) as well as l-ficolin levels. Median concentrations of l-ficolin and MBL were 2500 and 1124 ng/ml, respectively, while median LP activity was 272 mU/ml. After genotyping, 60.6% of babies were mbl-2 A/A, 35.4% were A/O and 4% were O/O genotypes. We found relative l-ficolin deficiency to be associated with prematurity, low birthweight and infections. l-Ficolin concentration correlated with gestational age and with birthweight, independently of gestational age. Preterm deliveries (<38 weeks) occurred more frequently among neonates with low LP activity but not with those having low serum MBL levels. Similarly, no association of serum MBL deficiency with low birthweight was found, but there was a correlation between LP activity and birthweight. Genotypes conferring very low serum MBL concentrations were associated with perinatal infections, and high-MBL-conferring genotypes were associated with prematurity. Our findings suggest that l-ficolin participates in host defence during the perinatal period and constitute the first evidence that relative l-ficolin deficiency may contribute to the adverse consequences of prematurity. Some similar trends

Informing the Human Plasma Protein Binding of Environmental Chemicals by Machine Learning in the Pharmaceutical Space: Applicability Domain and Limits of Predictability

EPA Science Inventory

The free fraction of a xenobiotic in plasma (Fub) is an important determinant of chemical adsorption, distribution, metabolism, elimination, and toxicity, yet experimental plasma protein binding data is scarce for environmentally relevant chemicals. The presented work explores th...

Potent haloperidol derivatives covalently binding to the dopamine D2 receptor.

PubMed

Schwalbe, Tobias; Kaindl, Jonas; Hübner, Harald; Gmeiner, Peter

2017-10-01

The dopamine D 2 receptor (D 2 R) is a common drug target for the treatment of a variety of neurological disorders including schizophrenia. Structure based design of subtype selective D 2 R antagonists requires high resolution crystal structures of the receptor and pharmacological tools promoting a better understanding of the protein-ligand interactions. Recently, we reported the development of a chemically activated dopamine derivative (FAUC150) designed to covalently bind the L94C mutant of the dopamine D 2 receptor. Using FAUC150 as a template, we elaborated the design and synthesis of irreversible analogs of the potent antipsychotic drug haloperidol forming covalent D 2 R-ligand complexes. The disulfide- and Michael acceptor-functionalized compounds showed significant receptor affinity and an irreversible binding profile in radioligand depletion experiments. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

PubMed

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.

Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease

PubMed Central

1992-01-01

Serum mannose-binding protein (MBP) is a C-type lectin that binds to terminal mannose and N-acetylglucosamine moieties present on surfaces of certain pathogens and activates the classical complement pathway. In the present study, we describe the mechanism underlying the activation triggered by MBP. The human serum MBP fraction was obtained by sequential affinity chromatography on mannan-Sepharose, anti-IgM- Sepharose and anti-MBP-Sepharose in the presence of calcium ions. This fraction contained a C1s-like serine protease as assessed by C4 consumption. The C1s-like serine protease, designated MBP-associated serine protease (MASP), was separated from MBP by rechromatography on anti-MBP-Sepharose in the presence of ethylenediaminetetraacetic acid. MASP exhibited both C4- and C2-consuming activities. The molecular mass of MASP was estimated to be 83 kD with two polypeptides of heavy (66 kD) and light (L) (31 kD) chains linked by disulfide bonds. The serine residue responsible for protease activity is located on the L chain. Reconstitution experiments using MASP and MBP revealed that combination of the two components restores C4- and C2-activating capacity on mannan. Based on analyses of molecular size, antigenicity, and 11 NH2- terminal amino acid sequences of the L chain, we conclude that MASP is a novel protein different from C1r or C1s. Our findings are not in accord with a proposed mechanism by which MBP utilizes the C1r2-C1s2 complex to initiate the classical complement pathway. PMID:1460414

Extraction process of palm kernel cake as a source of mannan for feed additive on poultry diet

NASA Astrophysics Data System (ADS)

Tafsin, M.; Hanafi, N. D.; Yusraini, E.

2017-05-01

Palm Kernel Cake (PKC) is a by-product of palm kernel oil extraction and found in large quantity in Indonesia. The inclusion of PKC on poultry diet are limited due to some nutritional problems such as anti-nutritional properties (mannan). On the other hand, mannan containing polysaccharides play in various biological functions particularly in enhancing the immune response and to control pathogen in poultry. The research objective to find out the extraction process of PKC and conducted at animal nutrition and Feed Science Laboratory, Agricultural Faculty, University of Sumatera Utara. Various extraction methode were used in this experiment, including fraction analysis used 7 number sieves, and followed by water and acetic acid extraction. The result indicated that PKC had different particle size according to sieve size and dominated by particle size 850 um. The analysis of sugar content indicated that each particle size had different characteristic on treatment by hot water extraction. The particle size 180—850 um had higher sugar content than coarse PKC (2000—3000 um). The total sugar content were recovered vary between 0.9—3,2% from PKC were extracted. Treatment grinding method followed by hot water extraction (100—120 °C, 1 h) increased total sugar content than previous treatments and reach 8% from PKC were extracted. Utilisation acetic acid decreased the total amount of total sugar from PKC were extracted. It is concluded that treatment by hot temperature (110—120 °C) for 1 h show highest yield to extract sugar from PKC.

A novel role for a major component of the vitamin D axis: vitamin D binding protein-derived macrophage activating factor induces human breast cancer cell apoptosis through stimulation of macrophages.

PubMed

Thyer, Lynda; Ward, Emma; Smith, Rodney; Fiore, Maria Giulia; Magherini, Stefano; Branca, Jacopo J V; Morucci, Gabriele; Gulisano, Massimo; Ruggiero, Marco; Pacini, Stefania

2013-07-08

The role of vitamin D in maintaining health appears greater than originally thought, and the concept of the vitamin D axis underlines the complexity of the biological events controlled by biologically active vitamin D (1,25(OH)(2)D3), its two binding proteins that are the vitamin D receptor (VDR) and the vitamin D-binding protein-derived macrophage activating factor (GcMAF). In this study we demonstrate that GcMAF stimulates macrophages, which in turn attack human breast cancer cells, induce their apoptosis and eventually phagocytize them. These results are consistent with the observation that macrophages infiltrated implanted tumors in mice after GcMAF injections. In addition, we hypothesize that the last 23 hydrophobic amino acids of VDR, located at the inner part of the plasma membrane, interact with the first 23 hydrophobic amino acids of the GcMAF located at the external part of the plasma membrane. This allows 1,25(OH)(2)D3 and oleic acid to become sandwiched between the two vitamin D-binding proteins, thus postulating a novel molecular mode of interaction between GcMAF and VDR. Taken together, these results support and reinforce the hypothesis that GcMAF has multiple biological activities that could be responsible for its anti-cancer effects, possibly through molecular interaction with the VDR that in turn is responsible for a multitude of non-genomic as well as genomic effects.

Light-up fluorescent probes utilizing binding behavior of perylenediimide derivatives to a hydrophobic pocket within DNA.

PubMed

Takada, Tadao; Yamaguchi, Kosato; Tsukamoto, Suguru; Nakamura, Mitsunobu; Yamana, Kazushige

2014-08-21

Here we study the binding behavior of perylenediimide () derivatives to a hydrophobic pocket created inside DNA and their photochemical properties capable of designing a light-up fluorescent sensor for short single-stranded DNA or RNA. The perylenediimide derivative with alkoxy groups () suppressing electron transfer quenching was examined. The bound randomly to DNA showed negligible fluorescence due to the aggregation-induced quenching, whereas the bound to the pocket as a monomeric form showed more than 100-fold fluorescence enhancement. Switching the binding states of the corresponded to a change in the fluorescence response for the hybridization event, which allowed us to design a fluorescent sensor of nucleic acids with a nanomolar detection limit.

Plasma and liver acetaminophen-protein adduct levels in mice after acetaminophen treatment: Dose–response, mechanisms, and clinical implications

DOE Office of Scientific and Technical Information (OSTI.GOV)

McGill, Mitchell R.; Lebofsky, Margitta; Norris, Hye-Ryun K.

2013-06-15

At therapeutic doses, acetaminophen (APAP) is a safe and effective analgesic. However, overdose of APAP is the principal cause of acute liver failure in the West. Binding of the reactive metabolite of APAP (NAPQI) to proteins is thought to be the initiating event in the mechanism of hepatotoxicity. Early work suggested that APAP-protein binding could not occur without glutathione (GSH) depletion, and likely only at toxic doses. Moreover, it was found that protein-derived APAP-cysteine could only be detected in serum after the onset of liver injury. On this basis, it was recently proposed that serum APAP-cysteine could be used asmore » diagnostic marker of APAP overdose. However, comprehensive dose–response and time course studies have not yet been done. Furthermore, the effects of co-morbidities on this parameter have not been investigated. We treated groups of mice with APAP at multiple doses and measured liver GSH and both liver and plasma APAP-protein adducts at various timepoints. Our results show that protein binding can occur without much loss of GSH. Importantly, the data confirm earlier work that showed that protein-derived APAP-cysteine can appear in plasma without liver injury. Experiments performed in vitro suggest that this may involve multiple mechanisms, including secretion of adducted proteins and diffusion of NAPQI directly into plasma. Induction of liver necrosis through ischemia–reperfusion significantly increased the plasma concentration of protein-derived APAP-cysteine after a subtoxic dose of APAP. While our data generally support the measurement of serum APAP-protein adducts in the clinic, caution is suggested in the interpretation of this parameter. - Highlights: • Extensive GSH depletion is not required for APAP-protein binding in the liver. • APAP-protein adducts appear in plasma at subtoxic doses. • Proteins are adducted in the cell and secreted out. • Coincidental liver injury increases plasma APAP-protein adducts at

Conformational analysis of (1. -->. 4)-. beta. -D-mannan triacetate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deslandes, Y.; Marchessault, R.H.; Bluhm, T.L.

1983-01-01

In wood, algae, and tubers, glucomannans have varying mannose-to-glucose ratios (M/G). Since diffraction on glucomannans of widely varying M/G do not show significant change in unit-cell base plane dimensions, the authors have suggested that isomorphous replacement may occur in glucomannans. To further investigate this point, it has been undertaken conformational analysis of glucomannan triacetate in which the X-ray fiber diagram suggests that two nonequivalent residues make up the asymmetric unit. X-ray fiber diagrams of the triacetate of glucomannan from Tubera salep show twofold symmetry along the chain axis with a fiber repeat of 1.6 nm. This implies that the asymmetricmore » unit is composed of two pyranose rings since the virtual bond length of a single pyranose ring cannot be greater than approximately 0.54 nm. By using empirical potential functions, it could be shown that the minimum internal energy of a mannan triacetate chain corresponds to a state where contiguous mannose triacetate units are not conformationally equivalent. This supports the hypothesis of mannobiose hexaacetate as the asymmetric unit. Furthermore, introduction of glucose triacetate into the backbone did not change the minimum energy conformation, thereby lending support to the isomorphous replacement concept in crystalline glucomannans. 19 references, 13 figures, 2 tables.« less

Population modelling to describe pharmacokinetics of amiodarone in rats: relevance of plasma protein and tissue depot binding.

PubMed

Campos Moreno, Eduardo; Merino Sanjuán, Matilde; Merino, Virginia; Nácher, Amparo; Martín Algarra, Rafael V; Casabó, Vicente G

2007-02-01

The objective of this paper was to characterize the disposition phase of AM in rats, after different high doses and modalities of i.v. administration. Three fitting programs, WINNONLIN, ADAPT II and NONMEM were employed. The two-stage fitting methods led to different results, none of which can adequately explain amiodarone's behaviour, although a great amount of data per subject is available. The non-linear mixed effect modelling approach allows satisfactory estimation of population pharmacokinetic parameters, and their respective variability. The best model to define the AM pharmacokinetic profile is a two-compartment model, with saturable and dynamic plasma protein binding and linear tissular depot dynamic binding. These results indicate that peripheral tissues act as depots, causing an important fall in AM plasma levels in the first moment after dosing. Later, the return of the drug from these depots causes a slow increase in serum concentration whenever the dose is reduced.

ITC-derived binding affinity may be biased due to titrant (nano)-aggregation. Binding of halogenated benzotriazoles to the catalytic domain of human protein kinase CK2

PubMed Central

Winiewska, Maria; Bugajska, Ewa

2017-01-01

The binding of four bromobenzotriazoles to the catalytic subunit of human protein kinase CK2 was assessed by two complementary methods: Microscale Thermophoresis (MST) and Isothermal Titration Calorimetry (ITC). New algorithm proposed for the global analysis of MST pseudo-titration data enabled reliable determination of binding affinities for two distinct sites, a relatively strong one with the Kd of the order of 100 nM and a substantially weaker one (Kd > 1 μM). The affinities for the strong binding site determined for the same protein-ligand systems using ITC were in most cases approximately 10-fold underestimated. The discrepancy was assigned directly to the kinetics of ligand nano-aggregates decay occurring upon injection of the concentrated ligand solution to the protein sample. The binding affinities determined in the reverse ITC experiment, in which ligands were titrated with a concentrated protein solution, agreed with the MST-derived data. Our analysis suggests that some ITC-derived Kd values, routinely reported together with PDB structures of protein-ligand complexes, may be biased due to the uncontrolled ligand (nano)-aggregation, which may occur even substantially below the solubility limit. PMID:28273138

Electrosynthesized MIPs for transferrin: Plastibodies or nano-filters?

PubMed

Zhang, Xiaorong; Yarman, Aysu; Erdossy, Júlia; Katz, Sagie; Zebger, Ingo; Jetzschmann, Katharina J; Altintas, Zeynep; Wollenberger, Ulla; Gyurcsányi, Róbert E; Scheller, Frieder W

2018-05-15

Molecularly imprinted polymer (MIP) nanofilms for transferrin (Trf) have been synthesized on gold surfaces by electro-polymerizing the functional monomer scopoletin in the presence of the protein target or around pre-adsorbed Trf. As determined by atomic force microscopy (AFM) the film thickness was comparable with the molecular dimension of the target. The target (re)binding properties of the electro-synthesized MIP films was evaluated by cyclic voltammetry (CV) and square wave voltammetry (SWV) through the target-binding induced permeability changes of the MIP nanofilms to the ferricyanide redox marker, as well as by surface plasmon resonance (SPR) and surface enhanced infrared absorption spectroscopy (SEIRAS) of the immobilized protein molecules. For Trf a linear concentration dependence in the lower micromolar range and an imprinting factor of ~5 was obtained by SWV and SPR. Furthermore, non-target proteins including the iron-free apo-Trf were discriminated by pronounced size and shape specificity. Whilst it is generally assumed that the rebinding of the target or of cross-reacting proteins exclusively takes place at the polymer here we considered also the interaction of the protein molecules with the underlying gold transducers. We demonstrate by SWV that adsorption of proteins suppresses the signal of the redox marker even at the bare gold surface and by SEIRAS that the treatment of the MIP with proteinase K or NaOH only partially removes the target protein. Therefore, we conclude that when interpreting binding of proteins to directly MIP-covered gold electrodes the interactions between the protein and the gold surface should also be considered. Copyright © 2018 Elsevier B.V. All rights reserved.

Technical note: Protozoa-specific antibodies raised in sheep plasma bind to their target protozoa in the rumen.

PubMed

Williams, Y J; Rea, S M; Popovski, S; Skillman, L C; Wright, A-D G

2014-12-01

Binding of IgG antibodies to Entodinium spp. in the rumen of sheep (Ovis aries) was investigated by adding IgG, purified from plasma, directly into the rumen. Plasma IgG was sourced from sheep that had or had not been immunized with a vaccine containing whole fixed Entodinium spp. cells. Ruminal fluid was sampled approximately 2 h after each antibody dosing. Binding of protozoa by a specific antibody was detected using an indirect fluorescent antibody test. An antibody titer in the ruminal fluid was determined by ELISA, and the concentration of ruminal fluid ammonia-N and ruminal pH were also determined. Entodinium spp. and total protozoa from IgG-infused sheep were enumerated by microscopic counts. Two-hourly additions of IgG maintained a low antibody titer in the rumen for 12 h and the binding of the antibody to the rumen protozoa was demonstrated. Increased ammonia-N concentrations and altered ruminal fluid pH patterns indicated that additional fermentation of protein was occurring in the rumen after addition of IgG. No reduction in numbers of Entodinium spp. was observed (P>0.05). Although binding of antibodies to protozoa has been demonstrated in the rumen, it is unclear how much cell death occurred. On the balance of probability, it would appear that the antibody was degraded or partially degraded, and the impact of this on protozoal populations and the measurement of a specific titer is also unclear.

Platelets Contain Tissue Factor Pathway Inhibitor-2 Derived from Megakaryocytes and Inhibits Fibrinolysis*

PubMed Central

Vadivel, Kanagasabai; Ponnuraj, Sathya-Moorthy; Kumar, Yogesh; Zaiss, Anne K.; Bunce, Matthew W.; Camire, Rodney M.; Wu, Ling; Evseenko, Denis; Herschman, Harvey R.; Bajaj, Madhu S.; Bajaj, S. Paul

2014-01-01

Tissue factor pathway inhibitor-2 (TFPI-2) is a homologue of TFPI-1 and contains three Kunitz-type domains and a basic C terminus region. The N-terminal domain of TFPI-2 is the only inhibitory domain, and it inhibits plasma kallikrein, factor XIa, and plasmin. However, plasma TFPI-2 levels are negligible (≤20 pm) in the context of influencing clotting or fibrinolysis. Here, we report that platelets contain significant amounts of TFPI-2 derived from megakaryocytes. We employed RT-PCR, Western blotting, immunohistochemistry, and confocal microscopy to determine that platelets, MEG-01 megakaryoblastic cells, and bone marrow megakaryocytes contain TFPI-2. ELISA data reveal that TFPI-2 binds factor V (FV) and partially B-domain-deleted FV (FV-1033) with Kd ∼9 nm and binds FVa with Kd ∼100 nm. Steady state analysis of surface plasmon resonance data reveal that TFPI-2 and TFPI-1 bind FV-1033 with Kd ∼36–48 nm and bind FVa with Kd ∼252–456 nm. Further, TFPI-1 (but not TFPI-1161) competes with TFPI-2 in binding to FV. These data indicate that the C-terminal basic region of TFPI-2 is similar to that of TFPI-1 and plays a role in binding to the FV B-domain acidic region. Using pull-down assays and Western blots, we show that TFPI-2 is associated with platelet FV/FVa. TFPI-2 (∼7 nm) in plasma of women at the onset of labor is also, in part, associated with FV. Importantly, TFPI-2 in platelets and in plasma of pregnant women inhibits FXIa and tissue-type plasminogen activator-induced clot fibrinolysis. In conclusion, TFPI-2 in platelets from normal or pregnant subjects and in plasma from pregnant women binds FV/Va and regulates intrinsic coagulation and fibrinolysis. PMID:25262870

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pidatala, Venkataramana R.; Mahboubi, Amir; Mortimer, Jenny C.

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts of purified material. Here, we describe a simple method for gaining detailed polysaccharide structural information, including resolution of structural isomers. For polysaccharide analysis by gel electrophoresis (PACE), plant cell wall material is hydrolyzed with glycosyl hydrolases specific to the polysaccharide of interest (e.g., mannanases for mannan). Large format polyacrylamide gels are then used to separate the released oligosaccharides, which have been fluorescently labeled. Gels can be visualized with a modified gel imaging system (see Table of Materials). The resulting oligosaccharidemore » fingerprint can either be compared qualitatively or, with replication, quantitatively. Linkage and branching information can be established using additional glycosyl hydrolases (e.g., mannosidases and galactosidases). Whilst this protocol describes a method for analyzing glucomannan structure, it can be applied to any polysaccharide for which characterized glycosyl hydrolases exist. Alternatively, it can be used to characterize novel glycosyl hydrolases using defined polysaccharide substrates.« less

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

DOE PAGES

Pidatala, Venkataramana R.; Mahboubi, Amir; Mortimer, Jenny C.

2017-10-16

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts of purified material. Here, we describe a simple method for gaining detailed polysaccharide structural information, including resolution of structural isomers. For polysaccharide analysis by gel electrophoresis (PACE), plant cell wall material is hydrolyzed with glycosyl hydrolases specific to the polysaccharide of interest (e.g., mannanases for mannan). Large format polyacrylamide gels are then used to separate the released oligosaccharides, which have been fluorescently labeled. Gels can be visualized with a modified gel imaging system (see Table of Materials). The resulting oligosaccharidemore » fingerprint can either be compared qualitatively or, with replication, quantitatively. Linkage and branching information can be established using additional glycosyl hydrolases (e.g., mannosidases and galactosidases). Whilst this protocol describes a method for analyzing glucomannan structure, it can be applied to any polysaccharide for which characterized glycosyl hydrolases exist. Alternatively, it can be used to characterize novel glycosyl hydrolases using defined polysaccharide substrates.« less

Munc13-4 Is a Rab11-binding Protein That Regulates Rab11-positive Vesicle Trafficking and Docking at the Plasma Membrane.

PubMed

Johnson, Jennifer L; He, Jing; Ramadass, Mahalakshmi; Pestonjamasp, Kersi; Kiosses, William B; Zhang, Jinzhong; Catz, Sergio D

2016-02-12

The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Differential proteomics analysis of the surface heterogeneity of dextran iron oxide nanoparticles and the implications for their in vivo clearance

PubMed Central

Simberg, Dmitri; Park, Ji-Ho; Karmali, Priya P.; Zhang, Wan-Ming; Merkulov, Sergei; McCrae, Keith; Bhatia, Sangeeta; Sailor, Michael; Ruoslahti, Erkki

2009-01-01

In order to understand the role of plasma proteins in the rapid liver clearance of dextran-coated superparamagnetic iron oxide (SPIO) in vivo, we analyzed the full repertoire of SPIO-binding blood proteins using novel two-dimensional differential mass spectrometry approach. The identified proteins showed specificity for surface domains of the nanoparticles: mannan-binding lectins bound to the dextran coating, histidine-rich glycoprotein and kininogen bound to the iron oxide part, and the complement lectin and contact clotting factors were secondary binders. Nanoparticle clearance studies in knockout mice suggested that these proteins, as well as several previously identified opsonins, do not play a significant role in the SPIO clearance. However, both the dextran coat and the iron oxide core remained accessible to specific probes after incubation of SPIO in plasma, suggesting that the nanoparticle surface could be available for recognition by macrophages, regardless of protein coating. These data provide guidance to rational design of bioinert, long-circulating nanoparticles. PMID:19394687

Differential proteomics analysis of the surface heterogeneity of dextran iron oxide nanoparticles and the implications for their in vivo clearance.

PubMed

Simberg, Dmitri; Park, Ji-Ho; Karmali, Priya P; Zhang, Wan-Ming; Merkulov, Sergei; McCrae, Keith; Bhatia, Sangeeta N; Sailor, Michael; Ruoslahti, Erkki

2009-08-01

In order to understand the role of plasma proteins in the rapid liver clearance of dextran-coated superparamagnetic iron oxide (SPIO) in vivo, we analyzed the full repertoire of SPIO-binding blood proteins using novel two-dimensional differential mass spectrometry approach. The identified proteins showed specificity for surface domains of the nanoparticles: mannan-binding lectins bound to the dextran coating, histidine-rich glycoprotein and kininogen bound to the iron oxide part, and the complement lectin and contact clotting factors were secondary binders. Nanoparticle clearance studies in knockout mice suggested that these proteins, as well as several previously identified opsonins, do not play a significant role in the SPIO clearance. However, both the dextran coat and the iron oxide core remained accessible to specific probes after incubation of SPIO in plasma, suggesting that the nanoparticle surface could be available for recognition by macrophages, regardless of protein coating. These data provide guidance to rational design of bioinert, long-circulating nanoparticles.

Novel vaccines targeting dendritic cells by coupling allergoids to nonoxidized mannan enhance allergen uptake and induce functional regulatory T cells through programmed death ligand 1.

PubMed

Sirvent, Sofía; Soria, Irene; Cirauqui, Cristina; Cases, Bárbara; Manzano, Ana I; Diez-Rivero, Carmen M; Reche, Pedro A; López-Relaño, Juan; Martínez-Naves, Eduardo; Cañada, F Javier; Jiménez-Barbero, Jesús; Subiza, Javier; Casanovas, Miguel; Fernández-Caldas, Enrique; Subiza, José Luis; Palomares, Oscar

2016-08-01

Allergen immunotherapy (AIT) is the only curative treatment for allergy. AIT faces pitfalls related to efficacy, security, duration, and patient compliance. Novel vaccines overcoming such inconveniences are in demand. We sought to study the immunologic mechanisms of action for novel vaccines targeting dendritic cells (DCs) generated by coupling glutaraldehyde-polymerized grass pollen allergoids to nonoxidized mannan (PM) compared with glutaraldehyde-polymerized allergoids (P) or native grass pollen extracts (N). Skin prick tests and basophil activation tests with N, P, or PM were performed in patients with grass pollen allergy. IgE-blocking experiments, flow cytometry, confocal microscopy, cocultures, suppression assays, real-time quantitative PCR, ELISAs, and ELISpot assays were performed to assess allergen capture by human DCs and T-cell responses. BALB/c mice were immunized with PM, N, or P. Antibody levels, cytokine production by splenocytes, and splenic forkhead box P3 (FOXP3)(+) regulatory T (Treg) cells were quantified. Experiments with oxidized PM were also performed. PM displays in vivo hypoallergenicity, induces potent blocking antibodies, and is captured by human DCs much more efficiently than N or P by mechanisms depending on mannose receptor- and dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-mediated internalization. PM endorses human DCs to generate functional FOXP3(+) Treg cells through programmed death ligand 1. Immunization of mice with PM induces a shift to nonallergic responses and increases the frequency of splenic FOXP3(+) Treg cells. Mild oxidation impairs these effects in human subjects and mice, demonstrating the essential role of preserving the carbohydrate structure of mannan. Allergoids conjugated to nonoxidized mannan represent suitable vaccines for AIT. Our findings might also be of the utmost relevance to development of therapeutic interventions in other immune tolerance-related diseases. Copyright �

The binding capability of plasma phospholipid transfer protein, but not HDL pool size, is critical to repress LPS induced inflammation.

PubMed

Yu, Yang; Cui, Yingjie; Zhao, Yanan; Liu, Shuai; Song, Guohua; Jiao, Peng; Li, Bin; Luo, Tian; Guo, Shoudong; Zhang, Xiangjian; Wang, Hao; Jiang, Xian-Cheng; Qin, Shucun

2016-02-09

Phospholipid transfer protein (PLTP) participates in high density lipoprotein (HDL) metabolism. Increased plasma PLTP activity was observed in lipopolysaccharide (LPS) triggered acute inflammatory diseases. This study aimed to determine the exact role of PLTP in LPS induced inflammation. HDL pool size was shrunk both in PLTP deficient mice (PLTP-/-) and PLTP transgenic mice (PLTP-Tg). PLTP displayed a strong protective effect on lethal endotoxemia in mice survival study. Furthermore, after LPS stimulation, the expression of pro-inflammatory cytokines were increased in bone marrow derived macrophage (BMDM) from PLTP-/-, while decreased in BMDM from PLTP-Tg compared with BMDM from wild-type mice (WT). Moreover, LPS induced nuclear factor kappa-B (NFκB) activation was enhanced in PLTP-/- BMDM or PLTP knockdown RAW264.7. Conversely, PLTP overexpression countered the NFκB activation in LPS challenged BMDM. Additionally, the activation of toll like receptor 4 (TLR4) induced by LPS showed no alteration in PLTP-/- BMDM. Finally, PLTP could bind to LPS, attenuate the pro-inflammatory effects of LPS, and improve the cell viability in vitro. To sum up, these findings elucidated that PLTP repressed LPS induced inflammation due to extracellular LPS binding capability, and the protective effects were not related to HDL pool size in mice.

Humoral markers of active Epstein-Barr virus infection associate with anti-extractable nuclear antigen autoantibodies and plasma galectin-3 binding protein in systemic lupus erythematosus.

PubMed

Rasmussen, N S; Nielsen, C T; Houen, G; Jacobsen, S

2016-12-01

We investigated if signs of active Epstein-Barr virus and cytomegalovirus infections associate with certain autoantibodies and a marker of type I interferon activity in patients with systemic lupus erythematosus. IgM and IgG plasma levels against Epstein-Barr virus early antigen diffuse and cytomegalovirus pp52 were applied as humoral markers of ongoing/recently active Epstein-Barr virus and cytomegalovirus infections, respectively. Plasma galectin-3 binding protein served as a surrogate marker of type I interferon activity. The measurements were conducted in 57 systemic lupus erythematosus patients and 29 healthy controls using ELISAs. Regression analyses and univariate comparisons were performed for associative evaluation between virus serology, plasma galectin-3 binding protein and autoantibodies, along with other clinical and demographic parameters. Plasma galectin-3 binding protein concentrations were significantly higher in systemic lupus erythematosus patients (P = 0.009) and associated positively with Epstein-Barr virus early antigen diffuse-directed antibodies and the presence of autoantibodies against extractable nuclear antigens in adjusted linear regressions (B = 2.02 and 2.02, P = 0.02 and P = 0.002, respectively). Furthermore, systemic lupus erythematosus patients with anti-extractable nuclear antigens had significantly higher antibody levels against Epstein-Barr virus early antigen diffuse (P = 0.02). Our study supports a link between active Epstein-Barr virus infections, positivity for anti-extractable nuclear antigens and increased plasma galectin-3 binding protein concentrations/type I interferon activity in systemic lupus erythematosus patients. © The Author(s) 2016.

Neutrophil-derived S100 calcium-binding proteins A8/A9 promote reticulated thrombocytosis and atherogenesis in diabetes

PubMed Central

Kraakman, Michael J.; Lee, Man K.S.; Al-Sharea, Annas; Dragoljevic, Dragana; Barrett, Tessa J.; Montenont, Emilie; Basu, Debapriya; Heywood, Sarah; Kammoun, Helene L.; Flynn, Michelle; Whillas, Alexandra; Hanssen, Nordin M.J.; Febbraio, Mark A.; Westein, Erik; Chin-Dusting, Jaye; Cooper, Mark E.; Berger, Jeffrey S.; Goldberg, Ira J.; Nagareddy, Prabhakara R.; Murphy, Andrew J.

2017-01-01

Platelets play a critical role in atherogenesis and thrombosis-mediated myocardial ischemia, processes that are accelerated in diabetes. Whether hyperglycemia promotes platelet production and whether enhanced platelet production contributes to enhanced atherothrombosis remains unknown. Here we found that in response to hyperglycemia, neutrophil-derived S100 calcium-binding proteins A8/A9 (S100A8/A9) interact with the receptor for advanced glycation end products (RAGE) on hepatic Kupffer cells, resulting in increased production of IL-6, a pleiotropic cytokine that is implicated in inflammatory thrombocytosis. IL-6 acts on hepatocytes to enhance the production of thrombopoietin, which in turn interacts with its cognate receptor c-MPL on megakaryocytes and bone marrow progenitor cells to promote their expansion and proliferation, resulting in reticulated thrombocytosis. Lowering blood glucose using a sodium-glucose cotransporter 2 inhibitor (dapagliflozin), depleting neutrophils or Kupffer cells, or inhibiting S100A8/A9 binding to RAGE (using paquinimod), all reduced diabetes-induced thrombocytosis. Inhibiting S100A8/A9 also decreased atherogenesis in diabetic mice. Finally, we found that patients with type 2 diabetes have reticulated thrombocytosis that correlates with glycated hemoglobin as well as increased plasma S100A8/A9 levels. These studies provide insights into the mechanisms that regulate platelet production and may aid in the development of strategies to improve on current antiplatelet therapies and to reduce cardiovascular disease risk in diabetes. PMID:28504650

Surface display of metal binding domain derived from PbrR on Escherichia coli specifically increases lead(II) adsorption.

PubMed

Hui, Chang-Ye; Guo, Yan; Yang, Xue-Qin; Zhang, Wen; Huang, Xian-Qing

2018-05-01

To improve the Pb 2+ biosorption capacity of the potential E. coli biosorbent, a putative Pb 2+ binding domain (PbBD) derived from PbrR was efficiently displayed on to the E. coli cell surface. The PbBD was obtained by truncating the N-terminal DNA-binding domain and C-terminal redundant amino acid residues of the Pb 2+ -sensing transcriptional factor PbrR. Whole-cell sorbents were constructed with the full-length PbrR and PbBD of PbrR genetically engineered onto the surface of E. coli cells using Lpp-OmpA as the anchor. Followed by a 1.71-fold higher display of PbBD than PbrR, the presence of PbBD on the surface of E. coli cells enabled a 1.92-fold higher Pb 2+ biosorption than that found in PbrR-displayed cells. Specific Pb 2+ binding via PbBD was the same as Pb 2+ binding via the full-length PbrR, with no observable decline even in the presence of Zn 2+ and Cd 2+ . Since surface-engineered E. coli cells with PbBD increased the Pb 2+ binding capacity and did not affect the adsorption selectivity, this suggests that surface display of the metal binding domain derived from MerR-like proteins may be used for the bioremediation of specific toxic heavy metals.

Reliability of plasma lipopolysaccharide-binding protein (LBP) from repeated measures in healthy adults.

PubMed

Citronberg, Jessica S; Wilkens, Lynne R; Lim, Unhee; Hullar, Meredith A J; White, Emily; Newcomb, Polly A; Le Marchand, Loïc; Lampe, Johanna W

2016-09-01

Plasma lipopolysaccharide-binding protein (LBP), a measure of internal exposure to bacterial lipopolysaccharide, has been associated with several chronic conditions and may be a marker of chronic inflammation; however, no studies have examined the reliability of this biomarker in a healthy population. We examined the temporal reliability of LBP measured in archived samples from participants in two studies. In Study one, 60 healthy participants had blood drawn at two time points: baseline and follow-up (either three, six, or nine months). In Study two, 24 individuals had blood drawn three to four times over a seven-month period. We measured LBP in archived plasma by ELISA. Test-retest reliability was estimated by calculating the intraclass correlation coefficient (ICC). Plasma LBP concentrations showed moderate reliability in Study one (ICC 0.60, 95 % CI 0.43-0.75) and Study two (ICC 0.46, 95 % CI 0.26-0.69). Restricting the follow-up period improved reliability. In Study one, the reliability of LBP over a three-month period was 0.68 (95 % CI: 0.41-0.87). In Study two, the ICC of samples taken ≤seven days apart was 0.61 (95 % CI 0.29-0.86). Plasma LBP concentrations demonstrated moderate test-retest reliability in healthy individuals with reliability improving over a shorter follow-up period.

Docking, molecular dynamics, binding energy-MM-PBSA studies of naphthofuran derivatives to identify potential dual inhibitors against BACE-1 and GSK-3β.

PubMed

Kumar, Akhil; Srivastava, Gaurava; Negi, Arvind S; Sharma, Ashok

2018-01-19

BACE-1 and GSK-3β both are potential therapeutic drug targets for Alzheimer's disease. Recently, both these targets received attention for designing dual inhibitors. Till now only two scaffolds (triazinone and curcumin) derivatives have been reported as BACE-1 and GSK-3β dual inhibitors. In our previous work, we have reported first in class dual inhibitor for BACE-1 and GSK-3β. In this study, we have explored other naphthofuran derivatives for their potential to inhibit BACE-1 and GSK-3β through docking, molecular dynamics, binding energy (MM-PBSA). These computational methods were performed to estimate the binding affinity of naphthofuran derivatives towards the BACE-1 and GSK-3β. In the docking results, two derivatives (NS7 and NS9) showed better binding affinity as compared to previously reported inhibitors. Hydrogen bond occupancy of NS7 and NS9 generated from MD trajectories showed good interaction with the flap residues Gln73, Thr72 of BACE-1 and Arg141, Thr138 residues of GSK-3β. MM-PBSA and energy decomposition per residue revealed different components of binding energy and relative importance of amino acid involved in binding. The results showed that the binding of inhibitors was majorly governed by the hydrophobic interactions and suggesting that hydrophobic interactions might be the key to design dual inhibitors for BACE1-1 and GSK-3β. Distance between important pair of amino acid residues indicated that BACE-1 and GSK-3β adopt closed conformation and become inactive after ligand binding. The results suggested that naphthofuran derivatives might act as dual inhibitor against BACE-1 and GSK-3β.

A Novel Role for a Major Component of the Vitamin D Axis: Vitamin D Binding Protein-Derived Macrophage Activating Factor Induces Human Breast Cancer Cell Apoptosis through Stimulation of Macrophages

PubMed Central

Thyer, Lynda; Ward, Emma; Smith, Rodney; Fiore, Maria Giulia; Magherini, Stefano; Branca, Jacopo J. V.; Morucci, Gabriele; Gulisano, Massimo; Ruggiero, Marco; Pacini, Stefania

2013-01-01

The role of vitamin D in maintaining health appears greater than originally thought, and the concept of the vitamin D axis underlines the complexity of the biological events controlled by biologically active vitamin D (1,25(OH)(2)D3), its two binding proteins that are the vitamin D receptor (VDR) and the vitamin D-binding protein-derived macrophage activating factor (GcMAF). In this study we demonstrate that GcMAF stimulates macrophages, which in turn attack human breast cancer cells, induce their apoptosis and eventually phagocytize them. These results are consistent with the observation that macrophages infiltrated implanted tumors in mice after GcMAF injections. In addition, we hypothesize that the last 23 hydrophobic amino acids of VDR, located at the inner part of the plasma membrane, interact with the first 23 hydrophobic amino acids of the GcMAF located at the external part of the plasma membrane. This al1ows 1,25(OH)(2)D3 and oleic acid to become sandwiched between the two vitamin D-binding proteins, thus postulating a novel molecular mode of interaction between GcMAF and VDR. Taken together, these results support and reinforce the hypothesis that GcMAF has multiple biological activities that could be responsible for its anti-cancer effects, possibly through molecular interaction with the VDR that in turn is responsible for a multitude of non-genomic as well as genomic effects. PMID:23857228

The postprandial plasma rye fingerprint includes benzoxazinoid-derived phenylacetamide sulfates.

PubMed

Hanhineva, Kati; Keski-Rahkonen, Pekka; Lappi, Jenni; Katina, Kati; Pekkinen, Jenna; Savolainen, Otto; Timonen, Oskari; Paananen, Jussi; Mykkänen, Hannu; Poutanen, Kaisa

2014-07-01

The bioavailability of whole-grain rye-derived phytochemicals has not yet been comprehensively characterized, and different baking and manufacturing processes can modulate the phytochemical composition of breads and other rye products. The aim of our study was to find key differences in the phytochemical profile of plasma after the consumption of 3 breads containing rye bran when compared with a plain white wheat bread control. Plasma metabolite profiles of 12 healthy middle-aged men and women were analyzed using LC quadrupole time-of-flight mass spectrometry metabolomics analysis while fasting and at 60 min, 120 min, 240 min, and 24 h after consuming a meal that contained either 100% whole-grain sourdough rye bread or white wheat bread enriched with native unprocessed rye bran or bioprocessed rye bran. White wheat bread was used as the control. The meals were served in random order after a 12-h overnight fast, with at least 3 d between each occasion. Two sulfonated phenylacetamides, hydroxy-N-(2-hydroxyphenyl) acetamide and N-(2-hydroxyphenyl) acetamide, potentially derived from the benzoxazinoid metabolites, were among the most discriminant postprandial plasma biomarkers distinguishing intake of breads containing whole-meal rye or rye bran from the control white wheat bread. Furthermore, subsequent metabolite profiling analysis of the consumed breads indicated that different bioprocessing/baking techniques involving exposure to microbial metabolism (e.g., sourdough fermentation) have a central role in modulating the phytochemical content of the whole-grain and bran-rich breads. © 2014 American Society for Nutrition.

Mechanisms of extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway in depressive disorder☆

PubMed Central

Wang, Hongyan; Zhang, Yingquan; Qiao, Mingqi

2013-01-01

The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway plays an important role in the mechanism of action of antidepressant drugs and has dominated recent studies on the pathogenesis of depression. In the present review we summarize the known roles of extracellular signal-regulated kinase, cAMP response element-binding protein and brain-derived neurotrophic factor in the pathogenesis of depression and in the mechanism of action of antidepressant medicines. The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor pathway has potential to be used as a biological index to help diagnose depression, and as such it is considered as an important new target in the treatment of depression. PMID:25206732

Effects of Chitosan Derivative N-[(2-Hydroxy-3-Trimethylammonium)Propyl]Chloride on Anticoagulant Activity of Guinea Pig Plasma.

PubMed

Drozd, N N; Shagdarova, B Ts; Il'ina, A V; Varlamov, V P

2017-07-01

Intravenous injection of protamine sulfate or quarternized chitosan derivative to guinea pigs after injection of 70 aIIa U/kg non-fractionated heparin shortened plasma clotting time (shown by partial activated thromboplastin time, thrombin time, and prothrombin time). Intravenous injection of protamine sulfate or quarternized chitosan derivative to guinea pigs after injection of 1 mg/kg (100 aXa U/kg) low-molecular-weight heparin (clexane) led to shortening of plasma clotting time in the ReaClot Heparin test and to prolongation of plasma amidolytic activity in the factor Xa chromogenic substrate test.

One-pot synthesis and sigma receptor binding studies of novel spirocyclic-2,6-diketopiperazine derivatives.

PubMed

Ghandi, Mehdi; Sherafat, Fatemeh; Sadeghzadeh, Masoud; Alirezapour, Behrouz

2016-06-01

New spirocyclic-2,6-diketopiperazine derivatives containing benzylpiperidine and cycloalkane moieties were synthesized by a one-pot two-step sequential Ugi/intramolecular N-amidation process in moderate to good yields. The in vitro ligand-binding profile studies performed on the sigma-1 and sigma-2 receptors revealed that the σ1 affinities and subtype selectivities of three spirocyclic piperidine derivatives are generally comparable to those of spirocycloalkane analogues. Compared to the low σ1 affinities obtained for cycloalkyl-substituted spirocyclic-2,6-diketopiperazines with n=2, those with n=1 proved to have optimal fitting with σ2 subtype by exhibiting higher affinities. Moreover, the best binding affinity and subtype selectivity was identified for compound 3c with Kiσ1=5.9±0.5nM and Kiσ2=563±21nM as well as 95-fold σ1/σ2 selectivity ratio, respectively. Copyright © 2016. Published by Elsevier Ltd.

Purification and identification of the fusicoccin binding protein from oat root plasma membrane

NASA Technical Reports Server (NTRS)

de Boer, A. H.; Watson, B. A.; Cleland, R. E.

1989-01-01

Fusicoccin (FC), a fungal phytotoxin, stimulates the H(+) -ATPase located in the plasma membrane (PM) of higher plants. The first event in the reaction chain leading to enhanced H(+) -efflux seems to be the binding of FC to a FC-binding protein (FCBP) in the PM. We solubilized 90% of the FCBP from oat (Avena sativa L. cv Victory) root PM in an active form with 1% octyl-glucoside. The FCBP was stabilized by the presence of protease inhibitors. The FCBP was purified by affinity chromatography using FC-linked adipic acid dihydrazide agarose (FC-AADA). Upon elution with 8 molar urea, two major protein bands on sodium dodecyl sulfate-polyaerylamide gel electrophoresis with molecular weights of 29,700 and 31,000 were obtained. Successive chromatography on BBAB Bio-Gel A, hexyl agarose, and FC-AADA resulted in the same two bands when the FC-AADA was eluted with sodium dodecyl sulfate. A direct correlation was made between 3H-FC-binding activity and the presence of the two protein bands. The stoichiometry of the 29,700 and 31,000 molecular weight bands was 1:2. This suggests that the FCBP occurs in the native form as a heterotrimer with an apparent molecular weight of approximately 92,000.

Urinary and plasma purine derivatives in fed and fasted llamas (Lama glama and L. guanacoe).

PubMed

Bakker, M L; Chen, X B; Kyle, D J; Orskov, E R; Bourke, D A

1996-02-01

The changes in urinary and plasma purine derivatives in response to fasting and level of feeding in llamas were examines. In one experiment, four llamas were gradually deprived of feed within 3 days and then fasted for 6 days. Daily urinary excretion of purine derivatives decreased with feed intake and leveled on the last 3 days of fasting at 177 +/- 26 mumol/kg W0.75. Allantoin and uric acid comprised 71% and 15% of total purine derivatives, respectively, in both fed and fasted states, but hypoxanthine plus xanthine increased from 9% to 36%. Plasma concentration of allantoin declined with feed intake reduction, but those of uric acid (217 mumol/l) and hypoxanthine plus xanthine (27 mumol/l) remained relatively unchanged. Concentration of uric acid was higher than that of allantoin, probably due to a high reabsorption of uric acid in renal tubules, which was measured as over 90%. In a second experiment, the four llamas were fed at 860 and 1740 g dry matter/d in a crossover design. Urinary total purine derivatives excretion responded to feed intake (10.4 vs 14.4 mmol/d), although the observed differences did not reach significance. Compared with some ruminant species, it appears that the llama resembles sheep regarding the magnitude of urinary purine derivatives excretion but is unique in maintaining a high concentration of uric acid in plasma, which could be part of the llama's adaptation to their environment.

Studies on identifying the binding sites of folate and its derivatives in Lactobacillus casei thymidylate synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maley, F.; Maley, G.F.

1983-01-01

It was shown that folate and its derivatives have a profound effect on stabilizing thymidylate synthase in vitro and in vivo, as a consequence of ternary formation between the folate, dUMP, or FdUMP, and the synthase. The degree to which complex formation is affected can be revealed qualitatively by circular dichroism and quantitatively by equilibrium dialysis using the Lactobacillus casei synthase. In contrast to the pteroylmonoglutamates, the pteroylpolyglutamates bind to thymidylate synthase in the absence of dUMP, but even their binding affinity is increased greatly by this nucleotide or its analogues. Similarly, treatment of the synthase with carboxypeptidase A preventsmore » the binding of the pteroylmonoglutamates and reduces the binding of the polyglutamates without affecting dUMP binding. The latter does not protect against carboxypeptidase inactivation but does potentiate the protective effect of the pteroylpolyglutamates. To determine the region of the synthase involved in the binding of the glutamate residues, Pte(/sup 14/C)GluGlu6 was activated by a water soluble carbodiimide in the presence and absence of dUMP. This folate derivative behaved as a competitive inhibitor of 5,10-CH/sub 2/H/sub 4/PteGlu, in contrast to methotrexate which was non-competitive. Separation of the five cyanogen bromide peptides from the L. casei synthase revealed 80% of the radioactivity to be associated with CNBr-2 and about 15% with CNBr-4. Chymotrypsin treatment of CNBr-2 yielded two /sup 14/C-labeled peaks on high performance liquid chromatography, with the slower migrating one being separated further into two peaks by Bio-gel P2 chromatography. All three peptides came from the same region of CNBr-2, encompassing residues 47-61 of the enzyme. From these studies it would appear that the residues most probably involved in the fixation of PteGlu7 are lysines 50 and 58. In contrast, methotrexate appeared to bind to another region of CNBr-2.« less

α-Mannan induces Th17-mediated pulmonary graft-versus-host disease in mice.

PubMed

Uryu, Hidetaka; Hashimoto, Daigo; Kato, Koji; Hayase, Eiko; Matsuoka, Satomi; Ogasawara, Reiki; Takahashi, Shuichiro; Maeda, Yoshinobu; Iwasaki, Hiromi; Miyamoto, Toshihiro; Saijo, Shinobu; Iwakura, Yoichiro; Hill, Geoffrey R; Akashi, Koichi; Teshima, Takanori

2015-05-07

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative therapy for various hematopoietic disorders. Graft-versus-host disease (GVHD) and infections are the major obstacles of HSCT, and their close relationship has been suggested. Although roles of bacterial and viral infections in the pathophysiology of GVHD are well described, impacts of fungal infection on GVHD remain to be elucidated. In mouse models of GVHD, injection of α-mannan (Mn), a major component of fungal cell wall, or heat-killed Candida albicans exacerbated GVHD, particularly in the lung. Mn-induced donor T-cell polarization toward Th17 and lung-specific chemokine environment in GVHD led to accumulation of Th17 cells in the lung. The detrimental effects of Mn on GVHD depended on donor IL-17A production and host C-type lectin receptor Dectin-2. These results suggest a previously unrecognized link between pulmonary GVHD and fungal infection after allogeneic HSCT. © 2015 by The American Society of Hematology.

Robustness of solvent/detergent treatment of plasma derivatives: a data collection from Plasma Protein Therapeutics Association member companies.

PubMed

Dichtelmüller, Herbert O; Biesert, Lothar; Fabbrizzi, Fabrizio; Gajardo, Rodrigo; Gröner, Albrecht; von Hoegen, Ilka; Jorquera, Juan I; Kempf, Christoph; Kreil, Thomas R; Pifat, Dominique; Osheroff, Wendy; Poelsler, Gerhard

2009-09-01

Solvent/detergent (S/D) treatment is an established virus inactivation technology that has been applied in the manufacture of medicinal products derived from human plasma for more than 20 years. Data on the inactivation of enveloped viruses by S/D treatment collected from seven Plasma Protein Therapeutics Association member companies demonstrate the robustness, reliability, and efficacy of this virus inactivation method. The results from 308 studies reflecting production conditions as well as technical variables significantly beyond the product release specification were evaluated for virus inactivation, comprising different combinations of solvent and detergent (tri(n-butyl) phosphate [TNBP]/Tween 80, TNBP/Triton X-100, TNBP/Na-cholate) and different products (Factor [F]VIII, F IX, and intravenous and intramuscular immunoglobulins). Neither product class, process temperature, protein concentration, nor pH value has a significant impact on virus inactivation. A variable that did appear to be critical was the concentration of solvent and detergent. The data presented here demonstrate the robustness of virus inactivation by S/D treatment for a broad spectrum of enveloped test viruses and process variables. Our data substantiate the fact that no transmission of viruses such as human immunodeficiency virus, hepatitis B virus, hepatitis C virus, or of other enveloped viruses was reported for licensed plasma derivatives since the introduction of S/D treatment.

In situ AFM imaging of apolipoprotein A-I directly derived from plasma HDL.

PubMed

Gan, Chaoye; Wang, Zhexuan; Chen, Yong

2017-04-01

The major apolipoproteins of plasma lipoproteins play vital roles in the structural integrity and physiological functions of lipoproteins. More than ten structural models of apolipoprotein A-I (apoA-I), the major apolipoprotein of high-density lipoprotein (HDL), have been developed successively. In these models, apoA-I was supposed to organize in a ring-shaped form. To date, however, there is no direct evidence under physiological condition. Here, atomic force microscopy (AFM) was used to in situ visualize the organization of apoA-I, which was exposed via depletion of the lipid component of plasma HDL pre-immobilized on functionalized mica sheets. For the first time, the ring-shaped coarse structure and three detailed structures (crescent-shaped, gapped "O"-shaped, and parentheses-shaped structures, respectively) of apoA-I in plasma HDL, which have the ability of binding scavenger receptors, were directly observed and quantitatively measured by AFM. The three detailed structures probably represent the different extents to which the lipid component of HDL was depleted. Data on lipid depletion of HDL may provide clues to understand lipid insertion of HDL. These data provide important information for the understanding of the structure/maturation of plasma HDL. Moreover, they suggest a powerful method for directly visualizing the major apolipoproteins of plasma lipoproteins or the protein component of lipoprotein-like lipid-protein complexes. Copyright © 2017 Elsevier B.V. All rights reserved.

Determination of the parameters of binding between lipopolysaccharide and chitosan and its N-acetylated derivative using a gravimetric piezoquartz biosensor.

PubMed

Naberezhnykh, G A; Gorbach, V I; Kalmykova, E N; Solov'eva, T F

2015-03-01

The interaction of endotoxin (lipopolysaccharide - LPS) with low molecular weight chitosan (5.5 kDa), its N-acylated derivative and chitoliposomes was studied using a gravimetric piezoelectric quartz crystal microbalance biosensor. The optimal conditions for the formation of a biolayer based on immobilized LPS on the resonator surface and its regeneration were elaborated. The association and dissociation rate constants for LPS binding to chitosans were determined and the affinity constants (Kaf) were calculated based on the data on changes in the oscillation frequency of the quartz crystal resonator. The Kaf values correlated with the ones obtained using other methods. The affinity of N-acylated chitosan binding to LPS was higher than that of the parent chitosan binding to LPS. Based on the results obtained, we suggest that water-soluble N-acylated derivatives of chitosan with low degree of substitution of amino groups could be useful compounds for endotoxin binding and neutralization. Copyright © 2015 Elsevier B.V. All rights reserved.

Platelet-rich plasma derived growth factors contribute to stem cell differentiation in musculoskeletal regeneration

NASA Astrophysics Data System (ADS)

Qian, Yun; Han, Qixin; Chen, Wei; Song, Jialin; Zhao, Xiaotian; Ouyang, Yuanming; Yuan, Weien; Fan, Cunyi

2017-10-01

Stem cell treatment and platelet-rich plasma (PRP) therapy are two significant issues in regenerative medicine. Stem cells such as bone marrow mesenchymal stem cells, adipose-derived stem cells and periodontal ligament stem cells can be successfully applied in the field of tissue regeneration. PRP, a natural product isolated from whole blood, can secrete multiple growth factors (GFs) for regulating physiological activities. These GFs can stimulate proliferation and differentiation of different stem cells in injury models. Therefore, combination of both agents receives wide expectations in regenerative medicine, especially in bone, cartilage and tendon repair. In this review, we thoroughly discussed the interaction and underlying mechanisms of platelet-rich plasma derived growth factors with stem cells, and assessed their functions in cell differentiation for musculoskeletal regeneration.

tRNA-derived short RNAs bind to Saccharomyces cerevisiae ribosomes in a stress-dependent manner and inhibit protein synthesis in vitro

PubMed Central

Kasprzyk, Marta; Twardowski, Tomasz

2016-01-01

Recently, a number of ribosome-associated non-coding RNAs (rancRNAs) have been discovered in all three domains of life. In our previous studies, we have described several types of rancRNAs in Saccharomyces cerevisiae, derived from many cellular RNAs, including mRNAs, rRNAs, tRNAs and snoRNAs. Here, we present the evidence that the tRNA fragments from simple eukaryotic organism S. cerevisiae directly bind to the ribosomes. Interestingly, rancRNA-tRFs in yeast are derived from both, 5′- and 3′-part of the tRNAs and both types of tRFs associate with the ribosomes in vitro. The location of tRFs within the ribosomes is distinct from classical A- and P-tRNA binding sites. Moreover, 3′-tRFs bind to the distinct site than 5′-tRFs. These interactions are stress dependent and as a consequence, provoke regulation of protein biosynthesis. We observe strong correlation between tRF binding to the ribosomes and inhibition of protein biosynthesis in particular environmental conditions. These results implicate the existence of an ancient and conserved mechanism of translation regulation with the involvement of ribosome-associating tRNA-derived fragments. PMID:27609601

Synthesis, DNA binding, topoisomerase inhibition and cytotoxic properties of 2-chloroethylnitrosourea derivatives of hoechst 33258.

PubMed

Bielawski, Krzysztof; Bielawska, Anna; Anchim, Tomasz; Wołczyński, Sławomir

2005-06-01

A number of novel 2-chloroethylnitrosourea derivatives of Hoechst 33258 were synthesized and examined for cytotoxicity in breast cancer cell cultures and for inhibition of topoisomerases I and II. Evaluation of the cytotoxicity of these compounds employing a MTT assay and inhibition of [3H]thymidine incorporation into DNA in both MDA-MB-231 and MCF-7 breast cancer cells demonstrated that these compounds were more active than Hoechst 33258. The DNA-binding ability of these compounds was evaluated by an ultrafiltration method using calf thymus DNA, poly(dA-dT)2 and poly(dG-dC)2, indicated that these compounds as well as Hoechst 33258 well interact with AT base pair compared with GC pair. Binding studies indicate that these compounds bind more tightly to double-stranded DNA than the parent compound Hoechst 33258. The degree to which these compounds inhibited cell growth breast cancer cells was generally consistent with their relative DNA binding affinity. Mechanistic studies revealed that these compounds act as topoisomerase I (topo I) or topoisomerase II (topo II) inhibitors in plasmid relaxation assays.

In vitro binding of the asialoglycoprotein receptor to the beta adaptin of plasma membrane coated vesicles.

PubMed Central

Beltzer, J P; Spiess, M

1991-01-01

The asialoglycoprotein (ASGP) receptor was used to probe total clathrin-coated vesicle proteins and purified adaptor proteins (APs) which had been fractionated by gel electrophoresis and transferred to nitrocellulose. The receptor was found to interact with proteins of approximately 100 kDa. The cytoplasmic domain of the ASGP receptor subunit H1 fused to dihydrofolate reductase competed for receptor binding to the 100 kDa polypeptide in the plasma membrane-type AP complexes (AP-2). A fusion protein containing the cytoplasmic domain of the endocytic mutant haemagglutinin HA-Y543 also competed, but a protein with the wild-type haemagglutinin sequence did not. This indicates that the observed interaction is specific for the cytoplasmic domain of the receptor and involves the tyrosine signal for endocytosis. When fractionated by gel electrophoresis in the presence of urea, the ASGP receptor binding polypeptide displayed a characteristic shift in electrophoretic mobility identifying it as the beta adaptin. Partial proteolysis of the AP-2 preparation followed by the receptor binding assay revealed that the aminoterminal domain of the beta adaptin contains the binding site for receptors. Images PMID:1935897

Cytochrome P450 binding studies of novel tacrine derivatives: Predicting the risk of hepatotoxicity.

PubMed

McEneny-King, Alanna; Osman, Wesseem; Edginton, Andrea N; Rao, Praveen P N

2017-06-01

The 1,2,3,4-tetrahydroacridine derivative tacrine was the first drug approved to treat Alzheimer's disease (AD). It is known to act as a potent cholinesterase inhibitor. However, tacrine was removed from the market due to its hepatotoxicity concerns as it undergoes metabolism to toxic quinonemethide species through the cytochrome P450 enzyme CYP1A2. Despite these challenges, tacrine serves as a useful template in the development of novel multi-targeting anti-AD agents. In this regard, we sought to evaluate the risk of hepatotoxicity in a series of C9 substituted tacrine derivatives that exhibit cholinesterase inhibition properties. The hepatotoxic potential of tacrine derivatives was evaluated using recombinant cytochrome (CYP) P450 CYP1A2 and CYP3A4 enzymes. Molecular docking studies were conducted to predict their binding modes and potential risk of forming hepatotoxic metabolites. Tacrine derivatives compound 1 (N-(3,4-dimethoxybenzyl)-1,2,3,4-tetrahydroacridin-9-amine) and 2 (6-chloro-N-(3,4-dimethoxybenzyl)-1,2,3,4-tetrahydroacridin-9-amine) which possess a C9 3,4-dimethoxybenzylamino substituent exhibited weak binding to CYP1A2 enzyme (1, IC 50 =33.0µM; 2, IC 50 =8.5µM) compared to tacrine (CYP1A2 IC 50 =1.5µM). Modeling studies show that the presence of a bulky 3,4-dimethoxybenzylamino C9 substituent prevents the orientation of the 1,2,3,4-tetrahydroacridine ring close to the heme-iron center of CYP1A2 thereby reducing the risk of forming hepatotoxic species. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

Uptake of oleate by isolated rat adipocytes is mediated by a 40-kDa plasma membrane fatty acid binding protein closely related to that in liver and gut

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schwieterman, W.; Sorrentino, D.; Potter, B.J.

1988-01-01

A portion of the hepatocellular uptake of nonesterified long-chain fatty acids is mediated by a specific 40-kDa plasma membrane fatty acid binding protein, which has also been isolated from the gut. To investigate whether a similar transport process exists in other tissues with high transmembrane fatty acid fluxes, initial rates (V/sub O/) of (/sup 3/H)-oleate uptake into isolated rat adipocytes were studied as a function of the concentration of unbound (/sup 3/H)oleate in the medium. V/sub O/ reached a maximum as the concentration of unbound oleate was increased and was significantly inhibited both by phloretin and by prior incubation ofmore » the cells with Pronase. A rabbit antibody to the rat liver plasma membrane fatty acid binding protein inhibited adipocyte fatty acid uptake by up to 63% in dose-dependent fashion. Inhibition was noncompetitive; at an immunoglobulin concentration of 250 ..mu..g/ml V/sub max/ was reduced from 2480 /plus minus/ 160 to 1870 /plus minus/ 80 pmol/min per 5 /times/ 10/sup 4/ adipocytes, with no change in K/sub m/. A basic kDa adipocyte plasma membrane fatty acid binding protein, isolated from crude adipocyte plasma membrane fractions, reacted strongly in both agar gel diffusion and electrophoretic blots with the antibody raised against the corresponding hepatic plasma membrane protein. These data indicate that the uptake of oleate by rat adipocytes is mediated by a 40-kDa plasma membrane fatty acid binding protein closely related to that in liver and gut.« less

Determination of geniposide in adjuvant arthritis rat plasma by ultra-high performance liquid chromatography tandem mass spectrometry method and its application to oral bioavailability and plasma protein binding ability studies.

PubMed

Chen, Jian; Wu, Hong; Xu, Guo-Bing; Dai, Miao-Miao; Hu, Shun-Li; Sun, Liang-Liang; Wang, Wei; Wang, Rong; Li, Shu-Pin; Li, Guo-Qiang

2015-04-10

A specific, sensitive and high throughput ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometric method (UHPLC-ESI-MS/MS) was established and validated to assay geniposide (GE), a promising anti-inflammatory drug, in adjuvant arthritis rat plasma: application to pharmacokinetic and oral bioavailability studies and plasma protein binding ability. Plasma samples were processed by de-proteinised with ice-cold methanol and separated on an ACQUITY UPLC™ HSS C18 column (100 mm × 2.1mm i.d., 1.8 μm particle size) at a gradient flow rate of 0.2 mL/min using acetonitrile-0.1% formic acid in water as mobile phase, and the total run time was 9 min. Mass detection was performed in selected reaction monitoring (SRM) mode with negative electro-spray ionization includes the addition of paeoniflorin (Pae) as an internal standard (IS). The mass transition ion-pair was followed as m/z 387.4 → 122.4 for GE and m/z 479.4 → 449.0 for IS. The calibration curves were linear over the concentration range of 2-50,000 ng/mL with lower limit of quantification of 2 ng/mL. The intra-day and inter-day precisions (RSD, %) of the assay were less than 8.4%, and the accuracy was within ± 6.4% in terms of relative error (RE). Extraction recovery, matrix effect and stability were satisfactory in adjuvant arthritis rat plasma. The UHPLC-ESI-MS/MS method was successfully applied to a pharmacokinetic study of GE after oral administration of depurated GE at 33, 66, 132 mg/kg and intravenous injection at 33, 66, 132 mg/kg in adjuvant arthritis (AA) rats. In addition, it was found that GE has rapid absorption and elimination, low absolute bioavailability, high plasma protein binding ability in AA rats after oral administration within the tested dosage range. It suggested that GE showed slow distribution into the intra- and extracellular space, and the binding rate was not proportionally dependent on plasma concentration of GE when the concentration of GE was

21 CFR 862.1685 - Thyroxine-binding globulin test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... globulin test system is a device intended to measure thyroxine (thyroid)-binding globulin (TBG), a plasma protein which binds thyroxine, in serum and plasma. Measurements obtained by this device are used in the...

21 CFR 862.1685 - Thyroxine-binding globulin test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... globulin test system is a device intended to measure thyroxine (thyroid)-binding globulin (TBG), a plasma protein which binds thyroxine, in serum and plasma. Measurements obtained by this device are used in the...

21 CFR 862.1685 - Thyroxine-binding globulin test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... globulin test system is a device intended to measure thyroxine (thyroid)-binding globulin (TBG), a plasma protein which binds thyroxine, in serum and plasma. Measurements obtained by this device are used in the...

21 CFR 862.1685 - Thyroxine-binding globulin test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... globulin test system is a device intended to measure thyroxine (thyroid)-binding globulin (TBG), a plasma protein which binds thyroxine, in serum and plasma. Measurements obtained by this device are used in the...

21 CFR 862.1685 - Thyroxine-binding globulin test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... globulin test system is a device intended to measure thyroxine (thyroid)-binding globulin (TBG), a plasma protein which binds thyroxine, in serum and plasma. Measurements obtained by this device are used in the...

Plasma binding of an alpha-blocking agent, nicergoline--affinity for serum albumin and native and modified alpha 1-acid glycoprotein.

PubMed

Robert, L; Migne, J; Santonja, R; Zini, R; Schmid, K; Tillement, J P

1983-06-01

The binding of nicergoline, an alpha-blocking drug, by human plasma proteins was studied using gel filtration, polyacrylamide gel electrophoresis, and equilibrium dialysis techniques. 3H-labeled nicergoline added to plasma was eluted together with two major protein fractions, one containing mainly serum albumin, the other glycoproteins such as alpha 1-acid glycoprotein (alpha 1-AG). Equilibrium dialysis experiments with pure human serum albumin and alpha 1-AG as well as with its chemically modified forms, desialylated, carboxymethylated, and both desialylated and carboxymethylated alpha 1-AG gave the following results: nicergoline has about a 4-fold higher affinity for alpha 1-AG than for serum albumin. There are two binding sites per molecule on serum albumin and one on alpha 1-AG. The binding parameters of alpha 1-AG were not significantly modified by desialylation or carboxymethylation. Only desialylated and carboxymethylated alpha 1-AG showed a decreased binding for nicergoline, suggesting conformational modifications induced by these combined treatments. The fact that desialylated alpha 1-AG keeps its affinity for nicergoline suggests the possibility of a selective introduction of this drug in cells possessing the Ashwell-type specific receptor for desialylated alpha 1-AG, for instance hepatocytes. Increased serum alpha 1-AG concentration induced by inflammatory reactions will also modify the distribution of bound nicergoline between serum albumin and alpha 1-AG and as a consequence its half-life and cell distribution.

A successful experience of the Iranian blood transfusion organization in improving accessibility and affordability of plasma derived medicine.

PubMed

Chegini, Azita; Torab, Seyed Ardeshir; Pourfatollah, Ali Akbar

2017-02-01

Plasma is the liquid part of blood. It is estimated 21.6 million liters of plasma collect from Whole blood annually. From these plasma, 4.2 million liters transfuse, 8.1 million liters fractionate, 9.3 million liters waste. Nowadays, blood products and PDM (plasma derived medicine) consider as essential medicine in modern health care and transfusion medicine. Iranian blood transfusion organization as a non-profit organization was established in 1974 in order to centralize all blood transfusion activities from donor recruitment to distribution of blood components to hospitals. Iran is the only country in EMR region with the rate of 20-29.9 blood donations per 1000 population and reached 100% voluntary non-remunerated blood donation in 2007. RBCs and platelets demand are much more than FFPs so the IBTO was faced the surplus plasma that could cause surplus plasma wastage. Simultaneously, hospitals need more plasma derived medicine especially albumin, IVIG, factor VIII, factor IX. IBTO was faced the challenges such as Fractionators selection, Plasma volume shipment, Contract duration, Product profile, Multiple External audits, Cold chain maintenance, Transporting plasma across international borders, NAT test. To overcome plasma wastage and storage of PDM. IBTO involved toll manufacturing in 2005 and not only prevents plasma wastage but also save MOH (ministry of health) budget. Copyright © 2016. Published by Elsevier Ltd.

A Novel Phosphatidylinositol 4,5-Bisphosphate Binding Domain Mediates Plasma Membrane Localization of ExoU and Other Patatin-like Phospholipases*

PubMed Central

Tyson, Gregory H.; Halavaty, Andrei S.; Kim, Hyunjin; Geissler, Brett; Agard, Mallory; Satchell, Karla J.; Cho, Wonhwa; Anderson, Wayne F.; Hauser, Alan R.

2015-01-01

Bacterial toxins require localization to specific intracellular compartments following injection into host cells. In this study, we examined the membrane targeting of a broad family of bacterial proteins, the patatin-like phospholipases. The best characterized member of this family is ExoU, an effector of the Pseudomonas aeruginosa type III secretion system. Upon injection into host cells, ExoU localizes to the plasma membrane, where it uses its phospholipase A2 activity to lyse infected cells. The targeting mechanism of ExoU is poorly characterized, but it was recently found to bind to the phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a marker for the plasma membrane of eukaryotic cells. We confirmed that the membrane localization domain (MLD) of ExoU had a direct affinity for PI(4,5)P2, and we determined that this binding was required for ExoU localization. Previously uncharacterized ExoU homologs from Pseudomonas fluorescens and Photorhabdus asymbiotica also localized to the plasma membrane and required PI(4,5)P2 for this localization. A conserved arginine within the MLD was critical for interaction of each protein with PI(4,5)P2 and for localization. Furthermore, we determined the crystal structure of the full-length P. fluorescens ExoU and found that it was similar to that of P. aeruginosa ExoU. Each MLD contains a four-helical bundle, with the conserved arginine exposed at its cap to allow for interaction with the negatively charged PI(4,5)P2. Overall, these findings provide a structural explanation for the targeting of patatin-like phospholipases to the plasma membrane and define the MLD of ExoU as a member of a new class of PI(4,5)P2 binding domains. PMID:25505182

Increased volume of distribution for recombinant activated factor VII and longer plasma-derived factor VII half-life may explain their long lasting prophylactic effect.

PubMed

Mathijssen, Natascha C J; Masereeuw, Rosalinde; Holme, Pal Andre; van Kraaij, Marian G J; Laros-van Gorkom, Britta A P; Peyvandi, Flora; van Heerde, Waander L

2013-08-01

Prophylaxis with plasma-derived or recombinant activated factor VII is beneficial in severe factor VII deficiency. To understand why prophylactic treatment with both products is efficacious, we conducted a pharmacokinetic study. Ten factor VII deficient patients were treated with either recombinant activated (20 μg/kg) or plasma-derived (25 IU/kg) factor VII in a cross-over design. Pharmacokinetic parameters were analyzed through activated factor VII activity, factor VII clotting activity, and factor VII antigen levels on depicted time points. Factor VII activity half-lifes, determined by non-compartmental and one-compartmental analysis (results in brackets), were shorter for recombinant activated (1.4h; 0.7h) than for plasma-derived factor VII (6.8h; 3.2h); both recombinant activated (5.1h; 2.1h and plasma-derived factor VII (5.8h; 3.2h) resulted in longer half-lives of factor VII antigen. Activated factor VII half-lives (based on activated factor VII activity levels) were significantly higher compared to factor VII clotting activity (1.6h; 0.9h). Volumes of distribution were significantly higher for activated factor VII (236 ml/kg; 175 ml/kg, measured by activated factor VII) as compared to plasma-derived factor VII (206 ml/kg; 64 ml/kg, measured by factor FVII activity), suggesting a plasma- and extracellular fluid distribution for recombinant activated factor VII. Recombinant activated factor VII showed significantly shorter half-lifes than plasma-derived factor VII. Volumes of distribution were significantly higher for treatment with recombinant activated factor VII. The longer half-life for plasma-derived factor VII, compared to recombinant activated factor VII, and the increased volume of distribution for recombinant activated factor VII, compared to plasma-derived factor VII may further elucidate the beneficial effect of prophylactic treatment of both products. Copyright © 2013 Elsevier Ltd. All rights reserved.

Alzheimer risk genes modulate the relationship between plasma apoE and cortical PiB binding

DOE PAGES

Lazaris, Andreas; Hwang, Kristy S.; Goukasian, Naira; ...

2015-10-15

Objective: We investigated the association between apoE protein plasma levels and brain amyloidosis and the effect of the top 10 Alzheimer disease (AD) risk genes on this association. Methods: Our dataset consisted of 18 AD, 52 mild cognitive impairment, and 3 cognitively normal Alzheimer's Disease Neuroimaging Initiative 1 (ADNI1) participants with available [ 11C]-Pittsburgh compound B (PiB) and peripheral blood protein data. We used cortical pattern matching to study associations between plasma apoE and cortical PiB binding and the effect of carrier status for the top 10 AD risk genes. Results: Low plasma apoE was significantly associated with high PiBmore » SUVR, except in the sensorimotor and entorhinal cortex. For BIN1 rs744373, the association was observed only in minor allele carriers. For CD2AP rs9349407 and CR1 rs3818361, the association was preserved only in minor allele noncarriers. We did not find evidence for modulation by CLU, PICALM, ABCA7, BIN1, and MS4A6A. Conclusions: Our data show that BIN1 rs744373, CD2AP rs9349407, and CR1 rs3818361 genotypes modulate the association between apoE protein plasma levels and brain amyloidosis, implying a potential epigenetic/downstream interaction.« less

A mass balance approach for calculation of recovery and binding enables the use of ultrafiltration as a rapid method for measurement of plasma protein binding for even highly lipophilic compounds.

PubMed

Wang, Changguang; Williams, Noelle S

2013-03-05

The aim of this study is to further validate the use of ultrafiltration (UF) as a method for determining plasma protein binding (PPB) by demonstrating that non-specific binding (NSB) is not a limitation, even for highly lipophilic compounds, because NSB sites on the apparatus are passivated in the presence of plasma. Mass balance theory was used to calculate recovery of 20 commercial and seven investigational compounds during ultrafiltration in the presence and absence of plasma. PPB was also measured using this mass balance approach for comparison to PPB determined by rapid equilibrium dialysis (RED) and as found in the literature. Compound recovery during UF was dramatically different in the presence and absence of plasma for compounds with high NSB in PBS only. A comparison of PPB calculated by ultrafiltration with literature values or calculated by RED gave concordant results. Discrepancies could be explained by changes in pH, insufficient time to equilibrium, or compound instability during RED, problems which were circumvented by ultrafiltration. Therefore, NSB, as measured by the traditional incubation of compound in PBS, need not be an issue when choosing UF as a PPB assay method. It is more appropriate to calculate compound recovery from the device in plasma as measured by mass balance to determine the suitability of the method for an individual compound. The speed with which UF can be conducted additionally avoids changes in pH or compound loss that can occur with other methods. The mass balance approach to UF is thus a preferred method for rapid determination of PPB. Copyright © 2012 Elsevier B.V. All rights reserved.

Comparative analyses of two thermophilic enzymes exhibiting both beta-1,4 mannosidic and beta-1,4 glucosidic cleavage activities from Caldanaerobius polysaccharolyticus.

PubMed

Han, Yejun; Dodd, Dylan; Hespen, Charles W; Ohene-Adjei, Samuel; Schroeder, Charles M; Mackie, Roderick I; Cann, Isaac K O

2010-08-01

The hydrolysis of polysaccharides containing mannan requires endo-1,4-beta-mannanase and 1,4-beta-mannosidase activities. In the current report, the biochemical properties of two endo-beta-1,4-mannanases (Man5A and Man5B) from Caldanaerobius polysaccharolyticus were studied. Man5A is composed of an N-terminal signal peptide (SP), a catalytic domain, two carbohydrate-binding modules (CBMs), and three surface layer homology (SLH) repeats, whereas Man5B lacks the SP, CBMs, and SLH repeats. To gain insights into how the two glycoside hydrolase family 5 (GH5) enzymes may aid the bacterium in energy acquisition and also the potential application of the two enzymes in the biofuel industry, two derivatives of Man5A (Man5A-TM1 [TM1 stands for truncational mutant 1], which lacks the SP and SLH repeats, and Man5A-TM2, which lacks the SP, CBMs, and SLH repeats) and the wild-type Man5B were biochemically analyzed. The Man5A derivatives displayed endo-1,4-beta-mannanase and endo-1,4-beta-glucanase activities and hydrolyzed oligosaccharides with a degree of polymerization (DP) of 4 or higher. Man5B exhibited endo-1,4-beta-mannanase activity and little endo-1,4-beta-glucanase activity; however, this enzyme also exhibited 1,4-beta-mannosidase and cellodextrinase activities. Man5A-TM1, compared to either Man5A-TM2 or Man5B, had higher catalytic activity with soluble and insoluble polysaccharides, indicating that the CBMs enhance catalysis of Man5A. Furthermore, Man5A-TM1 acted synergistically with Man5B in the hydrolysis of beta-mannan and carboxymethyl cellulose. The versatility of the two enzymes, therefore, makes them a resource for depolymerization of mannan-containing polysaccharides in the biofuel industry. Furthermore, on the basis of the biochemical and genomic data, a molecular mechanism for utilization of mannan-containing nutrients by C. polysaccharolyticus is proposed.

Interactions of Indole Derivatives with β-Cyclodextrin: A Quantitative Structure-Property Relationship Study

PubMed Central

Šoškić, Milan; Porobić, Ivana

2016-01-01

Retention factors for 31 indole derivatives, most of them with auxin activity, were determined by high-performance liquid chromatography, using bonded β-cyclodextrin as a stationary phase. A three-parameter QSPR (quantitative structure-property relationship) model, based on physico-chemical and structural descriptors was derived, which accounted for about 98% variations in the retention factors. The model suggests that the indole nucleus occupies the relatively apolar cavity of β-cyclodextrin while the carboxyl group of the indole -3-carboxylic acids makes hydrogen bonds with the hydroxyl groups of β-cyclodextrin. The length and flexibility of the side chain containing carboxyl group strongly affect the binding of these compounds to β-cyclodextrin. Non-acidic derivatives, unlike the indole-3-carboxylic acids, are poorly retained on the column. A reasonably well correlation was found between the retention factors of the indole-3-acetic acids and their relative binding affinities for human serum albumin, a carrier protein in the blood plasma. A less satisfactory correlation was obtained when the retention factors of the indole derivatives were compared with their affinities for auxin-binding protein 1, a plant auxin receptor. PMID:27124734

Low abdominal NIRS values and elevated plasma intestinal fatty acid-binding protein in a premature piglet model of necrotizing enterocolitis

USDA-ARS?s Scientific Manuscript database

To identify early markers of necrotizing enterocolitis (NEC), we hypothesized that continuous abdominal near-infrared spectroscopy (A-NIRS) measurement of splanchnic tissue oxygen saturation and intermittent plasma intestinal fatty-acid binding protein (pI-FABP) measured every 6 hours can detect NEC...

Culture Medium Supplements Derived from Human Platelet and Plasma: Cell Commitment and Proliferation Support

PubMed Central

Muraglia, Anita; Nguyen, Van Thi; Nardini, Marta; Mogni, Massimo; Coviello, Domenico; Dozin, Beatrice; Strada, Paolo; Baldelli, Ilaria; Formica, Matteo; Cancedda, Ranieri; Mastrogiacomo, Maddalena

2017-01-01

Present cell culture medium supplements, in most cases based on animal sera, are not fully satisfactory especially for the in vitro expansion of cells intended for human cell therapy. This paper refers to (i) an heparin-free human platelet lysate (PL) devoid of serum or plasma components (v-PL) and (ii) an heparin-free human serum derived from plasma devoid of PL components (Pl-s) and to their use as single components or in combination in primary or cell line cultures. Human mesenchymal stem cells (MSC) primary cultures were obtained from adipose tissue, bone marrow, and umbilical cord. Human chondrocytes were obtained from articular cartilage biopsies. In general, MSC expanded in the presence of Pl-s alone showed a low or no proliferation in comparison to cells grown with the combination of Pl-s and v-PL. Confluent, growth-arrested cells, either human MSC or human articular chondrocytes, treated with v-PL resumed proliferation, whereas control cultures, not supplemented with v-PL, remained quiescent and did not proliferate. Interestingly, signal transduction pathways distinctive of proliferation were activated also in cells treated with v-PL in the absence of serum, when cell proliferation did not occur, indicating that v-PL could induce the cell re-entry in the cell cycle (cell commitment), but the presence of serum proteins was an absolute requirement for cell proliferation to happen. Indeed, Pl-s alone supported cell growth in constitutively activated cell lines (U-937, HeLa, HaCaT, and V-79) regardless of the co-presence of v-PL. Plasma- and plasma-derived serum were equally able to sustain cell proliferation although, for cells cultured in adhesion, the Pl-s was more efficient than the plasma from which it was derived. In conclusion, the cells expanded in the presence of the new additives maintained their differentiation potential and did not show alterations in their karyotype. PMID:29209609

Effects of competitive red blood cell binding and reduced hematocrit on the blood and plasma levels of (/sup 14/C)Indapamide in the rat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lettieri, J.T.; Portelli, S.T.

1983-02-01

The effects of chlorthalidone and acetazolamide on the red blood cell binding of indapamide were investigated. Both drugs caused a substantial decrease in the amount of indapamide bound to the erythrocytes in vitro. This effect was demonstrated by a change in the indapamide blood/plasma ratio from approximately 6 in control samples, to a value of 1 when either of the displacing agents was added. Coadministration of acetazolamide with /sup 14/C-labeled indapamide to rats, resulted in a 5-fold drop in the blood levels of total radioactivity, relative to rats dosed with (/sup 14/C)indapamide alone. Concomitantly, there was a 2-fold increase inmore » the plasma levels of total radioactivity after acetazolamide coadministration. In rats whose hematocrits had been reduced by extensive bleeding, there were only minor alterations in the blood/plasma partitioning of (/sup 14/C)indapamide. Thus, chlorthalidone and acetazolamide were able to displace indapamide from erythrocytes in vitro and in vivo, possibly by competition at a carbonic anhydrase binding site. The pharmacokinetics of drugs which are extensively bound to erythrocytes may be significantly altered by the presence of other agents capable of competitive binding.« less

Heterogeneous Binding and Central Nervous System Distribution of the Multitargeted Kinase Inhibitor Ponatinib Restrict Orthotopic Efficacy in a Patient-Derived Xenograft Model of Glioblastoma.

PubMed

Laramy, Janice K; Kim, Minjee; Gupta, Shiv K; Parrish, Karen E; Zhang, Shuangling; Bakken, Katrina K; Carlson, Brett L; Mladek, Ann C; Ma, Daniel J; Sarkaria, Jann N; Elmquist, William F

2017-11-01

This study investigated how differences in drug distribution and free fraction at different tumor and tissue sites influence the efficacy of the multikinase inhibitor ponatinib in a patient-derived xenograft model of glioblastoma (GBM). Efficacy studies in GBM6 flank (heterotopic) and intracranial (orthotopic) models showed that ponatinib is effective in the flank but not in the intracranial model, despite a relatively high brain-to-plasma ratio. In vitro binding studies indicated that flank tumor had a higher free (unbound) drug fraction than normal brain. The total and free drug concentrations, along with the tissue-to-plasma ratio (Kp) and its unbound derivative (Kp,uu), were consistently higher in the flank tumor than the normal brain at 1 and 6 hours after a single dose in GBM6 flank xenografts. In the orthotopic xenografts, the intracranial tumor core displayed higher Kp and Kp,uu values compared with the brain-around-tumor (BAT). The free fractions and the total drug concentrations, hence free drug concentrations, were consistently higher in the core than in the BAT at 1 and 6 hours postdose. The delivery disadvantages in the brain and BAT were further evidenced by the low total drug concentrations in these areas that did not consistently exceed the in vitro cytotoxic concentration (IC 50 ). Taken together, the regional differences in free drug exposure across the intracranial tumor may be responsible for compromising efficacy of ponatinib in orthotopic GBM6. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Roxarsone binding to soil-derived dissolved organic matter: Insights from multi-spectroscopic techniques.

PubMed

Fu, Qing-Long; He, Jian-Zhou; Blaney, Lee; Zhou, Dong-Mei

2016-07-01

The fate and transport of roxarsone (ROX), a widely used organoarsenic feed additive, in soil is significantly influenced by the ubiquitous presence of soil-derived dissolved organic matter (DOM). In this study, fluorescence quenching titration and two-dimensional correlation spectroscopy (2D-COS) were employed to study ROX binding to DOM. Binding mechanisms were revealed by fluorescence lifetime measurement and Fourier transform infrared spectroscopy (FTIR). Humic- and protein-like fluorophores were identified in the excitation-emission matrix and synchronous fluorescence spectra of DOM. The conditional stability constant (log KC) for ROX binding to DOM was found to be 5.06, indicating that ROX was strongly bound to DOM. The binding order of ROX to DOM fluorophores revealed by 2D-COS followed the sequence of protein-like fluorophore ≈ the longer wavelength excited humic-like (L-humic-like) fluorophore > the shorter wavelength excited humic-like (S-humic-like) fluorophore. 2D-COS resolved issues with peak overlapping and allowed further exploration of the interaction between ROX and DOM. Results of fluorescence lifetime and FTIR spectra demonstrated that ROX interacted with DOM through the hydroxyl, amide II, carboxyl, aliphatic CH, and NO2 groups, yielding stable DOM-ROX complexes. The strong interaction between ROX and DOM implies that DOM plays an important role in the environmental fate of ROX in soil. Copyright © 2016 Elsevier Ltd. All rights reserved.

Sex-hormone-binding globulin.

PubMed

Anderson, D C

1974-01-01

A review was made to understand how plasma binding protein might influence sex-hormone action in target tissues. Steroids are predominately bound to plasma proteins and only unbound steroids enter the cells. Sex-hormone-binding globulin (SHBG) binds to both the main circulating steroid T and E2 but changes in SHBG concentrations exert significant results. Increased SHBG levels increase estrogen production and decreases T activity; whereas, increased androgens increase T action and inhibit SHBG production. These disturbances in hormone maintenance may lead to abnormal adult sex differentiation such as hirsutism and forms of hynaecomastia. By developing SHBG concentration measurement methods-responses of hirsutism to glucocorticoid or estrogem may be assessed. In addition, the effect of thyroid hormones on SHBG may also have therapeutic implications in endocrine disease.

Unmatter Plasma revisited

NASA Astrophysics Data System (ADS)

Smarandache, Florentin

2017-10-01

Unmmatter Plasma is a novel form of plasma, exclusively made of matter and its antimatter counterpart. The electron-positron beam plasma was generated in the laboratory in the beginning of 2015. This experimental fact shows that unmatter, a new form of matter that is formed by matter and antimatter bind together (mathematically predicted since 2004) really exists. That is the electron-positron plasma experiment of 2015 is the experimentum crucis verifying the mathematically predicted unmatter. Unmatter is formed by combinations of matter and antimatter that bind together, or by long-range mixture of matter and antimatter forming a weakly-coupled phase. Binding and bound state means that the interaction is sufficiently strong to tie together the particles of a system, therefore hindering them from becoming free. For example, a usual liquid is a bound state of molecules, while a gas is an un-bounded where the molecules can move freely in successive collisions.

What can be learned in the snake antivenom field from the developments in human plasma derived products?

PubMed

Burnouf, Thierry

2018-05-01

Human plasma-derived medicinal products and snake antivenom immunoglobulins are unique and complex therapeutic protein products. Human plasma products are obtained by fractionating large pools of plasma collected from blood plasma donors. They comprise a wide range of protein products, including polyvalent and hyperimmune immunoglobulins, coagulation factors, albumin, and various protease inhibitors that are transfused to patients affected by congenital or acquired protein deficiencies, immunological disorders, or metabolic diseases. Snake antivenoms are manufactured from pools of plasma collected from animals, typically horses, which have been immunized against snake venoms. Transfusing antivenoms is the cornerstone therapy to treat patients affected by snakebite envenoming. Over the last thirty years, much technical and regulatory evolution has been implemented to ensure that this class of biologicals meets modern quality requirements. The purpose of this review is to compare the main developments that took place in plasma production, protein fractionation, pathogen safety, quality control, preclinical and clinical studies, and regulations of these products. We also analyze whether both fields have been influencing and cross-fertilizing each other technically and in regulatory aspects to reach modern safety and efficacy standards at global levels, and how experience in the human plasma fractionation industry can further impact the manufacture of snake antivenom and that of other animal-derived antisera. Copyright © 2018 Elsevier Ltd. All rights reserved.

Effect of bile salt binding or protease inactivation on plasma cholecystokinin and gallbladder responses to bombesin.

PubMed

Thimister, P W; Hopman, W P; Sloots, C E; Rosenbusch, G; Tangerman, A; Willems, H L; Lamers, C B; Jansen, J B

1994-12-01

Bombesin-stimulated plasma cholecystokinin levels decrease after an initial increase despite continuous infusion of bombesin. The aim of this study was to determine if a feedback mechanism, mediated by bile salts or proteolytic enzymes, is responsible for this decline. Bombesin (1.0 ng.kg-1.min-1) was infused into volunteers for 180 minutes on separate occasions. Cholestyramine, colestipol, camostate, or saline were perfused intraduodenally during the second hour of the tests. Cholestyramine was also administered without infusion of bombesin. Colestipol and cholestyramine, dependent on their bile salt-binding capacity, markedly enhanced (P < 0.05) bombesin-stimulated plasma cholecystokinin from 2.1 +/- 0.5 pmol/L to 6.4 +/- 2.2 pmol/L and 12.1 +/- 3.3 pmol/L (P < 0.05 vs. colestipol), respectively, and further decreased gallbladder volume (P < 0.05) from 9.4 +/- 1.6 mL to 2.0 +/- 0.4 mL and 2.2 +/- 0.5 mL, respectively. The protease inhibitor camostate had no effect. Bile salt precipitation also enhanced plasma pancreatic polypeptide responses (P < 0.01) but did not alter gastrin responses. Plasma cholecystokinin responses to cholestyramine without bombesin infusion varied considerably, but increments were highly correlated to decreases in gallbladder volume (r = 0.91; P < 0.005). Bile salt sequestration but not protease inactivation enhances plasma cholecystokinin and gallbladder responses to bombesin infusion in humans.

Synergistic effects of dendritic cell targeting and laser-microporation on enhancing epicutaneous skin vaccination efficacy.

PubMed

Machado, Yoan; Duinkerken, Sanne; Hoepflinger, Veronika; Mayr, Melissa; Korotchenko, Evgeniia; Kurtaj, Almedina; Pablos, Isabel; Steiner, Markus; Stoecklinger, Angelika; Lübbers, Joyce; Schmid, Maximillian; Ritter, Uwe; Scheiblhofer, Sandra; Ablinger, Michael; Wally, Verena; Hochmann, Sarah; Raninger, Anna M; Strunk, Dirk; van Kooyk, Yvette; Thalhamer, Josef; Weiss, Richard

2017-11-28

Due to its unique immunological properties, the skin is an attractive target tissue for allergen-specific immunotherapy. In our current work, we combined a dendritic cell targeting approach with epicutaneous immunization using an ablative fractional laser to generate defined micropores in the upper layers of the skin. By coupling the major birch pollen allergen Bet v 1 to mannan from S. cerevisiae via mild periodate oxidation we generated hypoallergenic Bet-mannan neoglycoconjugates, which efficiently targeted CD14 + dendritic cells and Langerhans cells in human skin explants. Mannan conjugation resulted in sustained release from the skin and retention in secondary lymphoid organs, whereas unconjugated antigen showed fast renal clearance. In a mouse model, Bet-mannan neoglycoconjugates applied via laser-microporated skin synergistically elicited potent humoral and cellular immune responses, superior to intradermal injection. The induced antibody responses displayed IgE-blocking capacity, highlighting the therapeutic potential of the approach. Moreover, application via micropores, but not by intradermal injection, resulted in a mixed TH1/TH17-biased immune response. Our data clearly show that applying mannan-neoglycoconjugates to an organ rich in dendritic cells using laser-microporation is superior to intradermal injection. Due to their low IgE binding capacity and biodegradability, mannan neoglycoconjugates therefore represent an attractive formulation for allergen-specific epicutaneous immunotherapy. Copyright © 2017 Elsevier B.V. All rights reserved.

Mechanical properties of epoxy composites with plasma-modified rice-husk-derived nanosilica

NASA Astrophysics Data System (ADS)

Hubilla, Fatima Athena D.; Panghulan, Glenson R.; Pechardo, Jason; Vasquez, Magdaleno R., Jr.

2018-01-01

In this study, we explored the use of rice-husk-derived nanosilica (nSiO2) as fillers in epoxy resins. The nSiO2 was irradiated with a capacitively coupled 13.56 MHz radio frequency (RF) plasma using an admixture of argon (Ar) and hexamethyldisiloxane (HMDSO) or 1,7-octadiene (OD) monomers. The plasma-polymerized nSiO2 was loaded at various concentrations (1-5%) into the epoxy matrix. Surface hydrophobicity of the plasma-treated nSiO2-filled composites increased, which is attributed to the attachment of functional groups from the monomer gases on the silica surface. Microhardness increased by at least 10% upon the inclusion of plasma-modified nSiO2 compared with pristine nSiO2-epoxy composites. Likewise, hardness increased with increasing loading volume, with the HMDSO-treated silica composite recording the highest increase. Elastic moduli of the composites also showed an increase of at least 14% compared with untreated nSiO2-filled composites. This work demonstrated the use of rice husk, an agricultural waste, as a nSiO2 source for epoxy resin fillers.

Structural and functional characterization of solute binding proteins for aromatic compounds derived from lignin: p-coumaric acid and related aromatic acids.

PubMed

Tan, Kemin; Chang, Changsoo; Cuff, Marianne; Osipiuk, Jerzy; Landorf, Elizabeth; Mack, Jamey C; Zerbs, Sarah; Joachimiak, Andrzej; Collart, Frank R

2013-10-01

Lignin comprises 15-25% of plant biomass and represents a major environmental carbon source for utilization by soil microorganisms. Access to this energy resource requires the action of fungal and bacterial enzymes to break down the lignin polymer into a complex assortment of aromatic compounds that can be transported into the cells. To improve our understanding of the utilization of lignin by microorganisms, we characterized the molecular properties of solute binding proteins of ATP-binding cassette transporter proteins that interact with these compounds. A combination of functional screens and structural studies characterized the binding specificity of the solute binding proteins for aromatic compounds derived from lignin such as p-coumarate, 3-phenylpropionic acid and compounds with more complex ring substitutions. A ligand screen based on thermal stabilization identified several binding protein clusters that exhibit preferences based on the size or number of aromatic ring substituents. Multiple X-ray crystal structures of protein-ligand complexes for these clusters identified the molecular basis of the binding specificity for the lignin-derived aromatic compounds. The screens and structural data provide new functional assignments for these solute-binding proteins which can be used to infer their transport specificity. This knowledge of the functional roles and molecular binding specificity of these proteins will support the identification of the specific enzymes and regulatory proteins of peripheral pathways that funnel these compounds to central metabolic pathways and will improve the predictive power of sequence-based functional annotation methods for this family of proteins. Copyright © 2013 Wiley Periodicals, Inc.

Structural and functional characterization of solute binding proteins for aromatic compounds derived from lignin: p-coumaric acid and related aromatic acids

PubMed Central

Tan, Kemin; Chang, Changsoo; Cuff, Marianne; Osipiuk, Jerzy; Landorf, Elizabeth; Mack, Jamey C.; Zerbs, Sarah; Joachimiak, Andrzej; Collart, Frank R.

2013-01-01

Lignin comprises 15.25% of plant biomass and represents a major environmental carbon source for utilization by soil microorganisms. Access to this energy resource requires the action of fungal and bacterial enzymes to break down the lignin polymer into a complex assortment of aromatic compounds that can be transported into the cells. To improve our understanding of the utilization of lignin by microorganisms, we characterized the molecular properties of solute binding proteins of ATP.binding cassette transporter proteins that interact with these compounds. A combination of functional screens and structural studies characterized the binding specificity of the solute binding proteins for aromatic compounds derived from lignin such as p-coumarate, 3-phenylpropionic acid and compounds with more complex ring substitutions. A ligand screen based on thermal stabilization identified several binding protein clusters that exhibit preferences based on the size or number of aromatic ring substituents. Multiple X-ray crystal structures of protein-ligand complexes for these clusters identified the molecular basis of the binding specificity for the lignin-derived aromatic compounds. The screens and structural data provide new functional assignments for these solute.binding proteins which can be used to infer their transport specificity. This knowledge of the functional roles and molecular binding specificity of these proteins will support the identification of the specific enzymes and regulatory proteins of peripheral pathways that funnel these compounds to central metabolic pathways and will improve the predictive power of sequence-based functional annotation methods for this family of proteins. PMID:23606130

Effect of increasing dietary fiber on plasma levels of colon-derived solutes in hemodialysis patients.

PubMed

Sirich, Tammy L; Plummer, Natalie S; Gardner, Christopher D; Hostetter, Thomas H; Meyer, Timothy W

2014-09-05

Numerous uremic solutes are derived from the action of colon microbes. Two such solutes, indoxyl sulfate and p-cresol sulfate, have been associated with adverse outcomes in renal failure. This study tested whether increasing dietary fiber in the form of resistant starch would lower the plasma levels of these solutes in patients on hemodialysis. Fifty-six patients on maintenance hemodialysis were randomly assigned to receive supplements containing resistant starch (n=28) or control starch (n=28) daily for 6 weeks in a study conducted between October 2010 and May 2013. Of these, 40 patients (20 in each group) completed the study and were included in the final analysis. Plasma indoxyl sulfate and p-cresol sulfate levels were measured at baseline and week 6. Increasing dietary fiber for 6 weeks significantly reduced the unbound, free plasma level of indoxyl sulfate (median -29% [25th percentile, 75th percentile, -56, -12] for fiber versus -0.4% [-20, 34] for control, P=0.02). The reduction in free plasma levels of indoxyl sulfate was accompanied by a reduction in free plasma levels of p-cresol sulfate (r=0.81, P<0.001). However, the reduction of p-cresol sulfate levels was of lesser magnitude and did not achieve significance (median -28% [-46, 5] for fiber versus 4% [-28, 36] for control, P=0.05). Increasing dietary fiber in hemodialysis patients may reduce the plasma levels of the colon-derived solutes indoxyl sulfate and possibly p-cresol sulfate without the need to intensify dialysis treatments. Further studies are required to determine whether such reduction provides clinical benefits. Copyright © 2014 by the American Society of Nephrology.

The Magnaporthe oryzae Alt A 1-like protein MoHrip1 binds to the plant plasma membrane.

PubMed

Zhang, Yi; Liang, Yingbo; Dong, Yijie; Gao, Yuhan; Yang, Xiufen; Yuan, Jingjing; Qiu, Dewen

2017-10-07

MoHrip1, a protein isolated from Magnaporthe oryzae, belongs to the Alt A 1 (AA1) family. mohrip1 mRNA levels showed inducible expression throughout the infection process in rice. To determine the location of MoHrip1 in M. oryzae, a mohrip1-gfp mutant was generated. Fluorescence microscopy observations and western blotting analysis showed that MoHrip1 was both present in the secretome and abundant in the fungal cell wall. To obtain MoHrip1 protein, we carried out high-yield expression of MoHrip1 in Pichia pastoris. Treatment of tobacco plants with MoHrip1 induced the formation of necrosis, accumulation of reactive oxygen species and expression of several defense-related genes, as well as conferred disease resistance. By fusion to green fluorescent protein, we showed that MoHrip1 was able to bind to the tobacco and rice plant plasma membrane, causing rapid morphological changes at the cellular level, such as cell shrinkage and chloroplast disorganization. These findings indicate that MoHrip1 is a microbe-associated molecular pattern that is perceived by the plant immune system. This is the first study on an AA1 family protein that can bind to the plant plasma membrane. Copyright © 2017 Elsevier Inc. All rights reserved.

Structure and Function of Lipopolysaccharide Binding Protein

NASA Astrophysics Data System (ADS)

Schumann, Ralf R.; Leong, Steven R.; Flaggs, Gail W.; Gray, Patrick W.; Wright, Samuel D.; Mathison, John C.; Tobias, Peter S.; Ulevitch, Richard J.

1990-09-01

The primary structure of lipopolysaccharide binding protein (LBP), a trace plasma protein that binds to the lipid A moiety of bacterial lipopolysaccharides (LPSs), was deduced by sequencing cloned complementary DNA. LBP shares sequence identity with another LPS binding protein found in granulocytes, bactericidal/permeability-increasing protein, and with cholesterol ester transport protein of the plasma. LBP may control the response to LPS under physiologic conditions by forming high-affinity complexes with LPS that bind to monocytes and macrophages, which then secrete tumor necrosis factor. The identification of this pathway for LPS-induced monocyte stimulation may aid in the development of treatments for diseases in which Gram-negative sepsis or endotoxemia are involved.

Solubility and binding properties of PEGylated lysozyme derivatives with increasing molecular weight on hydrophobic-interaction chromatographic resins.

PubMed

Müller, Egbert; Josic, Djuro; Schröder, Tim; Moosmann, Anna

2010-07-09

Dynamic binding capacities and resolution of PEGylated lysozyme derivatives with varying molecular weights of poly (ethylene) glycol (PEG) with 5 kDa, 10 kDa and 30 kDa for HIC resins and columns are presented. To find the optimal range for the operating conditions, solubility studies were performed by high-throughput analyses in a 96-well plate format, and optimal salt concentrations and pH values were determined. The solubility of PEG-proteins was strongly influenced by the length of the PEG moiety. Large differences in the solubilities of PEGylated lysozymes in two different salts, ammonium sulfate and sodium chloride were found. Solubility of PEGylated lysozyme derivatives in ammonium sulfate decreases with increased length of attached PEG chains. In sodium chloride all PEGylated lysozyme derivatives are fully soluble in a concentration range between 0.1 mg protein/ml and 10 mg protein/ml. The binding capacities for PEGylated lysozyme to HIC resins are dependent on the salt type and molecular weight of the PEG polymer. In both salt solutions, ammonium sulfate and sodium chloride, the highest binding capacity of the resin was found for 5 kDa PEGylated lysozyme. For both native lysozyme and 30 kDa mono-PEGylated lysozyme the binding capacities were lower. In separation experiments on a TSKgel Butyl-NPR hydrophobic-interaction column with ammonium sulfate as mobile phase, the elution order was: native lysozyme, 5 kDa mono-PEGylated lysozyme and oligo-PEGylated lysozyme. This elution order was found to be reversed when sodium chloride was used. Furthermore, the resolution of the three mono-PEGylated forms was not possible with this column and ammonium sulfate as mobile phase. In 4 M sodium chloride a resolution of all PEGylated lysozyme forms was achieved. A tentative explanation for these phenomena can be the increased solvation of the PEG polymers in sodium chloride which changes the usual attractive hydrophobic forces in ammonium sulfate to more repulsive

Blood Plasma-Derived Anti-Glycan Antibodies to Sialylated and Sulfated Glycans Identify Ovarian Cancer Patients

PubMed Central

Pochechueva, Tatiana; Chinarev, Alexander; Schoetzau, Andreas; Fedier, André; Bovin, Nicolai V.; Hacker, Neville F.; Jacob, Francis; Heinzelmann-Schwarz, Viola

2016-01-01

Altered levels of naturally occurring anti-glycan antibodies (AGA) circulating in human blood plasma are found in different pathologies including cancer. Here the levels of AGA directed against 22 negatively charged (sialylated and sulfated) glycans were assessed in high-grade serous ovarian cancer (HGSOC, n = 22) patients and benign controls (n = 31) using our previously developed suspension glycan array (SGA). Specifically, the ability of AGA to differentiate between controls and HGSOC, the most common and aggressive type of ovarian cancer with a poor outcome was determined. Results were compared to CA125, the commonly used ovarian cancer biomarker. AGA to seven glycans that significantly (P<0.05) differentiated between HGSOC and control were identified: AGA to top candidates SiaTn and 6-OSulfo-TF (both IgM) differentiated comparably to CA125. The area under the curve (AUC) of a panel of AGA to 5 glycans (SiaTn, 6-OSulfo-TF, 6-OSulfo-LN, SiaLea, and GM2) (0.878) was comparable to CA125 (0.864), but it markedly increased (0.985) when combined with CA125. AGA to SiaTn and 6-OSulfo-TF were also valuable predictors for HGSOC when CA125 values appeared inconclusive, i.e. were below a certain threshold. AGA-glycan binding was in some cases isotype-dependent and sensitive to glycosidic linkage switch (α2–6 vs. α2–3), to sialylation, and to sulfation of the glycans. In conclusion, plasma-derived AGA to sialylated and sulfated glycans including SiaTn and 6-OSulfo-TF detected by SGA present a valuable alternative to CA125 for differentiating controls from HGSOC patients and for predicting the likelihood of HGSOC, and may be potential HGSOC tumor markers. PMID:27764122

Blood Plasma-Derived Anti-Glycan Antibodies to Sialylated and Sulfated Glycans Identify Ovarian Cancer Patients.

PubMed

Pochechueva, Tatiana; Chinarev, Alexander; Schoetzau, Andreas; Fedier, André; Bovin, Nicolai V; Hacker, Neville F; Jacob, Francis; Heinzelmann-Schwarz, Viola

2016-01-01

Altered levels of naturally occurring anti-glycan antibodies (AGA) circulating in human blood plasma are found in different pathologies including cancer. Here the levels of AGA directed against 22 negatively charged (sialylated and sulfated) glycans were assessed in high-grade serous ovarian cancer (HGSOC, n = 22) patients and benign controls (n = 31) using our previously developed suspension glycan array (SGA). Specifically, the ability of AGA to differentiate between controls and HGSOC, the most common and aggressive type of ovarian cancer with a poor outcome was determined. Results were compared to CA125, the commonly used ovarian cancer biomarker. AGA to seven glycans that significantly (P<0.05) differentiated between HGSOC and control were identified: AGA to top candidates SiaTn and 6-OSulfo-TF (both IgM) differentiated comparably to CA125. The area under the curve (AUC) of a panel of AGA to 5 glycans (SiaTn, 6-OSulfo-TF, 6-OSulfo-LN, SiaLea, and GM2) (0.878) was comparable to CA125 (0.864), but it markedly increased (0.985) when combined with CA125. AGA to SiaTn and 6-OSulfo-TF were also valuable predictors for HGSOC when CA125 values appeared inconclusive, i.e. were below a certain threshold. AGA-glycan binding was in some cases isotype-dependent and sensitive to glycosidic linkage switch (α2-6 vs. α2-3), to sialylation, and to sulfation of the glycans. In conclusion, plasma-derived AGA to sialylated and sulfated glycans including SiaTn and 6-OSulfo-TF detected by SGA present a valuable alternative to CA125 for differentiating controls from HGSOC patients and for predicting the likelihood of HGSOC, and may be potential HGSOC tumor markers.

Differential effect of amyloid beta peptides on mitochondrial axonal trafficking depends on their state of aggregation and binding to the plasma membrane.

PubMed

Zhang, Liang; Trushin, Sergey; Christensen, Trace A; Tripathi, Utkarsh; Hong, Courtney; Geroux, Rachel E; Howell, Kyle G; Poduslo, Joseph F; Trushina, Eugenia

2018-06-01

Inhibition of mitochondrial axonal trafficking by amyloid beta (Aβ) peptides has been implicated in early pathophysiology of Alzheimer's Disease (AD). Yet, it remains unclear whether the loss of motility inevitably induces the loss of mitochondrial function, and whether restoration of axonal trafficking represents a valid therapeutic target. Moreover, while some investigations identify Aβ oligomers as the culprit of trafficking inhibition, others propose that fibrils play the detrimental role. We have examined the effect of a panel of Aβ peptides with different mutations found in familial AD on mitochondrial motility in primary cortical mouse neurons. Peptides with higher propensity to aggregate inhibit mitochondrial trafficking to a greater extent with fibrils inducing the strongest inhibition. Binding of Aβ peptides to the plasma membrane was sufficient to induce trafficking inhibition where peptides with reduced plasma membrane binding and internalization had lesser effect on mitochondrial motility. We also found that Aβ peptide with Icelandic mutation A673T affects axonal trafficking of mitochondria but has very low rates of plasma membrane binding and internalization in neurons, which could explain its relatively low toxicity. Inhibition of mitochondrial dynamics caused by Aβ peptides or fibrils did not instantly affect mitochondrial bioenergetic and function. Our results support a mechanism where inhibition of axonal trafficking is initiated at the plasma membrane by soluble low molecular weight Aβ species and is exacerbated by fibrils. Since trafficking inhibition does not coincide with the loss of mitochondrial function, restoration of axonal transport could be beneficial at early stages of AD progression. However, strategies designed to block Aβ aggregation or fibril formation alone without ensuring the efficient clearance of soluble Aβ may not be sufficient to alleviate the trafficking phenotype. Copyright © 2018 The Authors. Published by

Differential effect of amyloid beta peptides on mitochondrial axonal trafficking depends on their state of aggregation and binding to the plasma membrane

PubMed Central

Zhang, Liang; Trushin, Sergey; Christensen, Trace A.; Tripathi, Utkarsh; Hong, Courtney; Geroux, Rachel E.; Howell, Kyle G.; Poduslo, Joseph F.; Trushina, Eugenia

2018-01-01

Inhibition of mitochondrial axonal trafficking by amyloid beta (Aβ) peptides has been implicated in early pathophysiology of Alzheimer’s Disease (AD). Yet, it remains unclear whether the loss of motility inevitably induces the loss of mitochondrial function, and whether restoration of axonal trafficking represents a valid therapeutic target. Moreover, while some investigations identify Aβ oligomers as the culprit of trafficking inhibition, others propose that fibrils play the detrimental role. We have examined the effect of a panel of Aβ peptides with different mutations found in familial AD on mitochondrial motility in primary cortical mouse neurons. Peptides with higher propensity to aggregate inhibit mitochondrial trafficking to a greater extent with fibrils inducing the strongest inhibition. Binding of Aβ peptides to the plasma membrane was sufficient to induce trafficking inhibition where peptides with reduced plasma membrane binding and internalization had lesser effect on mitochondrial motility. We also found that Aβ peptide with Icelandic mutation A673T affects axonal trafficking of mitochondria but has very low rates of plasma membrane binding and internalization in neurons, which could explain its relatively low toxicity. Inhibition of mitochondrial dynamics caused by Aβ peptides or fibrils did not instantly affect mitochondrial bioenergetic and function. Our results support a mechanism where inhibition of axonal trafficking is initiated at the plasma membrane by soluble low molecular weight Aβ species and is exacerbated by fibrils. Since trafficking inhibition does not coincide with the loss of mitochondrial function, restoration of axonal transport could be beneficial at early stages of AD progression. However, strategies designed to block Aβ aggregation or fibril formation alone without ensuring the efficient clearance of soluble Aβ may not be sufficient to alleviate the trafficking phenotype. PMID:29477640

Core Binding Site of a Thioflavin-T-Derived Imaging Probe on Amyloid β Fibrils Predicted by Computational Methods.

PubMed

Kawai, Ryoko; Araki, Mitsugu; Yoshimura, Masashi; Kamiya, Narutoshi; Ono, Masahiro; Saji, Hideo; Okuno, Yasushi

2018-05-16

Development of new diagnostic imaging probes for Alzheimer's disease, such as positron emission tomography (PET) and single photon emission computed tomography (SPECT) probes, has been strongly desired. In this study, we investigated the most accessible amyloid β (Aβ) binding site of [ 123 I]IMPY, a Thioflavin-T-derived SPECT probe, using experimental and computational methods. First, we performed a competitive inhibition assay with Orange-G, which recognizes the KLVFFA region in Aβ fibrils, suggesting that IMPY and Orange-G bind to different sites in Aβ fibrils. Next, we precisely predicted the IMPY binding site on a multiple-protofilament Aβ fibril model using computational approaches, consisting of molecular dynamics and docking simulations. We generated possible IMPY-binding structures using docking simulations to identify candidates for probe-binding sites. The binding free energy of IMPY with the Aβ fibril was calculated by a free energy simulation method, MP-CAFEE. These computational results suggest that IMPY preferentially binds to an interfacial pocket located between two protofilaments and is stabilized mainly through hydrophobic interactions. Finally, our computational approach was validated by comparing it with the experimental results. The present study demonstrates the possibility of computational approaches to screen new PET/SPECT probes for Aβ imaging.

Binding of phosphatidic acid to 14-3-3 proteins hampers their ability to activate the plant plasma membrane H+-ATPase.

PubMed

Camoni, Lorenzo; Di Lucente, Cristina; Pallucca, Roberta; Visconti, Sabina; Aducci, Patrizia

2012-08-01

Phosphatidic acid is a phospholipid second messenger implicated in various cellular processes in eukaryotes. In plants, production of phosphatidic acid is triggered in response to a number of biotic and abiotic stresses. Here, we show that phosphatidic acid binds to 14-3-3 proteins, a family of regulatory proteins which bind client proteins in a phosphorylation-dependent manner. Binding of phosphatidic acid involves the same 14-3-3 region engaged in protein target binding. Consequently, micromolar phosphatidic acid concentrations significantly hamper the interaction of 14-3-3 proteins with the plasma membrane H(+)-ATPase, a well characterized plant 14-3-3 target, thus inhibiting the phosphohydrolitic enzyme activity. Moreover, the proton pump is inhibited when endogenous PA production is triggered by phospholipase D and the G protein agonist mastoparan-7. Hence, our data propose a possible mechanism involving PA that regulates 14-3-3-mediated cellular processes in response to stress. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

Determination of plasma albumin concentration in healthy and diseased turtles: a comparison of protein electrophoresis and the bromcresol green dye-binding method.

PubMed

Müller, Kerstin; Brunnberg, Leo

2010-03-01

In reptile medicine, plasma chemistry analysis is widely used for the evaluation of an individual's health status. The standard method for the determination of plasma albumin concentration is protein electrophoresis combined with the determination of total protein concentration, but the bromcresol green (BCG) dye-binding method is also used. The reliability of the BCG method for the measurement of albumin concentration in reptiles is unknown. The aim of this study was to compare the plasma albumin values of turtles obtained by protein electrophoresis and the BCG method. Between March 2008 and September 2008, heparinized plasma samples from 16 clinically healthy and 10 diseased turtles of different species were collected. Plasma albumin concentrations were measured by protein electrophoresis and by the BCG method. The results of the 2 methods were compared using Passing-Bablok regression and Bland-Altman plots. Albumin concentration measured by BCG was weakly correlated with the corresponding protein electrophoretic values in all turtles (r(s)=.610, P<.001) and in healthy turtles evaluated separately (r(s)=.700, P=.003), whereas in diseased turtles no such correlation was found (r(s)=.374, P=.287). The albumin concentration measured with the 2 different methods differed significantly in all turtles (P=.009; Wilcoxon's test) and in healthy turtles (P=.005) but not in diseased animals (P=.241). In the Bland-Altman plot a systematic error was found between the 2 methods in diseased turtles. Measurement of albumin by the BCG dye-binding method may lead to inaccurate results for plasma albumin concentration, especially in ill turtles. Therefore, for health assessment in turtles, albumin should be measured by protein electrophoresis.

Increase of EPA-derived hydroxy, epoxy and dihydroxy fatty acid levels in human plasma after a single dose of long-chain omega-3 PUFA.

PubMed

Schuchardt, Jan Philipp; Schneider, Inga; Willenberg, Ina; Yang, Jun; Hammock, Bruce D; Hahn, Andreas; Schebb, Nils Helge

2014-06-01

Several supplementation studies with long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA) describe an increase of EPA-derived hydroxy, epoxy and dihydroxy fatty acids in blood, while changes in levels of other LC n-3 and n-6 PUFA-derived oxylipins were minor. In order to investigate the kinetics of changes in oxylipin levels in response to LC n-3 PUFA ingestion, we conducted a single dose treatment study with healthy subjects. In the present kinetic study, we compared patterns of hydroxy, epoxy and dihydroxy fatty acids in plasma of 6 healthy men before and after 6, 8, 24, and 48h of fish oil (1008mg EPA and 672mg DHA) ingestion. Levels of EPA- as well as other LC PUFA-derived hydroxy, epoxy and dihydroxy fatty acids were analyzed in plasma by LC-MS. Additionally, levels of these oxylipins were compared with their parent PUFA levels in plasma phospholipids. All EPA-derived oxylipin levels were significantly increased 6h after LC n-3 PUFA ingestion and gradually drop thereafter reaching the baseline levels about 48h after treatment. The relative increase in EPA plasma phospholipid levels highly correlated with the increase of plasma EPA-derived oxylipin levels at different time points. In contrast, plasma levels of arachidonic acid- and DHA-derived oxylipins as well as parent PUFA levels in plasma phospholipids were hardly changed. Our findings demonstrate that a single dose of LC n-3 PUFAs can rapidly induce a shift in the EPA oxylipin profile of healthy subjects within a few hours. Taking the high biological activity of the EPA-derived epoxy fatty acids into account, even short-term treatment with LC n-3 PUFAs may cause systemic effects, which warrant further investigation. Copyright © 2014 Elsevier Inc. All rights reserved.

Effect of conglutinin on phagocytic activity of bovine granulocytes.

PubMed

Dec, M; Wernicki, A; Puchalski, A; Urban-Chmiel, R; Radej, S

2012-01-01

In the present study we investigated the effect of bovine conglutinin on the phagocytic activity of leukocytes. We measured both the chemotactic activity of conglutinin and its effect on the internalization of zymosan particles and E. coli by granulocytes. We also assessed the binding of conglutinin to various microorganisms isolated from clinical cases in cattle. We showed that conglutinin binds strongly to the surface of yeast cells and to mannan-rich zymosan particles, while weak binding was observed in the case of the bacterial strains tested, including those whose O antigen is composed of mannan. Conglutinin (1-10 microg/ml) neither acts as a chemotactic factor for peripheral blood leukocytes nor affects ingestion of E. coli by granulocytes. However, as flow cytometry based assay showed, conglutinin (0.1-1 microg/ml) increased ingestion of zymosan expressed as mean fluorescence intensity (MFI) of positive cells.

A novel exopolysaccharide elicitor from endophytic fungus Gilmaniella sp. AL12 on volatile oils accumulation in Atractylodes lancea

NASA Astrophysics Data System (ADS)

Chen, Fei; Ren, Cheng-Gang; Zhou, Tong; Wei, Yu-Jia; Dai, Chuan-Chao

2016-10-01

Endophytes and plants can establish specific long-term symbiosis through the accumulation of secondary metabolites. Previous studies have shown that the endophytic fungus Gilmaniella sp. AL12 can stimulate Atractylodes lancea to produce volatile oils. The purpose of this report is to investigate key factors involved in the stimulation of A. lancea by AL12 and reveal the mechanism. We identified the active component from AL12 as an extracellular mannan with a polymerization degree of 26-42. Differential membrane proteomics of A. lancea was performed by 2D electrophoresis. The results showed that there were significant differences in the expression of 83 proteins. Based on these results, we conclude that AL12 secreted mannan contributes to the antagonistic balance seen in interactions between AL12 and A. lancea. One portion of the mannan was degraded to mannose for hexokinase activation, promoting photosynthesis and energy metabolism, with a potential metabolic fluxes flowing towards terpenoid biosynthesis. The other portion of the mannan directly enhanced autoimmunity of A. lancea through G protein-mediated signal transduction and the mannan-binding lectin pathway. Volatile oil accumulation was ultimately promoted in subsequent defense reactions. This study provides a new perspective on the regulation of secondary metabolites by endophytic fungal elicitors in medicinal plants.

A novel exopolysaccharide elicitor from endophytic fungus Gilmaniella sp. AL12 on volatile oils accumulation in Atractylodes lancea.

PubMed

Chen, Fei; Ren, Cheng-Gang; Zhou, Tong; Wei, Yu-Jia; Dai, Chuan-Chao

2016-10-05

Endophytes and plants can establish specific long-term symbiosis through the accumulation of secondary metabolites. Previous studies have shown that the endophytic fungus Gilmaniella sp. AL12 can stimulate Atractylodes lancea to produce volatile oils. The purpose of this report is to investigate key factors involved in the stimulation of A. lancea by AL12 and reveal the mechanism. We identified the active component from AL12 as an extracellular mannan with a polymerization degree of 26-42. Differential membrane proteomics of A. lancea was performed by 2D electrophoresis. The results showed that there were significant differences in the expression of 83 proteins. Based on these results, we conclude that AL12 secreted mannan contributes to the antagonistic balance seen in interactions between AL12 and A. lancea. One portion of the mannan was degraded to mannose for hexokinase activation, promoting photosynthesis and energy metabolism, with a potential metabolic fluxes flowing towards terpenoid biosynthesis. The other portion of the mannan directly enhanced autoimmunity of A. lancea through G protein-mediated signal transduction and the mannan-binding lectin pathway. Volatile oil accumulation was ultimately promoted in subsequent defense reactions. This study provides a new perspective on the regulation of secondary metabolites by endophytic fungal elicitors in medicinal plants.

Synthesis, crystal structure, DFT calculation and DNA binding studies of new water-soluble derivatives of dppz

NASA Astrophysics Data System (ADS)

Aminzadeh, Mohammad; Eslami, Abbas; Kia, Reza; Aleeshah, Roghayeh

2017-10-01

Diquaternarization of dipyrido-[2,3-a:2‧,3‧-c]-phenazine,(dppz) and its analogous dipyrido-[2,3-a:2‧,3‧-c]-dimethylphenazine,(dppx) using 1,3-dibromopropane afford new water-soluble derivatives of phenazine, propylene-bipyridyldiylium-phenazine (1) and propylene-bipyridyldiylium-dimethylphenazine (2). The compounds have been characterized by means of FT-IR, NMR, elemental analysis and conductometric measurements and their structure were determined by X-ray crystallography. The experimental studies on the compounds have been accompanied computationally by Density Functional Theory (DFT) calculations. The DNA binding properties of both compounds to calf thymus DNA (ctDNA) were investigated by UV-Vis absorption and emission methods. The expanded UV-Vis spectral data matrix was analyzed by multivariate curve resolution-alternating least squares (MCR-ALS) technique to obtain the concentration profile and pure spectra of all reaction species which existed in the interaction procedure. Multivariate curve resolution may help us to give a better understanding of the 1(Cl)2-ctDNA and 2(Cl)2-ctDNA interaction mechanism. The results suggest that both compounds bind tightly to DNA through intercalation mechanism and the DNA binding affinity of 2 is slightly lower than that of 1 due to steric hindrance of the methyl group. Also, thermal denaturation studies reveal that these compounds show strong affinity for binding with calf thymus DNA. The thermodynamic parameters of the DNA binding process were obtained from the temperature dependence of the binding constants and the results showed that binding of both compounds to DNA is an enthalpically driven process that is in agreement with proposed DNA intercalation capability of these compounds.

Plasma glial cell line-derived neurotrophic factor in patients with major depressive disorder: a preliminary study.

PubMed

Lee, Bun-Hee; Hong, Jin-Pyo; Hwang, Jung-A; Na, Kyoung-Sae; Kim, Won-Joong; Trigo, Jose; Kim, Yong-Ku

2016-02-01

Some clinical studies have reported reduced peripheral glial cell line-derived neurotrophic factor (GDNF) level in elderly patients with major depressive disorder (MDD). We verified whether a reduction in plasma GDNF level was associated with MDD. Plasma GDNF level was measured in 23 healthy control subjects and 23 MDD patients before and after 6 weeks of treatment. Plasma GDNF level in MDD patients at baseline did not differ from that in healthy controls. Plasma GDNF in MDD patients did not differ significantly from baseline to the end of treatment. GDNF level was significantly lower in recurrent-episode MDD patients than in first-episode patients before and after treatment. Our findings revealed significantly lower plasma GDNF level in recurrent-episode MDD patients, although plasma GDNF levels in MDD patients and healthy controls did not differ significantly. The discrepancy between our study and previous studies might arise from differences in the recurrence of depression or the ages of the MDD patients.

Copolymers enhance selective bacterial community colonization for potential root zone applications.

PubMed

Pham, Vy T H; Murugaraj, Pandiyan; Mathes, Falko; Tan, Boon K; Truong, Vi Khanh; Murphy, Daniel V; Mainwaring, David E

2017-11-21

Managing the impact of anthropogenic and climate induced stress on plant growth remains a challenge. Here we show that polymeric hydrogels, which maintain their hydrous state, can be designed to exploit functional interactions with soil microorganisms. This microbial enhancement may mitigate biotic and abiotic stresses limiting productivity. The presence of mannan chains within synthetic polyacrylic acid (PAA) enhanced the dynamics and selectivity of bacterial ingress in model microbial systems and soil microcosms. Pseudomonas fluorescens exhibiting high mannan binding adhesins showed higher ingress and localised microcolonies throughout the polymeric network. In contrast, ingress of Bacillus subtilis, lacking adhesins, was unaltered by mannan showing motility comparable to bulk liquids. Incubation within microcosms of an agricultural soil yielded hydrogel populations significantly increased from the corresponding soil. Bacterial diversity was markedly higher in mannan containing hydrogels compared to both control polymer and soil, indicating enhanced selectivity towards microbial families that contain plant beneficial species. Here we propose functional polymers applied to the potential root zone which can positively influence rhizobacteria colonization and potentially plant growth as a new approach to stress tolerance.

Characterization of complexes between phenethylamine enantiomers and β-cyclodextrin derivatives by capillary electrophoresis-Determination of binding constants and complex mobilities.

PubMed

Wahl, Joachim; Furuishi, Takayuki; Yonemochi, Etsuo; Meinel, Lorenz; Holzgrabe, Ulrike

2017-04-01

To optimize chiral separation conditions and to improve the knowledge of enantioseparation, it is important to know the binding constants K between analytes and cyclodextrins and the electrophoretic mobilities of the temporarily formed analyte-cyclodextrin-complexes. K values for complexes between eight phenethylamine enantiomers, namely ephedrine, pseudoephedrine, methylephedrine and norephedrine, and four different β-cyclodextrin derivatives were determined by affinity capillary electrophoresis. The binding constants were calculated from the electrophoretic mobility values of the phenethylamine enantiomers at increasing concentrations of cyclodextrins in running buffer. Three different linear plotting methods (x-reciprocal, y-reciprocal, double reciprocal) and nonlinear regression were used for the determination of binding constants with β-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, methyl-β-cyclodextrin and 6-O-α-maltosyl-β-cyclodextrin. The cyclodextrin concentration in a 50 mM phosphate buffer pH 3.0 was varied from 0 to 12 mM. To investigate the influence of the binding constant values on the enantioseparation the observed electrophoretic selectivities were compared with the obtained K values and the calculated enantiomer-cyclodextrin-complex mobilities. The different electrophoretic mobilities of the temporarily formed complexes were crucial factors for the migration order and enantioseparation of ephedrine derivatives. To verify the apparent binding constants determined by capillary electrophoresis, a titration process using ephedrine enantiomers and β-cyclodextrin was carried out. Furthermore, the isothermal titration calorimetry measurements gave information about the thermal properties of the complexes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of sequence-structure RNA binding motifs for SELEX-derived aptamers.

PubMed

Hoinka, Jan; Zotenko, Elena; Friedman, Adam; Sauna, Zuben E; Przytycka, Teresa M

2012-06-15

Systematic Evolution of Ligands by EXponential Enrichment (SELEX) represents a state-of-the-art technology to isolate single-stranded (ribo)nucleic acid fragments, named aptamers, which bind to a molecule (or molecules) of interest via specific structural regions induced by their sequence-dependent fold. This powerful method has applications in designing protein inhibitors, molecular detection systems, therapeutic drugs and antibody replacement among others. However, full understanding and consequently optimal utilization of the process has lagged behind its wide application due to the lack of dedicated computational approaches. At the same time, the combination of SELEX with novel sequencing technologies is beginning to provide the data that will allow the examination of a variety of properties of the selection process. To close this gap we developed, Aptamotif, a computational method for the identification of sequence-structure motifs in SELEX-derived aptamers. To increase the chances of identifying functional motifs, Aptamotif uses an ensemble-based approach. We validated the method using two published aptamer datasets containing experimentally determined motifs of increasing complexity. We were able to recreate the author's findings to a high degree, thus proving the capability of our approach to identify binding motifs in SELEX data. Additionally, using our new experimental dataset, we illustrate the application of Aptamotif to elucidate several properties of the selection process.

Identification of sequence–structure RNA binding motifs for SELEX-derived aptamers

PubMed Central

Hoinka, Jan; Zotenko, Elena; Friedman, Adam; Sauna, Zuben E.; Przytycka, Teresa M.

2012-01-01

Motivation: Systematic Evolution of Ligands by EXponential Enrichment (SELEX) represents a state-of-the-art technology to isolate single-stranded (ribo)nucleic acid fragments, named aptamers, which bind to a molecule (or molecules) of interest via specific structural regions induced by their sequence-dependent fold. This powerful method has applications in designing protein inhibitors, molecular detection systems, therapeutic drugs and antibody replacement among others. However, full understanding and consequently optimal utilization of the process has lagged behind its wide application due to the lack of dedicated computational approaches. At the same time, the combination of SELEX with novel sequencing technologies is beginning to provide the data that will allow the examination of a variety of properties of the selection process. Results: To close this gap we developed, Aptamotif, a computational method for the identification of sequence–structure motifs in SELEX-derived aptamers. To increase the chances of identifying functional motifs, Aptamotif uses an ensemble-based approach. We validated the method using two published aptamer datasets containing experimentally determined motifs of increasing complexity. We were able to recreate the author's findings to a high degree, thus proving the capability of our approach to identify binding motifs in SELEX data. Additionally, using our new experimental dataset, we illustrate the application of Aptamotif to elucidate several properties of the selection process. Contact: przytyck@ncbi.nlm.nih.gov, Zuben.Sauna@fda.hhs.gov PMID:22689764

Novel Triazole-Quinoline Derivatives as Selective Dual Binding Site Acetylcholinesterase Inhibitors.

PubMed

Mantoani, Susimaire P; Chierrito, Talita P C; Vilela, Adriana F L; Cardoso, Carmen L; Martínez, Ana; Carvalho, Ivone

2016-02-05

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder worldwide. Currently, the only strategy for palliative treatment of AD is to inhibit acetylcholinesterase (AChE) in order to increase the concentration of acetylcholine in the synaptic cleft. Evidence indicates that AChE also interacts with the β-amyloid (Aβ) protein, acting as a chaperone and increasing the number and neurotoxicity of Aβ fibrils. It is known that AChE has two binding sites: the peripheral site, responsible for the interactions with Aβ, and the catalytic site, related with acetylcholine hydrolysis. In this work, we reported the synthesis and biological evaluation of a library of new tacrine-donepezil hybrids, as a potential dual binding site AChE inhibitor, containing a triazole-quinoline system. The synthesis of hybrids was performed in four steps using the click chemistry strategy. These compounds were evaluated as hAChE and hBChE inhibitors, and some derivatives showed IC50 values in the micro-molar range and were remarkably selective towards hAChE. Kinetic assays and molecular modeling studies confirm that these compounds block both catalytic and peripheral AChE sites. These results are quite interesting since the triazole-quinoline system is a new structural scaffold for AChE inhibitors. Furthermore, the synthetic approach is very efficient for the preparation of target compounds, allowing a further fruitful new chemical library optimization.

Thorium binding by biochar fibres derived from Luffa Cylindrica after controlled surface oxidation

NASA Astrophysics Data System (ADS)

Liatsou, Ioanna; Christodoulou, Eleni; Paschalidis, Ioannis

2017-04-01

Controlled surface modification of biochar fibres derived from Luffa Cylindrica sponges has been carried out by nitric acid and the degree of oxidation could be controlled by changing the acid concentration or the reaction time. The extent of surface oxidation has been quantified by acid-base titration and FTIR-spectroscopy. Furthermore, thorium binding has been studied as a function of various parameters and the experimental results show that even under strong acidic conditions the relative sorption is above 70% and the sorption capacity of the biochar fibres for Th(IV) at pH 3 is qmax= 70 gṡkg-1.

Synthesis and characterization of a Eu-DTPA-PEGO-MSH(4) derivative for evaluation of binding of multivalent molecules to melanocortin receptors.

PubMed

Xu, Liping; Vagner, Josef; Alleti, Ramesh; Rao, Venkataramanarao; Jagadish, Bhumasamudram; Morse, David L; Hruby, Victor J; Gillies, Robert J; Mash, Eugene A

2010-04-15

A labeled variant of MSH(4), a tetrapeptide that binds to the human melanocortin 4 receptor (hMC4R) with low microM affinity, was prepared by solid-phase synthesis methods, purified, and characterized. The labeled ligand, Eu-DTPA-PEGO-His-dPhe-Arg-Trp-NH(2), exhibited a K(d) for hMC4R of 9.1+/-1.4 microM, approximately 10-fold lower affinity than the parental ligand. The labeled MSH(4) derivative was employed in a competitive binding assay to characterize the interactions of hMC4R with monovalent and divalent MSH(4) constructs derived from squalene. The results were compared with results from a similar assay that employed a more potent labeled ligand, Eu-DTPA-NDP-alpha-MSH. While results from the latter assay reflected only statistical effects, results from the former assay reflected a mixture of statistical, proximity, and/or cooperative binding effects. Copyright 2010 Elsevier Ltd. All rights reserved.

Quantitative structure activity relationships from optimised ab initio bond lengths: steroid binding affinity and antibacterial activity of nitrofuran derivatives

NASA Astrophysics Data System (ADS)

Smith, P. J.; Popelier, P. L. A.

2004-02-01

The present day abundance of cheap computing power enables the use of quantum chemical ab initio data in Quantitative Structure-Activity Relationships (QSARs). Optimised bond lengths are a new such class of descriptors, which we have successfully used previously in representing electronic effects in medicinal and ecological QSARs (enzyme inhibitory activity, hydrolysis rate constants and pKas). Here we use AM1 and HF/3-21G* bond lengths in conjunction with Partial Least Squares (PLS) and a Genetic Algorithm (GA) to predict the Corticosteroid-Binding Globulin (CBG) binding activity of the classic steroid data set, and the antibacterial activity of nitrofuran derivatives. The current procedure, which does not require molecular alignment, produces good r2 and q2 values. Moreover, it highlights regions in the common steroid skeleton deemed relevant to the active regions of the steroids and nitrofuran derivatives.

Peptide p5 binds both heparinase-sensitive glycosaminoglycans and fibrils in patient-derived AL amyloid extracts.

PubMed

Martin, Emily B; Williams, Angela; Heidel, Eric; Macy, Sallie; Kennel, Stephen J; Wall, Jonathan S

2013-06-21

In previously published work, we have described heparin-binding synthetic peptides that preferentially recognize amyloid deposits in a mouse model of reactive systemic (AA) amyloidosis and can be imaged by using positron and single photon emission tomographic imaging. We wanted to extend these findings to the most common form of visceral amyloidosis, namely light chain (AL); however, there are no robust experimental animal models of AL amyloidosis. To further define the binding of the lead peptide, p5, to AL amyloid, we characterized the reactivity in vitro of p5 with in situ and patient-derived AL amyloid extracts which contain both hypersulfated heparan sulfate proteoglycans as well as amyloid fibrils. Histochemical staining demonstrated that the peptide specifically localized with tissue-associated AL amyloid deposits. Although we anticipated that p5 would undergo electrostatic interactions with the amyloid-associated glycosaminoglycans expressing heparin-like side chains, no significant correlation between peptide binding and glycosaminoglycan content within amyloid extracts was observed. In contrast, following heparinase I treatment, although overall binding was reduced, a positive correlation between peptide binding and amyloid fibril content became evident. This interaction was further confirmed using synthetic light chain fibrils that contain no carbohydrates. These data suggest that p5 can bind to both the sulfated glycosaminoglycans and protein fibril components of AL amyloid. Understanding these complex electrostatic interactions will aid in the optimization of synthetic peptides for use as amyloid imaging agents and potentially as therapeutics for the treatment of amyloid diseases. Copyright © 2013 Elsevier Inc. All rights reserved.

New Binding Mode to TNF-Alpha Revealed by Ubiquitin-Based Artificial Binding Protein

PubMed Central

Hoffmann, Andreas; Kovermann, Michael; Lilie, Hauke; Fiedler, Markus; Balbach, Jochen; Rudolph, Rainer; Pfeifer, Sven

2012-01-01

A variety of approaches have been employed to generate binding proteins from non-antibody scaffolds. Utilizing a beta-sheet of the human ubiquitin for paratope creation we obtained binding proteins against tumor necrosis factor (TNF)-alpha. The bioactive form of this validated pharmacological target protein is a non-covalently linked homo-trimer. This structural feature leads to the observation of a certain heterogeneity concerning the binding mode of TNF-alpha binding molecules, for instance in terms of monomer/trimer specificity. We analyzed a ubiquitin-based TNF-alpha binder, selected by ribosome display, with a particular focus on its mode of interaction. Using enzyme-linked immunosorbent assays, specific binding to TNF-alpha with nanomolar affinity was observed. In isothermal titration calorimetry we obtained comparable results regarding the affinity and detected an exothermic reaction with one ubiquitin-derived binding molecule binding one TNF-alpha trimer. Using NMR spectroscopy and other analytical methods the 1∶3 stoichiometry could be confirmed. Detailed binding analysis showed that the interaction is affected by the detergent Tween-20. Previously, this phenomenon was reported only for one other type of alternative scaffold-derived binding proteins – designed ankyrin repeat proteins – without further investigation. As demonstrated by size exclusion chromatography and NMR spectroscopy, the presence of the detergent increases the association rate significantly. Since the special architecture of TNF-alpha is known to be modulated by detergents, the access to the recognized epitope is indicated to be restricted by conformational transitions within the target protein. Our results suggest that the ubiquitin-derived binding protein targets a new epitope on TNF-alpha, which differs from the epitopes recognized by TNF-alpha neutralizing antibodies. PMID:22363609

Assay of free captopril in human plasma as monobromobimane derivative, using RPLC/(+)ESI/MS/MS: validation aspects and bioequivalence evaluation.

PubMed

Medvedovici, Andrei; Albu, Florin; Sora, Iuliana Daniela; Udrescu, Stefan; Galaon, Toma; David, Victor

2009-10-01

A sensitive method for determination of free captopril as monobromobimane derivative in plasma samples is discussed. The internal standard (IS) was 5-methoxy-1H-benzimidazole-2-thiol. Derivatization with monobromobimane immediately after blood collection and plasma preparation prevents oxidation of captopril to the corresponding disulfide compound and enhances the ionization yield. Consequently, derivatization enhances sample stability and detection sensitivity. Addition of the internal standard was made immediately after plasma preparation. The internal standard was also derivatized by monobromobimane, as it contains a thiol functional group. Preparation of plasma samples containing captopril and IS derivatives was based upon protein precipitation through addition of acetonitrile, in a volumetric ratio 1:2. The reversed-phase liquid chromatographic separation was achieved on a rapid resolution cartridge Zorbax SB-C(18), monitored through positive electrospray ionization and tandem MS detection using the multiple-reaction monitoring mode. Transitions were 408-362 amu for the captopril derivative and 371-260 amu for the internal standard derivative. The kinetics of captopril oxidation to the corresponding disulfide compound in plasma matrix was also studied using the proposed method. A linear log-log calibration was obtained over the concentration interval 2.5-750 ng/mL. A low limit of quantitation in the 2.5 ng/mL range was obtained. The analytical method was fully validated and successfully applied in a three-way, three-period, single-dose (50 mg), block-randomized bioequivalence study for two pharmaceutical formulations (captopril LPH 25 and 50 mg) against the comparator Capoten 50 mg. Copyright (c) 2009 John Wiley & Sons, Ltd.

The development and amino acid binding ability of nano-materials based on azo derivatives: theory and experiment.

PubMed

Shang, Xuefang; Du, Jinge; Yang, Wancai; Liu, Yun; Fu, Zhiyuan; Wei, Xiaofang; Yan, Ruifang; Yao, Ningcong; Guo, Yaping; Zhang, Jinlian; Xu, Xiufang

2014-05-01

Two nano-material-containing azo groups have been designed and developed, and the binding ability of nano-materials with various amino acids has been characterized by UV-vis and fluorescence titrations. Results indicated that two nano-materials showed the strongest binding ability for homocysteine among twenty normal kinds of amino acids (alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, arginine, glycine, serine, threonine, asparagine, phenylalanine, histidine, tryptophan, proline, lysine, glutamine, tyrosine and homocysteine). The reason for the high sensitivity for homocysteine was that two nano-materials containing an aldehyde group reacted with SH in homocysteine and afforded very stable thiazolidine derivatives. Theoretical investigation further illustrated the possible binding mode in host-guest interaction and the roles of molecular frontier orbitals in molecular interplay. Thus, the two nano-materials can be used as optical sensors for the detection of homocysteine. Copyright © 2014 Elsevier B.V. All rights reserved.

Novel bis-(−)-nor-meptazinol derivatives act as dual binding site AChE inhibitors with metal-complexing property

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Wei; NPFPC Key Laboratory of Contraceptives and Devices, Shanghai Institute of Planned Parenthood Research, 2140 Xietu Road, Shanghai 200032; Li, Juan

The strategy of dual binding site acetylcholinesterase (AChE) inhibition along with metal chelation may represent a promising direction for multi-targeted interventions in the pathophysiological processes of Alzheimer's disease (AD). In the present study, two derivatives (ZLA and ZLB) of a potent dual binding site AChE inhibitor bis-(−)-nor-meptazinol (bis-MEP) were designed and synthesized by introducing metal chelating pharmacophores into the middle chain of bis-MEP. They could inhibit human AChE activity with IC{sub 50} values of 9.63 μM (for ZLA) and 8.64 μM (for ZLB), and prevent AChE-induced amyloid-β (Aβ) aggregation with IC{sub 50} values of 49.1 μM (for ZLA) and 55.3more » μM (for ZLB). In parallel, molecular docking analysis showed that they are capable of interacting with both the catalytic and peripheral anionic sites of AChE. Furthermore, they exhibited abilities to complex metal ions such as Cu(II) and Zn(II), and inhibit Aβ aggregation triggered by these metals. Collectively, these results suggest that ZLA and ZLB may act as dual binding site AChEIs with metal-chelating potency, and may be potential leads of value for further study on disease-modifying treatment of AD. -- Highlights: ► Two novel bis-(−)-nor-meptazinol derivatives are designed and synthesized. ► ZLA and ZLB may act as dual binding site AChEIs with metal-chelating potency. ► They are potential leads for disease-modifying treatment of Alzheimer's disease.« less

Reversible covalent binding of neratinib to human serum albumin in vitro.

PubMed

Chandrasekaran, Appavu; Shen, Li; Lockhead, Susan; Oganesian, Aram; Wang, Jianyao; Scatina, JoAnn

2010-12-01

Neratinib (HKI-272), an irreversible inhibitor of Her 2 tyrosine kinase, is currently in development as an alternative for first and second line therapy in metastatic breast cancer patients who overexpress Her 2. Following incubation of [(14)C]neratinib in control human plasma at 37°C for 6 hours, about 60% to 70% of the radioactivity was not extractable, due to covalent binding to albumin. In this study, factors that could potentially affect the covalent binding of neratinib to plasma proteins, specifically to albumin were investigated. When [(14)C]neratinib was incubated at 10 μg/mL in human serum albumin (HSA) or control human plasma, the percent binding increased with time; the highest percentages of binding (46 and 67%, respectively) were observed at 6 hours, the longest duration of incubation examined. Binding increased with increasing temperature; the highest percentages of binding to HSA or human plasma (59 and 78%) were observed at 45°C, the highest temperature tested. The binding also increased with increasing pH of incubation; the highest percentages of binding (56 and 65%) were observed at pH 8.5, the highest pH value tested. The percentages of binding were similar (53% to 57%) when a wide range of concentrations of [(14)C]neratinib (50 ng/mL to 10 μg/mL) were incubated with human plasma at 37°C for 6 hours, indicating that the binding was independent of the substrate concentration, especially in the therapeutic range (50 to 200 ng/mL). When human plasma proteins containing covalently bound [(14)C]neratinb were suspended in a 10 fold volume of phosphate buffer at pH 4.0, 6.0, 7.4, and 8.5, and further incubated at 37°C for ~ 16 hours, about 45%, 44%, 32%, and 12% of the total radioactivity, respectively, was released as unchanged [(14)C]neratinib, indicating that the binding is reversible in nature, with more released at pH 7.4 and below. In conclusion, the covalent binding of neratinib to serum albumin is pH, time and temperature dependent, but

AFM force spectroscopy reveals how subtle structural differences affect the interaction strength between Candida albicans and DC-SIGN.

PubMed

te Riet, Joost; Reinieren-Beeren, Inge; Figdor, Carl G; Cambi, Alessandra

2015-11-01

The fungus Candida albicans is the most common cause of mycotic infections in immunocompromised hosts. Little is known about the initial interactions between Candida and immune cell receptors, such as the C-type lectin dendritic cell-specific intracellular cell adhesion molecule-3 (ICAM-3)-grabbing non-integrin (DC-SIGN), because a detailed characterization at the structural level is lacking. DC-SIGN recognizes specific Candida-associated molecular patterns, that is, mannan structures present in the cell wall of Candida. The molecular recognition mechanism is however poorly understood. We postulated that small differences in mannan-branching may result in considerable differences in the binding affinity. Here, we exploit atomic force microscope-based dynamic force spectroscopy with single Candida cells to gain better insight in the carbohydrate recognition capacity of DC-SIGN. We demonstrate that slight differences in the N-mannan structure of Candida, that is, the absence or presence of a phosphomannan side chain, results in differences in the recognition by DC-SIGN as follows: (i) it contributes to the compliance of the outer cell wall of Candida, and (ii) its presence results in a higher binding energy of 1.6 kB T. The single-bond affinity of tetrameric DC-SIGN for wild-type C. albicans is ~10.7 kB T and a dissociation constant kD of 23 μM, which is relatively strong compared with other carbohydrate-protein interactions described in the literature. In conclusion, this study shows that DC-SIGN specifically recognizes mannan patterns on C. albicans with high affinity. Knowledge on the binding pocket of DC-SIGN and its pathogenic ligands will lead to a better understanding of how fungal-associated carbohydrate structures are recognized by receptors of the immune system and can ultimately contribute to the development of new anti-fungal drugs. Copyright © 2015 John Wiley & Sons, Ltd.

Analytic second derivative of the energy for density-functional tight-binding combined with the fragment molecular orbital method

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakata, Hiroya, E-mail: hiroya.nakata.gt@kyocera.jp; Nishimoto, Yoshio; Fedorov, Dmitri G.

2016-07-28

The analytic second derivative of the energy is developed for the fragment molecular orbital (FMO) method combined with density-functional tight-binding (DFTB), enabling simulations of infrared and Raman spectra of large molecular systems. The accuracy of the method is established in comparison to full DFTB without fragmentation for a set of representative systems. The performance of the FMO-DFTB Hessian is discussed for molecular systems containing up to 10 041 atoms. The method is applied to the study of the binding of α-cyclodextrin to polyethylene glycol, and the calculated IR spectrum of an epoxy amine oligomer reproduces experiment reasonably well.

A new method of derived equatorial plasma bubbles motion by tracing OI 630 nm emission all-sky images

NASA Astrophysics Data System (ADS)

Li, M.; Yu, T.; Chunliang, X.; Zuo, X.; Liu, Z.

2017-12-01

A new method for estimating the equatorial plasma bubbles (EPBs) motions from airglow emission all-sky images is presented in this paper. This method, which is called 'cloud-derived wind technology' and widely used in satellite observation of wind, could reasonable derive zonal and meridional velocity vectors of EPBs drifts by tracking a series of successive airglow 630.0 nm emission images. Airglow emission images data are available from an all sky airglow camera in Hainan Fuke (19.5°N, 109.2°E) supported by China Meridional Project, which can receive the 630.0nm emission from the ionosphere F region at low-latitudes to observe plasma bubbles. A series of pretreatment technology, e.g. image enhancement, orientation correction, image projection are utilized to preprocess the raw observation. Then the regions of plasma bubble extracted from the images are divided into several small tracing windows and each tracing window can find a target window in the searching area in following image, which is considered as the position tracing window moved to. According to this, velocities in each window are calculated by using the technology of cloud-derived wind. When applying the cloud-derived wind technology, the maximum correlation coefficient (MCC) and the histogram of gradient (HOG) methods to find the target window, which mean to find the maximum correlation and the minimum euclidean distance between two gradient histograms in respectively, are investigated and compared in detail. The maximum correlation method is fianlly adopted in this study to analyze the velocity of plasma bubbles because of its better performance than HOG. All-sky images from Hainan Fuke, between August 2014 and October 2014, are analyzed to investigate the plasma bubble drift velocities using MCC method. The data at different local time at 9 nights are studied and find that zonal drift velocity in different latitude at different local time ranges from 50 m/s to 180 m/s and there is a peak value at

Heparin-Binding Protein Measurement Improves the Prediction of Severe Infection With Organ Dysfunction in the Emergency Department

PubMed Central

Arnold, Ryan; Boyd, John H.; Zindovic, Marko; Zindovic, Igor; Lange, Anna; Paulsson, Magnus; Nyberg, Patrik; Russell, James A.; Pritchard, David; Christensson, Bertil; Åkesson, Per

2015-01-01

Objectives: Early identification of patients with infection and at risk of developing severe disease with organ dysfunction remains a difficult challenge. We aimed to evaluate and validate the heparin-binding protein, a neutrophil-derived mediator of vascular leakage, as a prognostic biomarker for risk of progression to severe sepsis with circulatory failure in a multicenter setting. Design: A prospective international multicenter cohort study. Setting: Seven different emergency departments in Sweden, Canada, and the United States. Patients: Adult patients with a suspected infection and at least one of three clinical systemic inflammatory response syndrome criteria (excluding leukocyte count). Intervention: None. Measurements and Main Results: Plasma levels of heparin-binding protein, procalcitonin, C-reactive protein, lactate, and leukocyte count were determined at admission and 12–24 hours after admission in 759 emergency department patients with suspected infection. Patients were defined depending on the presence of infection and organ dysfunction. Plasma samples from 104 emergency department patients with suspected sepsis collected at an independent center were used to validate the results. Of the 674 patients diagnosed with an infection, 487 did not have organ dysfunction at enrollment. Of these 487 patients, 141 (29%) developed organ dysfunction within the 72-hour study period; 78.0% of the latter patients had an elevated plasma heparin-binding protein level (> 30 ng/mL) prior to development of organ dysfunction (median, 10.5 hr). Compared with other biomarkers, heparin-binding protein was the best predictor of progression to organ dysfunction (area under the receiver operating characteristic curve = 0.80). The performance of heparin-binding protein was confirmed in the validation cohort. Conclusion: In patients presenting at the emergency department, heparin-binding protein is an early indicator of infection-related organ dysfunction and a strong predictor

Decreased Plasma Brain-Derived Neurotrophic Factor and Vascular Endothelial Growth Factor Concentrations during Military Training

PubMed Central

Nibuya, Masashi; Ishida, Toru; Yamamoto, Tetsuo; Mukai, Yasuo; Mitani, Keiji; Tsumatori, Gentaro; Scott, Daniel; Shimizu, Kunio

2014-01-01

Decreased concentrations of plasma brain-derived neurotrophic factor (BDNF) and serum BDNF have been proposed to be a state marker of depression and a biological indicator of loaded psychosocial stress. Stress evaluations of participants in military mission are critically important and appropriate objective biological parameters that evaluate stress are needed. In military circumstances, there are several problems to adopt plasma BDNF concentration as a stress biomarker. First, in addition to psychosocial stress, military missions inevitably involve physical exercise that increases plasma BDNF concentrations. Second, most participants in the mission do not have adequate quality or quantity of sleep, and sleep deprivation has also been reported to increase plasma BDNF concentration. We evaluated plasma BDNF concentrations in 52 participants on a 9-week military mission. The present study revealed that plasma BDNF concentration significantly decreased despite elevated serum enzymes that escaped from muscle and decreased quantity and quality of sleep, as detected by a wearable watch-type sensor. In addition, we observed a significant decrease in plasma vascular endothelial growth factor (VEGF) during the mission. VEGF is also neurotrophic and its expression in the brain has been reported to be up-regulated by antidepressive treatments and down-regulated by stress. This is the first report of decreased plasma VEGF concentrations by stress. We conclude that decreased plasma concentrations of neurotrophins can be candidates for mental stress indicators in actual stressful environments that include physical exercise and limited sleep. PMID:24586790

Application of compound mixture of caprylic acid, iron and mannan oligosaccharide against Sparicotyle chrysophrii (Monogenea: Polyopisthocotylea) in gilthead sea bream, Sparus aurata.

PubMed

Rigos, George; Mladineo, Ivona; Nikoloudaki, Chrysa; Vrbatovic, Anamarija; Kogiannou, Dimitra

2016-08-05

We have evaluated the therapeutic effect of a compound mixture of caprylic acid (200 mg/kg fish), organic iron (0.2% of diet) and mannan oligosaccharide (0.4% of diet) in gilthead sea bream, Sparus aurata Linnaeus, infected with Sparicotyle chrysophrii Beneden et Hesse, 1863 in controlled conditions. One hundred and ten reared and S. chrysophrii-free fish (197 g) located in a cement tank were infected by the parasite two weeks following the addition of 150 S. chrysophrii-infected fish (70 g). Growth parameters and gill parasitic load were measured in treated against control fish after a ten-week-period. Differences in final weight, feed conversion ratio, specific growth rate and feed efficiency were not statistically significant between the experimental groups, suggesting no evident effect with respect to fish growth during the study period. Although the prevalence of S. chrysophrii was not affected by the mixture at the end of the experiment, the number of adults and larvae was significantly lower. The mean intensity encompassing the number of adults and larvae was 8.1 in treated vs 17.7 in control fish. Individual comparisons of gill arches showed that the preferred parasitism site for S. chrysophrii it the outermost or fourth gill arch, consistently apparent in fish fed the modified diet and in control fish. In conclusion, the combined application of caprylic acid, organic iron and mannan oligosaccharide can significantly affect the evolution of infection with S. chrysophrii in gilthead sea bream, being capable of reducing adult and larval stages of the monogenean. However, no difference in growth improvement was observed after the trial period, potentially leaving space for further optimisation of the added dietary compounds.

High-Affinity Binding of Silybin Derivatives to the Nucleotide-Binding Domain of a Leishmania tropica P-Glycoprotein-Like Transporter and Chemosensitization of a Multidrug-Resistant Parasite to Daunomycin

PubMed Central

Pérez-Victoria, José M.; Pérez-Victoria, F. Javier; Conseil, Gwenaëlle; Maitrejean, Mathias; Comte, Gilles; Barron, Denis; Di Pietro, Attilio; Castanys, Santiago; Gamarro, Francisco

2001-01-01

In order to overcome the multidrug resistance mediated by P-glycoprotein-like transporters in Leishmania spp., we have studied the effects produced by derivatives of the flavanolignan silybin and related compounds lacking the monolignol unit on (i) the affinity of binding to a recombinant C-terminal nucleotide-binding domain of the L. tropica P-glycoprotein-like transporter and (ii) the sensitization to daunomycin on promastigote forms of a multidrug-resistant L. tropica line overexpressing the transporter. Oxidation of the flavanonol silybin to the corresponding flavonol dehydrosilybin, the presence of the monolignol unit, and the addition of a hydrophobic substituent such as dimethylallyl, especially at position 8 of ring A, considerably increased the binding affinity. The in vitro binding affinity of these compounds for the recombinant cytosolic domain correlated with their modulation of drug resistance phenotype. In particular, 8-(3,3-dimethylallyl)-dehydrosilybin effectively sensitized multidrug-resistant Leishmania spp. to daunomycin. The cytosolic domains are therefore attractive targets for the rational design of inhibitors against P-glycoprotein-like transporters. PMID:11158738

Measurement of free glucocorticoids: quantifying corticosteroid-binding globulin binding affinity and its variation within and among mammalian species.

PubMed

Delehanty, Brendan; Hossain, Sabrina; Jen, Chao Ching; Crawshaw, Graham J; Boonstra, Rudy

2015-01-01

Plasma glucocorticoids (GCs) are commonly used as measures of stress in wildlife. A great deal of evidence indicates that only free GC (GC not bound by the specific binding protein, corticosteroid-binding globulin, CBG) leaves the circulation and exerts biological effects on GC-sensitive tissues. Free hormone concentrations are difficult to measure directly, so researchers estimate free GC using two measures: the binding affinity and the binding capacity in plasma. We provide an inexpensive saturation binding method for calculating the binding affinity (equilibrium dissociation constant, K d) of CBG that can be run without specialized laboratory equipment. Given that other plasma proteins, such as albumin, also bind GCs, the method compensates for this non-specific binding. Separation of bound GC from free GC was achieved with dextran-coated charcoal. The method provides repeatable estimates (12% coefficient of variation in the red squirrel, Tamiasciurus hudsonicus), and there is little evidence of inter-individual variation in K d (range 2.0-7.3 nM for 16 Richardson's ground squirrels, Urocitellus richardsonii). The K d values of 28 mammalian species we assessed were mostly clustered around a median of 4 nM, but five species had values between 13 and 61 nM. This pattern may be distinct from birds, for which published values are more tightly distributed (1.5-5.1 nM). The charcoal separation method provides a reliable and robust method for measuring the K d in a wide range of species. It uses basic laboratory equipment to provide rapid results at very low cost. Given the importance of CBG in regulating the biological activity of GCs, this method is a useful tool for physiological ecologists.

An APOC3 3'UTR variant associated with plasma triglycerides levels and coronary heart disease by creating a functional miR-4271 binding site.

PubMed

Hu, Sen-Lin; Cui, Guang-Lin; Huang, Jin; Jiang, Jian-Gang; Wang, Dao-Wen

2016-09-14

Apolipoprotein C-III (APOC3) is a key regulator of plasma triglycerides levels. Increasing evidence has shown that loss-of-function mutations in APOC3 is associated with reduction in plasma triglycerides levels and will confer a benefit in patients at high risk for cardiovascular disease. However, these favorable mutations were extremely distribution discrepant among different ethnics. In this study, the APOC3 gene was resequenced and we identified a common variant which located in the microRNA-binding site in APOC3 and would affect its expression and the risk of coronary heart disease (CHD). The molecular mechanism was explored. We found that the T allele of rs4225 suppressed APOC3 translation by facilitating miR-4271 binding, but not the G allele. Subjects carrying the GG genotype had higher plasma APOC3 levels (p for trend = 0.03) than those with the TT genotype. Furthermore, the T allele was significantly associated with decreased triglyceride levels [Beta (SE): -0.024 (0.020), P = 0.03]. Finally, the case-control study suggested that the TT genotype resulted in a significant reduction in overall CHD risk [OR, 0.89 (95% confidence interval, 0.77-0.98), P = 0.009]. In conclusion, our results provide evidence that the rs4225 in the 3'-UTR of APOC3 might contribute to the risk of CHD by interfering with miR-4271 binding.

Binding of urea and thiourea with a barbiturate derivative: experimental and theoretical approach.

PubMed

Dixit, Namrata; Shukla, P K; Mishra, P C; Mishra, Lallan; Roesky, Herbert W

2010-01-14

A barbiturate derivative [1,5-dihydro-5-[5-pyrimidine-2,4(1H,3H)-dionyl]-2H-chromeno[2,3-d] pyrimidine-2,4(3H)-dione)] (L1) possesses functionalities complementary to amide and thioamide. Hence its binding with urea and thiourea, is monitored using UV-vis and fluorescence titrations as well as isothermal titration calorimetry (ITC) study. Theoretical studies on hydrogen-bonded complexes of L1-urea and L1-thiourea in the gas phase, aqueous, and DMSO medium are carried out using density functional theory (DFT) at the B3LYP/6-31G** level. The theoretical calculations support the experimental results.

The yeast plasma membrane ATP binding cassette (ABC) transporter Aus1: purification, characterization, and the effect of lipids on its activity.

PubMed

Marek, Magdalena; Milles, Sigrid; Schreiber, Gabriele; Daleke, David L; Dittmar, Gunnar; Herrmann, Andreas; Müller, Peter; Pomorski, Thomas Günther

2011-06-17

The ATP binding cassette (ABC) transporter Aus1 is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and is required for sterol uptake. These observations suggest that Aus1 promotes the translocation of sterols across membranes, but the precise transport mechanism has yet to be identified. In this study, an extraction and purification procedure was developed to characterize the Aus1 transporter. The detergent-solubilized protein was able to bind and hydrolyze ATP. Mutagenesis of the conserved lysine to methionine in the Walker A motif abolished ATP hydrolysis. Likewise, ATP hydrolysis was inhibited by classical inhibitors of ABC transporters. Upon reconstitution into proteoliposomes, the ATPase activity of Aus1 was specifically stimulated by phosphatidylserine (PS) in a stereoselective manner. We also found that Aus1-dependent sterol uptake, but not Aus1 expression and trafficking to the plasma membrane, was affected by changes in cellular PS levels. These results suggest a direct interaction between Aus1 and PS that is critical for the activity of the transporter.

Release kinetics of platelet-derived and plasma-derived growth factors from autologous plasma rich in growth factors.

PubMed

Anitua, Eduardo; Zalduendo, Mari Mar; Alkhraisat, Mohammad Hamdan; Orive, Gorka

2013-10-01

Many studies have evaluated the biological effects of platelet rich plasma reporting the final outcomes on cell and tissues. However, few studies have dealt with the kinetics of growth factor delivery by plasma rich in growth factors. Venous blood was obtained from three healthy volunteers and processed with PRGF-Endoret technology to prepare autologous plasma rich in growth factors. The gel-like fibrin scaffolds were then incubated in triplicate, in a cell culture medium to monitor the release of PDGF-AB, VEGF, HGF and IGF-I during 8 days of incubation. A leukocyte-platelet rich plasma was prepared employing the same technology and the concentrations of growth factors and interleukin-1β were determined after 24h of incubation. After each period, the medium was collected, fibrin clot was destroyed and the supernatants were stored at -80°C until analysis. The growth factor delivery is diffusion controlled with a rapid initial release by 30% of the bioactive content after 1h of incubation and a steady state release when almost 70% of the growth factor content has been delivered. Autologous fibrin matrix retained almost 30% of the amount of the growth factors after 8 days of incubation. The addition of leukocytes to the formula of platelet rich plasma did not increase the concentration of the growth factors, while it drastically increased the presence of pro-inflammatory IL-1β. Further studies employing an in vitro inflammatory model would be interesting to study the difference in growth factors and pro-inflammatory cytokines between leukocyte-free and leukocyte-rich platelet rich plasma. Copyright © 2013 Elsevier GmbH. All rights reserved.

Characterisation of zinc-binding domains of peroxisomal RING finger proteins using size exclusion chromatography/inductively coupled plasma-mass spectrometry.

PubMed

Koellensperger, Gunda; Daubert, Simon; Erdmann, Ralf; Hann, Stephan; Rottensteiner, Hanspeter

2007-11-01

We determined the zinc binding stoichiometry of peroxisomal RING finger proteins by measuring sulfur/metal ratios using inductively coupled plasma-mass spectrometry coupled to size exclusion chromatography, a strategy that provides a fast and quantitative overview on the binding of metals in proteins. As a quality control, liquid chromatography-electrospray ionisation-time of flight-mass spectrometry was used to measure the molar masses of the intact proteins. The RING fingers of Pex2p, Pex10p, and Pex12p showed a stoichiometry of 2.0, 2.1, and 1.2 mol zinc/mol protein, respectively. Thus, Pex2p and Pex10p possess a typical RING domain with two coordinated zinc atoms, whereas that of Pex12p coordinates only a single zinc atom.

Thermodynamics and binding mode of novel structurally related 1,2,4-thiadiazole derivatives with native and modified cyclodextrins

NASA Astrophysics Data System (ADS)

Terekhova, Irina V.; Chislov, Mikhail V.; Brusnikina, Maria A.; Chibunova, Ekaterina S.; Volkova, Tatyana V.; Zvereva, Irina A.; Proshin, Alexey N.

2017-03-01

Study of complex formation of cyclodextrins with 1,2,4-thiadiazole derivatives intended for Alzheimer's disease treatment was carried out using 1H NMR, ITC and phase solubility methods. Structure of cyclodextrins and thiadiazoles affects the binding mode and thermodynamics of complexation. The larger cavity of β- and γ-cyclodextrins is more appropriate for deeper insertion of 1,2,4-thiadiazole derivatives which is accompanied by intensive dehydration and solvent reorganization. Benzene ring of the thiadiazoles is located inside macrocyclic cavity while piperidine ring is placed outside the cavity and can form H-bonds with cyclodextrin exterior. Complexation with cyclodextrins induces the enhancement of aqueous solubility of 1,2,4-thiadiazole derivatives.

Dietary fat and the diabetic state alter insulin binding and the fatty acyl composition of the adipocyte plasma membrane.

PubMed Central

Field, C J; Ryan, E A; Thomson, A B; Clandinin, M T

1988-01-01

Control and diabetic rats were fed on semi-purified high-fat diets providing a polyunsaturated/saturated fatty acid ratio (P/S) of 1.0 or 0.25, to examine the effect of diet on the fatty acid composition of major phospholipids of the adipocyte plasma membrane. Feeding the high-P/S diet (P/S = 1.0) compared with the low-P/S diet (P/S = 0.25) increased the content of polyunsaturated fatty acids in membrane phospholipids in both control and diabetic animals. The diabetic state decreased the content of polyunsaturated fatty acids, particularly arachidonic acid, in adipocyte membrane phospholipids. The decrease in arachidonic acid in membrane phospholipids of diabetic animals tended to be normalized to within the control values when high-P/S diets were given. For control animals, altered plasma-membrane composition was associated with change in insulin binding, suggesting that change in plasma-membrane composition may have physiological consequences for insulin-stimulated functions in the adipocyte. PMID:3052424

Reticulomics: Protein-Protein Interaction Studies with Two Plasmodesmata-Localized Reticulon Family Proteins Identify Binding Partners Enriched at Plasmodesmata, Endoplasmic Reticulum, and the Plasma Membrane.

PubMed

Kriechbaumer, Verena; Botchway, Stanley W; Slade, Susan E; Knox, Kirsten; Frigerio, Lorenzo; Oparka, Karl; Hawes, Chris

2015-11-01

The endoplasmic reticulum (ER) is a ubiquitous organelle that plays roles in secretory protein production, folding, quality control, and lipid biosynthesis. The cortical ER in plants is pleomorphic and structured as a tubular network capable of morphing into flat cisternae, mainly at three-way junctions, and back to tubules. Plant reticulon family proteins (RTNLB) tubulate the ER by dimerization and oligomerization, creating localized ER membrane tensions that result in membrane curvature. Some RTNLB ER-shaping proteins are present in the plasmodesmata (PD) proteome and may contribute to the formation of the desmotubule, the axial ER-derived structure that traverses primary PD. Here, we investigate the binding partners of two PD-resident reticulon proteins, RTNLB3 and RTNLB6, that are located in primary PD at cytokinesis in tobacco (Nicotiana tabacum). Coimmunoprecipitation of green fluorescent protein-tagged RTNLB3 and RTNLB6 followed by mass spectrometry detected a high percentage of known PD-localized proteins as well as plasma membrane proteins with putative membrane-anchoring roles. Förster resonance energy transfer by fluorescence lifetime imaging microscopy assays revealed a highly significant interaction of the detected PD proteins with the bait RTNLB proteins. Our data suggest that RTNLB proteins, in addition to a role in ER modeling, may play important roles in linking the cortical ER to the plasma membrane. © 2015 American Society of Plant Biologists. All Rights Reserved.

Food proteins and maturation of small intestinal microvillus membranes (MVM). I. Binding characteristics of cow's milk proteins and concanavalin A to MVM from newborn and adult rats.

PubMed

Stern, M; Gellermann, B

1988-01-01

To study maturational changes of food protein and lectin binding to rat small intestinal microvillus membranes (MVM), MVM were prepared from newborn and adult animals by a modified CaCl2 precipitation technique. Radiolabeled cow's milk proteins [alpha-lactalbumin, alpha-casein, beta-lactoglobulin, bovine serum albumin (BSA)] and the lectin concanavalin A (Con A) were used for incubations. Binding assays were done using miniature ultracentrifugation for separation of unbound material. Binding of Con A to MVM from newborn and adult rats was strong, specific, and saturable. Binding of Con A was inhibited by cold Con A and by the sugar ligand polymer mannan. Adult MVM bound more Con A than newborn preparations. Unlike Con A, binding of cow's milk proteins by MVM was weak, nonspecific, and noninhibitable. Newborn MVM bound more cow's milk proteins than adult controls. This was true for all the proteins tested (p less than 0.001). Binding rose with decreased molecular weight of cow's milk proteins, but molecular weight was not the only determining factor for binding. Trypsin treatment of MVM caused a marked increase of BSA binding in adult but not in newborn preparations. This finding indicated the importance of protein components of MVM for cow's milk protein binding. Maturational changes in protein-lipid interactions and membrane fluidity possibly influence nonspecific cow's milk protein binding to MVM. Differences in binding between newborns and adults were not directly related to maturational shifts in membrane glycosylation that are indicated by differential Con A binding. Increased cow's milk protein binding in newborn individuals might increase the potential risk to develop an adverse reaction to food proteins.

Amino Acid Derivatives as Palmitoylethanolamide Prodrugs: Synthesis, In Vitro Metabolism and In Vivo Plasma Profile in Rats

PubMed Central

Vacondio, Federica; Bassi, Michele; Silva, Claudia; Castelli, Riccardo; Carmi, Caterina; Scalvini, Laura; Lodola, Alessio; Vivo, Valentina; Flammini, Lisa; Barocelli, Elisabetta; Mor, Marco; Rivara, Silvia

2015-01-01

Palmitoylethanolamide (PEA) has antinflammatory and antinociceptive properties widely exploited in veterinary and human medicine, despite its poor pharmacokinetics. Looking for prodrugs that could progressively release PEA to maintain effective plasma concentrations, we prepared carbonates, esters and carbamates at the hydroxyl group of PEA. Chemical stability (pH 7.4) and stability in rat plasma and liver homogenate were evaluated by in vitro assays. Carbonates and carbamates resulted too labile and too resistant in plasma, respectively. Ester derivatives, prepared by conjugating PEA with various amino acids, allowed to modulate the kinetics of PEA release in plasma and stability in liver homogenate. L-Val-PEA, with suitable PEA release in plasma, and D-Val-PEA, with high resistance to hepatic degradation, were orally administered to rats and plasma levels of prodrugs and PEA were measured at different time points. Both prodrugs showed significant release of PEA, but provided lower plasma concentrations than those obtained with equimolar doses of PEA. Amino-acid esters of PEA are a promising class to develop prodrugs, even if they need further chemical optimization. PMID:26053855

Fatty acids and small organic compounds bind to mineralo-organic nanoparticles derived from human body fluids as revealed by metabolomic analysis

NASA Astrophysics Data System (ADS)

Martel, Jan; Wu, Cheng-Yeu; Hung, Cheng-Yu; Wong, Tsui-Yin; Cheng, Ann-Joy; Cheng, Mei-Ling; Shiao, Ming-Shi; Young, John D.

2016-03-01

Nanoparticles entering the human body instantly become coated with a ``protein corona'' that influences the effects and distribution of the particles in vivo. Yet, whether nanoparticles may bind to other organic compounds remains unclear. Here we use an untargeted metabolomic approach based on ultra-performance liquid chromatography and quadruple time-of-flight mass spectrometry to identify the organic compounds that bind to mineral nanoparticles formed in human body fluids (serum, plasma, saliva, and urine). A wide range of organic compounds is identified, including fatty acids, glycerophospholipids, amino acids, sugars, and amides. Our results reveal that, in addition to the proteins identified previously, nanoparticles harbor an ``organic corona'' containing several fatty acids which may affect particle-cell interactions in vivo. This study provides a platform to study the organic corona of biological and synthetic nanoparticles found in the human body.Nanoparticles entering the human body instantly become coated with a ``protein corona'' that influences the effects and distribution of the particles in vivo. Yet, whether nanoparticles may bind to other organic compounds remains unclear. Here we use an untargeted metabolomic approach based on ultra-performance liquid chromatography and quadruple time-of-flight mass spectrometry to identify the organic compounds that bind to mineral nanoparticles formed in human body fluids (serum, plasma, saliva, and urine). A wide range of organic compounds is identified, including fatty acids, glycerophospholipids, amino acids, sugars, and amides. Our results reveal that, in addition to the proteins identified previously, nanoparticles harbor an ``organic corona'' containing several fatty acids which may affect particle-cell interactions in vivo. This study provides a platform to study the organic corona of biological and synthetic nanoparticles found in the human body. Electronic supplementary information (ESI) available. See

Plasma membrane proteome analysis identifies a role of barley membrane steroid binding protein in root architecture response to salinity.

PubMed

Witzel, Katja; Matros, Andrea; Møller, Anders L B; Ramireddy, Eswarayya; Finnie, Christine; Peukert, Manuela; Rutten, Twan; Herzog, Andreas; Kunze, Gotthard; Melzer, Michael; Kaspar-Schoenefeld, Stephanie; Schmülling, Thomas; Svensson, Birte; Mock, Hans-Peter

2018-06-01

Although the physiological consequences of plant growth under saline conditions have been well described, understanding the core mechanisms conferring plant salt adaptation has only started. We target the root plasma membrane proteomes of two barley varieties, cvs. Steptoe and Morex, with contrasting salinity tolerance. In total, 588 plasma membrane proteins were identified by mass spectrometry, of which 182 were either cultivar or salinity stress responsive. Three candidate proteins with increased abundance in the tolerant cv. Morex were involved either in sterol binding (a GTPase-activating protein for the adenosine diphosphate ribosylation factor [ZIGA2], and a membrane steroid binding protein [MSBP]) or in phospholipid synthesis (phosphoethanolamine methyltransferase [PEAMT]). Overexpression of barley MSBP conferred salinity tolerance to yeast cells, whereas the knock-out of the heterologous AtMSBP1 increased salt sensitivity in Arabidopsis. Atmsbp1 plants showed a reduced number of lateral roots under salinity, and root-tip-specific expression of barley MSBP in Atmsbp1 complemented this phenotype. In barley, an increased abundance of MSBP correlates with reduced root length and lateral root formation as well as increased levels of auxin under salinity being stronger in the tolerant cv. Morex. Hence, we concluded the involvement of MSBP in phytohormone-directed adaptation of root architecture in response to salinity. © 2018 John Wiley & Sons Ltd.

Mannans and endo-β-mannanases (MAN) in Brachypodium distachyon: expression profiling and possible role of the BdMAN genes during coleorhiza-limited seed germination

PubMed Central

González-Calle, Virginia; Barrero-Sicilia, Cristina; Carbonero, Pilar; Iglesias-Fernández, Raquel

2015-01-01

Immunolocalization of mannans in the seeds of Brachypodium distachyon reveals the presence of these polysaccharides in the root embryo and in the coleorhiza in the early stages of germination (12h), decreasing thereafter to the point of being hardly detected at 27h. Concurrently, the activity of endo-β-mannanases (MANs; EC 3.2.1.78) that catalyse the hydrolysis of β-1,4 bonds in mannan polymers, increases as germination progresses. The MAN gene family is represented by six members in the Brachypodium genome, and their expression has been explored in different organs and especially in germinating seeds. Transcripts of BdMAN2, BdMAN4 and BdMAN6 accumulate in embryos, with a maximum at 24–30h, and are detected in the coleorhiza and in the root by in situ hybridization analyses, before root protrusion (germination sensu stricto). BdMAN4 is not only present in the embryo root and coleorhiza, but is abundant in the de-embryonated (endosperm) imbibed seeds, while BdMAN2 and BdMAN6 are faintly expressed in endosperm during post-germination (36–42h). BdMAN4 and BdMAN6 transcripts are detected in the aleurone layer. These data indicate that BdMAN2, BdMAN4 and BdMAN6 are important for germination sensu stricto and that BdMAN4 and BdMAN6 may also influence reserve mobilization. Whether the coleorhiza in monocots and the micropylar endosperm in eudicots have similar functions, is discussed. PMID:25922488

Plasma α-synuclein and cognitive impairment in the Parkinson's Associated Risk Syndrome: A pilot study.

PubMed

Wang, Hua; Atik, Anzari; Stewart, Tessandra; Ginghina, Carmen; Aro, Patrick; Kerr, Kathleen F; Seibyl, John; Jennings, Danna; Jensen, Poul Henning; Marek, Kenneth; Shi, Min; Zhang, Jing

2018-04-27

Plasma total and nervous system derived exosomal (NDE) α-synuclein have been determined as potential biomarkers of Parkinson's disease (PD). To explore the utility of plasma α-synuclein in the prodromal phase of PD, plasma total and NDE α-synuclein were evaluated in baseline and 2-year follow-up samples from 256 individuals recruited as part of the Parkinson's Associated Risk Syndrome (PARS) study. The results demonstrated that baseline and longitudinal increases in total α-synuclein predicted progression of cognitive decline in hyposmic individuals with dopamine transporter (DAT) binding reduction. On the other hand, a longitudinal decrease in NDE α-synuclein predicted worsening cognitive scores in hyposmic individuals with DAT binding reduction. Finally, in individuals with faster DAT progression, decreasing NDE/total α-synuclein ratio was associated with a larger reduction in DAT from baseline to follow-up. These results suggest that, though underlying mechanisms remain to be defined, alterations in plasma total and NDE α-synuclein concentrations are likely associated with PD progression, especially in the aspect of cognitive impairment, at early stages of the disease. Copyright © 2018. Published by Elsevier Inc.

Spectroscopic profiling and computational study of the binding of tschimgine: A natural monoterpene derivative, with calf thymus DNA

NASA Astrophysics Data System (ADS)

Khajeh, Masoumeh Ashrafi; Dehghan, Gholamreza; Dastmalchi, Siavoush; Shaghaghi, Masoomeh; Iranshahi, Mehrdad

2018-03-01

DNA is a major target for a number of anticancer substances. Interaction studies between small molecules and DNA are essential for rational drug designing to influence main biological processes and also introducing new probes for the assay of DNA. Tschimgine (TMG) is a monoterpene derivative with anticancer properties. In the present study we tried to elucidate the interaction of TMG with calf thymus DNA (CT-DNA) using different spectroscopic methods. UV-visible absorption spectrophotometry, fluorescence and circular dichroism (CD) spectroscopies as well as molecular docking study revealed formation of complex between TMG and CT-DNA. Binding constant (Kb) between TMG and DNA was 2.27 × 104 M- 1, that is comparable to groove binding agents. The fluorescence spectroscopic data revealed that the quenching mechanism of fluorescence of TMG by CT-DNA is static quenching. Thermodynamic parameters (ΔH < 0 and ΔS < 0) at different temperatures indicated that van der Waals forces and hydrogen bonds were involved in the binding process of TMG with CT-DNA. Competitive binding assay with methylene blue (MB) and Hoechst 33258 using fluorescence spectroscopy displayed that TMG possibly binds to the minor groove of CT-DNA. These observations were further confirmed by CD spectral analysis, viscosity measurements and molecular docking.

Plasma Levels of Fatty Acid-Binding Protein 4, Retinol-Binding Protein 4, High-Molecular-Weight Adiponectin, and Cardiovascular Mortality Among Men With Type 2 Diabetes: A 22-Year Prospective Study.

PubMed

Liu, Gang; Ding, Ming; Chiuve, Stephanie E; Rimm, Eric B; Franks, Paul W; Meigs, James B; Hu, Frank B; Sun, Qi

2016-11-01

To examine select adipokines, including fatty acid-binding protein 4, retinol-binding protein 4, and high-molecular-weight (HMW) adiponectin in relation to cardiovascular disease (CVD) mortality among patients with type 2 diabetes mellitus. Plasma levels of fatty acid-binding protein 4, retinol-binding protein 4, and HMW adiponectin were measured in 950 men with type 2 diabetes mellitus in the Health Professionals Follow-up Study. After an average of 22 years of follow-up (1993-2015), 580 deaths occurred, of whom 220 died of CVD. After multivariate adjustment for covariates, higher levels of fatty acid-binding protein 4 were significantly associated with a higher CVD mortality: comparing extreme tertiles, the hazard ratio and 95% confidence interval of CVD mortality was 1.78 (1.22-2.59; P trend=0.001). A positive association was also observed for HMW adiponectin: the hazard ratio (95% confidence interval) was 2.07 (1.42-3.06; P trend=0.0002), comparing extreme tertiles, whereas higher retinol-binding protein 4 levels were nonsignificantly associated with a decreased CVD mortality with an hazard ratio (95% confidence interval) of 0.73 (0.50-1.07; P trend=0.09). A Mendelian randomization analysis suggested that the causal relationships of HMW adiponectin and retinol-binding protein 4 would be directionally opposite to those observed based on the biomarkers, although none of the Mendelian randomization associations achieved statistical significance. These data suggest that higher levels of fatty acid-binding protein 4 and HMW adiponectin are associated with elevated CVD mortality among men with type 2 diabetes mellitus. Biological mechanisms underlying these observations deserve elucidation, but the associations of HMW adiponectin may partially reflect altered adipose tissue functionality among patients with type 2 diabetes mellitus. © 2016 American Heart Association, Inc.

Nucleic acid is a novel ligand for innate, immune pattern recognition collectins surfactant proteins A and D and mannose-binding lectin.

PubMed

Palaniyar, Nades; Nadesalingam, Jeya; Clark, Howard; Shih, Michael J; Dodds, Alister W; Reid, Kenneth B M

2004-07-30

Collectins are a family of innate immune proteins that contain fibrillar collagen-like regions and globular carbohydrate recognition domains (CRDs). The CRDs of these proteins recognize various microbial surface-specific carbohydrate patterns, particularly hexoses. We hypothesized that collectins, such as pulmonary surfactant proteins (SPs) SP-A and SP-D and serum protein mannose-binding lectin, could recognize nucleic acids, pentose-based anionic phosphate polymers. Here we show that collectins bind DNA from a variety of origins, including bacteria, mice, and synthetic oligonucleotides. Pentoses, such as arabinose, ribose, and deoxyribose, inhibit the interaction between SP-D and mannan, one of the well-studied hexose ligands for SP-D, and biologically relevant d-forms of the pentoses are better competitors than the l-forms. In addition, DNA and RNA polymer-related compounds, such as nucleotide diphosphates and triphosphates, also inhibit the carbohydrate binding ability of SP-D, or approximately 60 kDa trimeric recombinant fragments of SP-D that are composed of the alpha-helical coiled-coil neck region and three CRDs (SP-D(n/CRD)) or SP-D(n/CRD) with eight GXY repeats (SPD(GXY)(8)(n/CRD)). Direct binding and competition studies suggest that collectins bind nucleic acid via their CRDs as well as by their collagen-like regions, and that SP-D binds DNA more effectively than do SP-A and mannose-binding lectin at physiological salt conditions. Furthermore, the SP-D(GXY)(8)(n/CRD) fragments co-localize with DNA, and the protein competes the interaction between propidium iodide, a DNA-binding dye, and apoptotic cells. In conclusion, we show that collectins are a new class of proteins that bind free DNA and the DNA present on apoptotic cells by both their globular CRDs and collagen-like regions. Collectins may therefore play an important role in decreasing the inflammation caused by DNA in lungs and other tissues.

High-performance liquid chromatography measurement of hyperforin and its reduced derivatives in rodent plasma.

PubMed

Rozio, M; Fracasso, C; Riva, A; Morazzoni, P; Caccia, S

2005-02-25

A reverse-phase high-performance liquid chromatography method was developed for the determination of hyperforin and its reduced derivatives octahydrohyperforin and tetrahydrohyperforin in rodent plasma. The procedure includes solid-phase extraction from plasma using the Baker 3cc C8 cartridge, resolution on the Symmetry Shield RP8 column (150 mm x 4.6 mm, i.d. 3.5 microm) and UV absorbance detection at 300 nm. The assay was linear over a wide range, with an overall coefficient of variation less than 10% for all compounds. The precision and accuracy were within acceptable limits and the limit of quantitation was sufficient for studies preliminarily assessing the disposition of tetrahydrohyperforin and octahydrohyperforin in the mouse and rat.

The remarkable stability of chimeric, sialic acid-derived alpha/delta-peptides in human blood plasma.

PubMed

Saludes, Jonel P; Natarajan, Arutselvan; DeNardo, Sally J; Gervay-Hague, Jacquelyn

2010-05-01

Peptides are labile toward proteolytic enzymes, and structural modifications are often required to prolong their metabolic half-life and increase resistance. One modification is the incorporation of non-alpha-amino acids into the peptide to deter recognition by hydrolytic enzymes. We previously reported the synthesis of chimeric alpha/delta-peptides from glutamic acids (Glu) and the sialic acid derivative Neu2en. Conformational analyses revealed these constructs adopt secondary structures in water and may serve as conformational surrogates of polysialic acid. Polysialic acid is a tumor-associated polysaccharide and is correlated with cancer metastasis. Soluble polysialic acid is rapidly cleared from the blood limiting its potential for vaccine development. One motivation in developing structural surrogates of polysialic acid was to create constructs with increased bioavailability. Here, we report plasma stability profiles of Glu/Neu2en alpha/delta-peptides. DOTA was conjugated at the peptide N-termini by solid phase peptide synthesis, radiolabeled with (111)In, incubated in human blood plasma at 37 degrees C, and their degradation patterns monitored by cellulose acetate electrophoresis and radioactivity counting. Results indicate that these peptides exhibit a long half-life that is two- to three-orders of magnitude higher than natural alpha-peptides. These findings provide a viable platform for the synthesis of plasma stable, sialic acid-derived peptides that may find pharmaceutical application.

Functions of Intracellular Retinoid Binding-Proteins.

PubMed

Napoli, Joseph L

Multiple binding and transport proteins facilitate many aspects of retinoid biology through effects on retinoid transport, cellular uptake, metabolism, and nuclear delivery. These include the serum retinol binding protein sRBP (aka Rbp4), the plasma membrane sRBP receptor Stra6, and the intracellular retinoid binding-proteins such as cellular retinol-binding proteins (CRBP) and cellular retinoic acid binding-proteins (CRABP). sRBP transports the highly lipophilic retinol through an aqueous medium. The major intracellular retinol-binding protein, CRBP1, likely enhances efficient retinoid use by providing a sink to facilitate retinol uptake from sRBP through the plasma membrane or via Stra6, delivering retinol or retinal to select enzymes that generate retinyl esters or retinoic acid, and protecting retinol/retinal from excess catabolism or opportunistic metabolism. Intracellular retinoic acid binding-proteins (CRABP1 and 2, and FABP5) seem to have more diverse functions distinctive to each, such as directing retinoic acid to catabolism, delivering retinoic acid to specific nuclear receptors, and generating non-canonical actions. Gene ablation of intracellular retinoid binding-proteins does not cause embryonic lethality or gross morphological defects. Metabolic and functional defects manifested in knockouts of CRBP1, CRBP2 and CRBP3, however, illustrate their essentiality to health, and in the case of CRBP2, to survival during limited dietary vitamin A. Future studies should continue to address the specific molecular interactions that occur between retinoid binding-proteins and their targets and their precise physiologic contributions to retinoid homeostasis and function.

The functional dissection of the plasma corona of SiO₂-NPs spots histidine rich glycoprotein as a major player able to hamper nanoparticle capture by macrophages.

PubMed

Fedeli, Chiara; Segat, Daniela; Tavano, Regina; Bubacco, Luigi; De Franceschi, Giorgia; de Laureto, Patrizia Polverino; Lubian, Elisa; Selvestrel, Francesco; Mancin, Fabrizio; Papini, Emanuele

2015-11-14

A coat of strongly-bound host proteins, or hard corona, may influence the biological and pharmacological features of nanotheranostics by altering their cell-interaction selectivity and macrophage clearance. With the goal of identifying specific corona-effectors, we investigated how the capture of amorphous silica nanoparticles (SiO2-NPs; Ø = 26 nm; zeta potential = -18.3 mV) by human lymphocytes, monocytes and macrophages is modulated by the prominent proteins of their plasma corona. LC MS/MS analysis, western blotting and quantitative SDS-PAGE densitometry show that Histidine Rich Glycoprotein (HRG) is the most abundant component of the SiO2-NP hard corona in excess plasma from humans (HP) and mice (MP), together with minor amounts of the homologous Kininogen-1 (Kin-1), while it is remarkably absent in their Foetal Calf Serum (FCS)-derived corona. HRG binds with high affinity to SiO2-NPs (HRG Kd ∼2 nM) and competes with other plasma proteins for the NP surface, so forming a stable and quite homogeneous corona inhibiting nanoparticles binding to the macrophage membrane and their subsequent uptake. Conversely, in the case of lymphocytes and monocytes not only HRG but also several common plasma proteins can interchange in this inhibitory activity. The depletion of HRG and Kin-1 from HP or their plasma exhaustion by increasing NP concentration (>40 μg ml(-1) in 10% HP) lead to a heterogeneous hard corona, mostly formed by fibrinogen (Fibr), HDLs, LDLs, IgGs, Kallikrein and several minor components, allowing nanoparticle binding to macrophages. Consistently, the FCS-derived SiO2-NP hard corona, mainly formed by hemoglobin, α2 macroglobulin and HDLs but lacking HRG, permits nanoparticle uptake by macrophages. Moreover, purified HRG competes with FCS proteins for the NP surface, inhibiting their recruitment in the corona and blocking NP macrophage capture. HRG, the main component of the plasma-derived SiO2-NPs' hard corona, has antiopsonin characteristics and

An APOC3 3′UTR variant associated with plasma triglycerides levels and coronary heart disease by creating a functional miR-4271 binding site

PubMed Central

Hu, Sen-Lin; Cui, Guang-Lin; Huang, Jin; Jiang, Jian-Gang; Wang, Dao-Wen

2016-01-01

Apolipoprotein C-III (APOC3) is a key regulator of plasma triglycerides levels. Increasing evidence has shown that loss-of-function mutations in APOC3 is associated with reduction in plasma triglycerides levels and will confer a benefit in patients at high risk for cardiovascular disease. However, these favorable mutations were extremely distribution discrepant among different ethnics. In this study, the APOC3 gene was resequenced and we identified a common variant which located in the microRNA-binding site in APOC3 and would affect its expression and the risk of coronary heart disease (CHD). The molecular mechanism was explored. We found that the T allele of rs4225 suppressed APOC3 translation by facilitating miR-4271 binding, but not the G allele. Subjects carrying the GG genotype had higher plasma APOC3 levels (p for trend = 0.03) than those with the TT genotype. Furthermore, the T allele was significantly associated with decreased triglyceride levels [Beta (SE): −0.024 (0.020), P = 0.03]. Finally, the case-control study suggested that the TT genotype resulted in a significant reduction in overall CHD risk [OR, 0.89 (95% confidence interval, 0.77–0.98), P = 0.009]. In conclusion, our results provide evidence that the rs4225 in the 3′-UTR of APOC3 might contribute to the risk of CHD by interfering with miR-4271 binding. PMID:27624799

M-Ficolin Binds Selectively to the Capsular Polysaccharides of Streptococcus pneumoniae Serotypes 19B and 19C and of a Streptococcus mitis Strain

PubMed Central

Kjaer, Troels R.; Hansen, Annette G.; Sørensen, Uffe B. S.; Holm, Anne T.; Sørensen, Grith L.; Jensenius, Jens C.

2013-01-01

The three human ficolins (H-, L-, and M-ficolins) and mannan-binding lectin are pattern recognition molecules of the innate immune system mediating activation of the lectin pathway of the complement system. These four human proteins bind to some microorganisms and may be involved in the resolution of infections. We investigated binding selectivity by examining the binding of M-ficolin to a panel of more than 100 different streptococcal strains (Streptococcus pneumoniae and Streptococcus mitis), each expressing distinct polysaccharide structures. M-ficolin binding was observed for three strains only: strains of the pneumococcal serotypes 19B and 19C and a single S. mitis strain expressing a similar polysaccharide structure. The bound M-ficolin, in association with MASP-2, mediated the cleavage of complement factor C4. Binding to the bacteria was inhibitable by N-acetylglucosamine, indicating that the interaction with the bacterial surface takes place via the fibrinogen-like domain. The common N-acetylmannosamine residue present in the structures of the four capsular polysaccharides of group 19 is linked via a phosphodiester bond. This residue is apparently not a ligand for M-ficolin, since the lectin binds to two of the group 19 polysaccharides only. M-ficolin bound strongly to serotype 19B and 19C polysaccharides. In contrast to those of serotypes 19A and 19F, serotype 19B and 19C polysaccharides contain an extra N-acetylmannosamine residue linked via glycoside linkage only. Thus, this extra residue seems to be the M-ficolin ligand. In conclusion, we were able to demonstrate specific binding of M-ficolin to some capsular polysaccharides of the opportunistic pathogen S. pneumoniae and of the commensal bacterium S. mitis. PMID:23184524

Preclinical Pharmacokinetics, Tissue Distribution, and Plasma Protein Binding of Sodium (±)-5-Bromo-2-(α-Hydroxypentyl) Benzoate (BZP), an Innovative Potent Anti-ischemic Stroke Agent.

PubMed

Tian, Xin; Li, Hong-Meng; Wei, Jing-Yao; Liu, Bing-Jie; Zhang, Yu-Hai; Wang, Gao-Ju; Chang, Jun-Biao; Qiao, Hai-Ling

2016-01-01

Sodium (±)-5-bromo-2-(α-hydroxypentyl) benzoate (BZP) is a potential cardiovascular drug and exerts potent neuroprotective effect against transient and long-term ischemic stroke in rats. BZP could convert into 3-butyl-6-bromo-1(3H)-isobenzofuranone (Br-NBP) in vitro and in vivo. However, the pharmacokinetic profiles of BZP and Br-NBP still have not been evaluated. For the purpose of investigating the pharmacokinetic profiles, tissue distribution, and plasma protein binding of BZP and Br-NBP, a rapid, sensitive, and specific method based on liquid chromatography coupled to mass spectrometry (LC-MS/MS) has been developed for determination of BZP and Br-NBP in biological samples. The results indicated that BZP and Br-NBP showed a short elimination half-life, and pharmacokinetic profile in rats (3, 6, and 12 mg/kg; i.v.) and beagle dogs (1, 2, and 4 mg/kg; i.v.gtt) were obtained after single dosing of BZP. After multiple dosing of BZP, there was no significant accumulation of BZP and Br-NBP in the plasma of rats and beagle dogs. Following i.v. single dose (6 mg/kg) of BZP to rats, BZP and Br-NBP were distributed rapidly into all tissues examined, with the highest concentrations of BZP and Br-NBP in lung and kidney, respectively. The brain distribution of Br-NBP in middle cerebral artery occlusion (MCAO) rats was more than in normal rats (P < 0.05). The plasma protein binding degree of BZP at three concentrations (8000, 20,000, and 80,000 ng/mL) from rat, beagle dog, and human plasma were 98.1-98.7, 88.9-92.7, and 74.8-83.7% respectively. In conclusion, both BZP and Br-NBP showed short half-life, good dose-linear pharmacokinetic profile, wide tissue distribution, and different degree protein binding to various species plasma. This was the first preclinical pharmacokinetic investigation of BZP and Br-NBP in both rats and beagle dogs, which provided vital guidance for further preclinical research and the subsequent clinical trials.

Plasma treatments of dressings for wound healing: a review.

PubMed

Eswaramoorthy, Nithya; McKenzie, David R

2017-12-01

This review covers the use of plasma technology relevant to the preparation of dressings for wound healing. The current state of knowledge of plasma treatments that have potential to provide enhanced functional surfaces for rapid and effective healing is summarized. Dressings that are specialized to the needs of individual cases of chronic wounds such as diabetic ulcers are a special focus. A summary of the biology of wound healing and a discussion of the various types of plasmas that are suitable for the customizing of wound dressings are given. Plasma treatment allows the surface energy and air permeability of the dressing to be controlled, to ensure optimum interaction with the wound. Plasmas also provide control over the surface chemistry and in cases where the plasma creates energetic ion bombardment, activation with long-lived radicals that can bind therapeutic molecules covalently to the surface of the dressing. Therapeutic innovations enabled by plasma treatment include the attachment of microRNA or antimicrobial peptides. Bioactive molecules that promote subsequent cell adhesion and proliferation can also be bound, leading to the recruitment of cells to the dressing that may be stem cells or patient-derived cells. The presence of a communicating cell population expressing factors promotes healing.

Effects of mannan-oligosaccharides and Lactobacillus acidophilus supplementation on growth performance, nutrient utilization and faecal characteristics in Murrah buffalo calves.

PubMed

Sharma, A N; Kumar, S; Tyagi, A K

2018-06-01

A study of 120 days was undertaken to ascertain the effect of mannan-oligosaccharides (MOS) and Lactobacillus acidophilus supplementation on growth performance, nutrient utilization and faecal characteristics in Murrah buffalo calves. Twenty Murrah buffalo calves of 5-7 days old and 31 ± 2.0 kg of body weight (BW) were randomly assigned into four groups. Group I served as the control (CON) in which only basal diet (concentrate mixture and green fodder) was provided, without any supplementation. Mannan-oligosaccharides at 4 g/calf/day were supplemented as prebiotic to Group II (PRE), whereas Group III (PRO) received Lactobacillus acidophilus in the form of fermented milk as probiotic at 200 ml/calf/day having 10 8  CFU/ml and Group IV (SYN) was supplemented with both MOS and Lactobacillus acidophilus as synbiotic at similar dose. Final BW (kg), dry matter intake, average daily gain, feed conversion efficiency and structural growth measurements were improved (p < .05) in the treatment groups compared to control. Digestibility of neutral detergent fibre was higher (p < .05) in SYN followed by PRE and PRO than control. The faecal lactobacilli and bifidobacterium population was higher (p < .05) in all the supplemented groups with a concomitant reduction in faecal coliform count as compared to control. Faecal ammonia, lactate and pH were also altered favourably (p < .05) in all the supplemented groups as compared to CON. The faecal volatile fatty acids were higher (p < .05) in PRE, PRO and SYN group than CON. The incorporation of MOS and Lactobacillus acidophilus in diet either individually or in combination as synbiotic has the potential to improve the performance and faecal characteristics in Murrah buffalo calves; however, the observed responses among the treatment groups were more evident in the synbiotic fed group compared to individual supplementation of MOS and Lactobacillus acidophilus. © 2018 Blackwell Verlag GmbH.

A Lipoprotein Receptor Cluster IV Mutant Preferentially Binds Amyloid-β and Regulates Its Clearance from the Mouse Brain*

PubMed Central

Sagare, Abhay P.; Bell, Robert D.; Srivastava, Alaka; Sengillo, Jesse D.; Singh, Itender; Nishida, Yoichiro; Chow, Nienwen; Zlokovic, Berislav V.

2013-01-01

Soluble low density lipoprotein receptor-related protein-1 (sLRP1) binds ∼70% of amyloid β-peptide (Aβ) in human plasma. In Alzheimer disease (AD) and individuals with mild cognitive impairment converting to AD, plasma sLRP1 levels are reduced and sLRP1 is oxidized, which results in diminished Aβ peripheral binding and higher levels of free Aβ in plasma. Experimental studies have shown that free circulating Aβ re-enters the brain and that sLRP1 and/or its recombinant wild type cluster IV (WT-LRPIV) prevent Aβ from entering the brain. Treatment of Alzheimer APPsw+/0 mice with WT-LRPIV has been shown to reduce brain Aβ pathology. In addition to Aβ, LRPIV binds multiple ligands. To enhance LRPIV binding for Aβ relative to other LRP1 ligands, we generated a library of LRPIV-derived fragments and full-length LRPIV variants with glycine replacing aspartic acid residues 3394, 3556, and 3674 in the calcium binding sites. Compared with WT-LRPIV, a lead LRPIV-D3674G mutant had 1.6- and 2.7-fold higher binding affinity for Aβ40 and Aβ42 in vitro, respectively, and a lower binding affinity for other LRP1 ligands (e.g. apolipoprotein E2, E3, and E4 (1.3–1.8-fold), tissue plasminogen activator (2.7-fold), matrix metalloproteinase-9 (4.1-fold), and Factor Xa (3.8-fold)). LRPIV-D3674G cleared mouse endogenous brain Aβ40 and Aβ42 25–27% better than WT-LRPIV. A 3-month subcutaneous treatment of APPsw+/0 mice with LRPIV-D3674G (40 μg/kg/day) reduced Aβ40 and Αβ42 levels in the hippocampus, cortex, and cerebrospinal fluid by 60–80% and improved cerebral blood flow responses and hippocampal function at 9 months of age. Thus, LRPIV-D3674G is an efficient new Aβ clearance therapy. PMID:23580652

Infectious dengue vesicles derived from CD61+ cells in acute patient plasma exhibited a diaphanous appearance

PubMed Central

Hsu, Alan Yi-Hui; Wu, Shang-Rung; Tsai, Jih-Jin; Chen, Po-Lin; Chen, Ya-Ping; Chen, Tsai-Yun; Lo, Yu-Chih; Ho, Tzu-Chuan; Lee, Meed; Chen, Min-Ting; Chiu, Yen-Chi; Perng, Guey Chuen

2015-01-01

The levels of neutralizing antibody to a pathogen are an effective indicator to predict efficacy of a vaccine in trial. And yet not all the trial vaccines are in line with the theory. Using dengue virus (DENV) to investigate the viral morphology affecting the predictive value, we evaluated the viral morphology in acute dengue plasma compared to that of Vero cells derived DENV. The virions in plasma were infectious and heterogeneous in shape with a “sunny-side up egg” appearance, viral RNA was enclosed with CD61+ cell-derived membrane interspersed by the viral envelope protein, defined as dengue vesicles. The unique viral features were also observed from ex vivo infected human bone marrow. Dengue vesicles were less efficiently neutralized by convalescent patient serum, compared to virions produced from Vero cells. Our results exhibit a reason why potencies of protective immunity fail in vivo and significantly impact dengue vaccine and drug development. PMID:26657027

Identification and characterization of a calmodulin binding domain in the plasma membrane Ca2+-ATPase from Trypanosoma equiperdum.

PubMed

Ramírez-Iglesias, José Rubén; Pérez-Gordones, María Carolina; Del Castillo, Jesús Rafael; Mijares, Alfredo; Benaim, Gustavo; Mendoza, Marta

2018-05-09

The plasma membrane Ca 2+ -ATPase (PMCA) from trypanosomatids lacks a classical calmodulin (CaM) binding domain, although CaM stimulated activities have been detected by biochemical assays. Recently we proposed that the Trypanosoma equiperdum CaM-sensitive PMCA (TePMCA) contains a potential 1-18 CaM-binding motif at the C-terminal region of the pump. In the present study, we evaluated the potential CaM-binding motifs using CaM from Trypanosoma cruzi and either the recombinant full length TePMCA C-terminal sequence (P14) or synthetic peptides comprising different regions of the C-terminal domain. We demonstrated that P14 and a synthetic peptide corresponding to residues 1037-1062 (which contains the predicted 1-18 binding motif) competed efficiently for binding to TcCaM, exhibiting similar IC 50 s of 200 nM. A stable complex of this peptide and TcCaM was formed in the presence of Ca 2+ , as determined by native-polyacrylamide gel electrophoresis. A predicted structure obtained by molecular docking showed an interaction of the 1-18 binding motif with the Ca 2+ /CaM complex. Moreover, when the peptide was incubated with CaM and Ca 2+ , a blue shift in the tryptophan fluorescence spectrum (from 350 to 329 nm) was observed. Substitutions at W 1039 and F 1056 , strongly decreased both CaM-peptide interaction and the complex assembly. Our results demonstrated the presence of a functional 1-18 motif at the TePMCA C-terminal domain. Furthermore, on the basis of spectrofluorometric assays and the resulting structure modeled by docking we propose that the L 1042 and W 1060 residues might also participate as anchors to form a 1-4-18-22 motif. Copyright © 2018 Elsevier B.V. All rights reserved.

Diet-derived phenols in plasma and tissues and their implications for health.

PubMed

Clifford, M N

2004-12-01

This paper seeks to catalyse a reappraisal of the nature, fate and biological significance in humans of phenols, polyphenols and tannins (PPT) consumed in normal diets, and in particular questions the primacy of PPT radical-scavenging mechanisms for the supposed health benefits of diets rich in fruits and vegetables. PPT are classified by structure and function. Arguments are presented to show that cinnamates and derived polyphenols make significantly larger contributions to the total PPT intake than the flavonols and flavones upon which the vast majority of attention has been focussed previously. Daily intakes of total PPT may range from less than 100 mg to in excess of 2 g, and the critical importance of coffee and black tea as the major dietary sources is shown. Only some 5% of the dietary PPT is absorbed in the duodenum, and of this only some 5%, mainly flavanols, reaches the plasma unchanged, the balance being mammalian conjugates. Over 95% of the intake passes to the colon and is fermented by the gut microflora. A fraction of the resulting microbial metabolites is absorbed and appears in the plasma primarily as mammalian conjugates. Even following high intakes of PPT, the plasma metabolites collectively make a very small (less than 5%) and transient contribution to the total concentration of redox active substances in plasma. This explains the failure of most studies that sought to detect an increase in plasma antioxidant power after consuming a PPT-rich meal or supplement. The powerfully antioxidant PPT aglycones, much used in in vitro studies, do not reach the plasma. The redox potential of those unchanged PPT and PPT metabolites that reach the plasma enables them to scavenge damaging radicals, but the endogenous plasma antioxidants, especially ascorbate, are required for disposal of the resultant phenoxyl radicals. Black tea and coffee, the major sources of PPT, are poor sources of ascorbate. It is suggested that if diets rich in fruits and vegetables are

Spectroscopic profiling and computational study of the binding of tschimgine: A natural monoterpene derivative, with calf thymus DNA.

PubMed

Khajeh, Masoumeh Ashrafi; Dehghan, Gholamreza; Dastmalchi, Siavoush; Shaghaghi, Masoomeh; Iranshahi, Mehrdad

2018-03-05

DNA is a major target for a number of anticancer substances. Interaction studies between small molecules and DNA are essential for rational drug designing to influence main biological processes and also introducing new probes for the assay of DNA. Tschimgine (TMG) is a monoterpene derivative with anticancer properties. In the present study we tried to elucidate the interaction of TMG with calf thymus DNA (CT-DNA) using different spectroscopic methods. UV-visible absorption spectrophotometry, fluorescence and circular dichroism (CD) spectroscopies as well as molecular docking study revealed formation of complex between TMG and CT-DNA. Binding constant (K b ) between TMG and DNA was 2.27×10 4 M -1 , that is comparable to groove binding agents. The fluorescence spectroscopic data revealed that the quenching mechanism of fluorescence of TMG by CT-DNA is static quenching. Thermodynamic parameters (ΔH<0 and ΔS<0) at different temperatures indicated that van der Waals forces and hydrogen bonds were involved in the binding process of TMG with CT-DNA. Competitive binding assay with methylene blue (MB) and Hoechst 33258 using fluorescence spectroscopy displayed that TMG possibly binds to the minor groove of CT-DNA. These observations were further confirmed by CD spectral analysis, viscosity measurements and molecular docking. Copyright © 2017 Elsevier B.V. All rights reserved.

Label-free quantitative proteomic analysis of human plasma-derived microvesicles to find protein signatures of abdominal aortic aneurysms.

PubMed

Martinez-Pinna, Roxana; Gonzalez de Peredo, Anne; Monsarrat, Bernard; Burlet-Schiltz, Odile; Martin-Ventura, Jose Luis

2014-08-01

To find potential biomarkers of abdominal aortic aneurysms (AAA), we performed a differential proteomic study based on human plasma-derived microvesicles. Exosomes and microparticles isolated from plasma of AAA patients and control subjects (n = 10 each group) were analyzed by a label-free quantitative MS-based strategy. Homemade and publicly available software packages have been used for MS data analysis. The application of two kinds of bioinformatic tools allowed us to find differential protein profiles from AAA patients. Some of these proteins found by the two analysis methods belong to main pathological mechanisms of AAA such as oxidative stress, immune-inflammation, and thrombosis. Data analysis from label-free MS-based experiments requires the use of sophisticated bioinformatic approaches to perform quantitative studies from complex protein mixtures. The application of two of these bioinformatic tools provided us a preliminary list of differential proteins found in plasma-derived microvesicles not previously associated to AAA, which could help us to understand the pathological mechanisms related to this disease. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Intravenous iron-dextran: studies on unsaturated iron-binding capacity

PubMed Central

Cox, J. S. G.; Moss, G. F.; Bremner, I.; Reason, Janet

1968-01-01

A method is described for measuring the plasma unsaturated iron-binding capacity in the presence of very high concentrations of iron as iron-dextran. The procedure utilizes 59Fe to label the apotransferrin with subsequent separation of ionic iron from transferrin-bound iron on an ion exchange or Sephadex G.25 column. The unsaturated iron-binding capacity has been measured in rabbits and dogs after intravenous injection of iron-dextran and in human subjects after total dose infusion of iron-dextran. No evidence of saturation of the unsaturated iron-binding capacity was found even when the plasma iron values were greater than 40,000 μg Fe/100 ml. PMID:5697365

[Pharmacokinetic properties of platinium derivatives].

PubMed

Boisdron-Celle, M; Lebouil, A; Allain, P; Gamelin, E

2001-08-01

The three platinum derivatives currently available share many pharmacokinetic and pharmacodynamic (PK-PD) properties but present also some distinct characteristics, due to their structural differences. They result in different systemic PK-PD and metabolic behaviour and toxicity profile. Oxaliplatin is quickly transformed into dach-platinum, the active metabolite, by loosing oxalate chain. Eighty to eighty-eight per cent of platinum are bound to proteins, as for cisplatin, whereas carboplatin is less reactive. Cisplatin and oxaliplatin active metabolites, i.e. monoaquo platin and dach-platin quickly react with small proteins with sulfhydryl groups, such as glutathione, cysteine and methionine, and then with high molecular weight proteins, such as albumin and gammaglobulins through covalent link. Thus, their terminal half lives are long, about ten days, but no platinum accumulation has been reported in plasma with oxaliplatin, whereas after cisplatin administration, both total and ultrafiltrable platinum progressively accumulate in plasma. This difference may play a role in the lack of oxaliplatin nephrotoxicity and its more delayed and reversible neurotoxicity. On the other hand, carboplatin is more stable, less bound to proteins and is largely excreted inchanged in urine. This can explain that it passes more easily through the blood brain barrier. Erythrocytes represent an important deep compartment, especially for oxaliplatin, a little bit less for cisplatin. Oxaliplatin is trapped in erythrocytes through a covalent binding to globin. There, its half life is identical to that of erythrocytes. According to certain authors, this trapping would be involved in the incidence of anemia. On the contrary, carboplatin is quickly extruded from erythrocytes. The three derivatives kinetics in plasma present a wide interindividual variability, resulting in differences in term of toxicity and efficacy. For the three of them, plasma clearance is correlated to creatinine

Binding of sulphonated indigo derivatives to RepA-WH1 inhibits DNA-induced protein amyloidogenesis

PubMed Central

Gasset-Rosa, Fátima; Maté, María Jesús; Dávila-Fajardo, Cristina; Bravo, Jerónimo; Giraldo, Rafael

2008-01-01

The quest for inducers and inhibitors of protein amyloidogenesis is of utmost interest, since they are key tools to understand the molecular bases of proteinopathies such as Alzheimer, Parkinson, Huntington and Creutzfeldt–Jakob diseases. It is also expected that such molecules could lead to valid therapeutic agents. In common with the mammalian prion protein (PrP), the N-terminal Winged-Helix (WH1) domain of the pPS10 plasmid replication protein (RepA) assembles in vitro into a variety of amyloid nanostructures upon binding to different specific dsDNA sequences. Here we show that di- (S2) and tetra-sulphonated (S4) derivatives of indigo stain dock at the DNA recognition interface in the RepA-WH1 dimer. They compete binding of RepA to its natural target dsDNA repeats, found at the repA operator and at the origin of replication of the plasmid. Calorimetry points to the existence of a major site, with micromolar affinity, for S4-indigo in RepA-WH1 dimers. As revealed by electron microscopy, in the presence of inducer dsDNA, both S2/S4 stains inhibit the assembly of RepA-WH1 into fibres. These results validate the concept that DNA can promote protein assembly into amyloids and reveal that the binding sites of effector molecules can be targeted to inhibit amyloidogenesis. PMID:18285361

Molecular Characterization of Lipopolysaccharide Binding to Human α-1-Acid Glycoprotein

PubMed Central

Huang, Johnny X.; Azad, Mohammad A. K.; Yuriev, Elizabeth; Baker, Mark A.; Nation, Roger L.; Li, Jian; Cooper, Matthew A.; Velkov, Tony

2012-01-01

The ability of AGP to bind circulating lipopolysaccharide (LPS) in plasma is believed to help reduce the proinflammatory effect of bacterial lipid A molecules. Here, for the first time we have characterized human AGP binding characteristics of the LPS from a number of pathogenic Gram-negative bacteria: Escherichia coli, Salmonella typhimurium, Klebsiella pneumonia, Pseudomonas aeruginosa, and Serratia marcescens. The binding affinity and structure activity relationships (SAR) of the AGP-LPS interactions were characterized by surface plasma resonance (SPR). In order to dissect the contribution of the lipid A, core oligosaccharide and O-antigen polysaccharide components of LPS, the AGP binding affinity of LPS from smooth strains, were compared to lipid A, Kdo2-lipid A, Ra, Rd, and Re rough LPS mutants. The SAR analysis enabled by the binding data suggested that, in addition to the important role played by the lipid A and core components of LPS, it is predominately the unique species- and strain-specific carbohydrate structure of the O-antigen polysaccharide that largely determines the binding affinity for AGP. Together, these data are consistent with the role of AGP in the binding and transport of LPS in plasma during acute-phase inflammatory responses to invading Gram-negative bacteria. PMID:23316371

DNA binding of a proflavine derivative bearing a platinum hanging residue.

PubMed

Biagini, Silvia; Bianchi, Antonio; Biver, Tarita; Boggioni, Alessia; Nikolayenko, Igor V; Secco, Fernando; Venturini, Marcella

2011-04-01

New platinum(II) complex of 3,6-diamine-9-[6,6-bis(2-aminohethyl)-1,6-diaminohexyl]acridine, AzaPt, has been synthesised and characterised. Behaviour of AzaPt in solution (protonation and possible self-aggregation phenomena) has been investigated by spectral methods (absorbance and fluorescence) at I=0.1M and 25°C, and the equilibrium parameters of binding to calf thymus DNA have been established. Two different modes of DNA binding by the complex were detected, which depend on the polymer to dye molar ratio (P/D). At relatively low P/D values the mode was interpreted as binding by the polyamine residue external to the base pairs, while at high P/D values the binding corresponds to intercalation of the proflavine residue. Such interpretation is supported by the observed salt effect on binding and the temperature variation of the binding constants, which allowed estimating the ΔH and ΔS values contributions. Spectrophotometric analysis of the long time range binding revealed that AzaPt is involved in a slow reaction, interpreted as an attack by the platinum ion on the nucleobases. The time constant for such interaction was calculated and found to be the same order of magnitude as for processes responsible for the action of anti-tumour drugs that do covalently bind to polynucleotides. Copyright © 2010 Elsevier Inc. All rights reserved.

Brain-derived neurotrophic factor Val66met polymorphism and plasma levels in road traffic accident survivors.

PubMed

van den Heuvel, Leigh; Suliman, Sharain; Malan-Müller, Stefanie; Hemmings, Sian; Seedat, Soraya

2016-11-01

Alterations in brain-derived neurotrophic factor (BDNF) expression and release may play a role in the pathogenesis of post-traumatic stress disorder (PTSD). This study evaluated road traffic accident (RTA) survivors to determine whether PTSD and trauma-related factors were associated with plasma BDNF levels and BDNF Val66Met carrier status following RTA exposure. One hundred and twenty-three RTA survivors (mean age 33.2 years, SD = 10.6 years; 56.9% male) were assessed 10 (SD = 4.9) days after RTA exposure. Acute stress disorder (ASD), as assessed with the Acute Stress Disorder Scale, was present in 50 (42.0%) of the participants. Plasma BDNF levels were measured with enzyme-linked immunosorbent assay and BDNF Val66Met genotyping was performed. PTSD, as assessed with the Clinician-Administered PTSD Scale, was present in 10 (10.8%) participants at 6 months follow-up. Neither BDNF Val66Met genotype nor plasma BDNF was significantly associated with the presence or severity of ASD or PTSD. Plasma BDNF levels were, however, significantly correlated with the lifetime number of trauma exposures. In RTA survivors, plasma BDNF levels increased with increasing number of prior trauma exposures. Plasma BDNF may, therefore, be a marker of trauma load.

Plasma brain derived neurotrophic factor (BDNF) and response to ketamine in treatment-resistant depression.

PubMed

Haile, C N; Murrough, J W; Iosifescu, D V; Chang, L C; Al Jurdi, R K; Foulkes, A; Iqbal, S; Mahoney, J J; De La Garza, R; Charney, D S; Newton, T F; Mathew, S J

2014-02-01

Ketamine produces rapid antidepressant effects in treatment-resistant depression (TRD), but the magnitude of response varies considerably between individual patients. Brain-derived neurotrophic factor (BDNF) has been investigated as a biomarker of treatment response in depression and has been implicated in the mechanism of action of ketamine. We evaluated plasma BDNF and associations with symptoms in 22 patients with TRD enrolled in a randomized controlled trial of ketamine compared to an anaesthetic control (midazolam). Ketamine significantly increased plasma BDNF levels in responders compared to non-responders 240 min post-infusion, and Montgomery-Åsberg Depression Rating Scale (MADRS) scores were negatively correlated with BDNF (r=-0.701, p = 0.008). Plasma BDNF levels at 240 min post-infusion were highly negatively associated with MADRS scores at 240 min (r = -0.897, p=.002), 24 h (r = -0.791, p = 0.038), 48 h (r = -0.944, p = 0.001) and 72 h (r = -0.977, p = 0.010). No associations with BDNF were found for patients receiving midazolam. These data support plasma BDNF as a peripheral biomarker relevant to ketamine antidepressant response.

Detecting cis-regulatory binding sites for cooperatively binding proteins

PubMed Central

van Oeffelen, Liesbeth; Cornelis, Pierre; Van Delm, Wouter; De Ridder, Fedor; De Moor, Bart; Moreau, Yves

2008-01-01

Several methods are available to predict cis-regulatory modules in DNA based on position weight matrices. However, the performance of these methods generally depends on a number of additional parameters that cannot be derived from sequences and are difficult to estimate because they have no physical meaning. As the best way to detect cis-regulatory modules is the way in which the proteins recognize them, we developed a new scoring method that utilizes the underlying physical binding model. This method requires no additional parameter to account for multiple binding sites; and the only necessary parameters to model homotypic cooperative interactions are the distances between adjacent protein binding sites in basepairs, and the corresponding cooperative binding constants. The heterotypic cooperative binding model requires one more parameter per cooperatively binding protein, which is the concentration multiplied by the partition function of this protein. In a case study on the bacterial ferric uptake regulator, we show that our scoring method for homotypic cooperatively binding proteins significantly outperforms other PWM-based methods where biophysical cooperativity is not taken into account. PMID:18400778

Clinical role of protein binding of quinolones.

PubMed

Bergogne-Bérézin, Eugénie

2002-01-01

Protein binding of antibacterials in plasma and tissues has long been considered a component of their pharmacokinetic parameters, playing a potential role in distribution, excretion and therapeutic effectiveness. Since the beginning of the 'antibacterial era', this factor has been extensively analysed for all antibacterial classes, showing that wide variations of the degree of protein binding occur even in the same antibacterial class, as with beta-lactams. As the understanding of protein binding grew, the complexity of the binding system was increasingly perceived and its dynamic character described. Studies of protein binding of the fluoroquinolones have shown that the great majority of these drugs exhibit low protein binding, ranging from approximately 20 to 40% in plasma, and that they are bound predominantly to albumin. The potential role in pharmacokinetics-pharmacodynamics of binding of fluoroquinolones to plasma, tissue and intracellular proteins has been analysed, but it has not been established that protein binding has any significant direct or indirect impact on therapeutic effectiveness. Regarding the factors influencing the tissue distribution of antibacterials, physicochemical characteristics and the small molecular size of fluoroquinolones permit a rapid penetration into extravascular sites and intracellularly, with a rapid equilibrium being established between intravascular and extravascular compartments. The high concentrations of these drugs achieved in tissues, body fluids and intracellularly, in addition to their wide antibacterial spectrum, mean that fluoroquinolones have therapeutic effectiveness in a large variety of infections. The tolerability of quinolones has generally been reported as good, based upon long experience in using pefloxacin, ciprofloxacin and ofloxacin in clinical practice. Among more recently developed molecules, good tolerability has been reported for levofloxacin, moxifloxacin and gatifloxacin, but certain other new

Dust cloud evolution in sub-stellar atmospheres via plasma deposition and plasma sputtering

NASA Astrophysics Data System (ADS)

Stark, C. R.; Diver, D. A.

2018-04-01

Context. In contemporary sub-stellar model atmospheres, dust growth occurs through neutral gas-phase surface chemistry. Recently, there has been a growing body of theoretical and observational evidence suggesting that ionisation processes can also occur. As a result, atmospheres are populated by regions composed of plasma, gas and dust, and the consequent influence of plasma processes on dust evolution is enhanced. Aim. This paper aims to introduce a new model of dust growth and destruction in sub-stellar atmospheres via plasma deposition and plasma sputtering. Methods: Using example sub-stellar atmospheres from DRIFT-PHOENIX, we have compared plasma deposition and sputtering timescales to those from neutral gas-phase surface chemistry to ascertain their regimes of influence. We calculated the plasma sputtering yield and discuss the circumstances where plasma sputtering dominates over deposition. Results: Within the highest dust density cloud regions, plasma deposition and sputtering dominates over neutral gas-phase surface chemistry if the degree of ionisation is ≳10-4. Loosely bound grains with surface binding energies of the order of 0.1-1 eV are susceptible to destruction through plasma sputtering for feasible degrees of ionisation and electron temperatures; whereas, strong crystalline grains with binding energies of the order 10 eV are resistant to sputtering. Conclusions: The mathematical framework outlined sets the foundation for the inclusion of plasma deposition and plasma sputtering in global dust cloud formation models of sub-stellar atmospheres.

Characterization of membrane protein interactions in plasma membrane derived vesicles with quantitative imaging Förster resonance energy transfer.

PubMed

Sarabipour, Sarvenaz; Del Piccolo, Nuala; Hristova, Kalina

2015-08-18

Here we describe an experimental tool, termed quantitative imaging Förster resonance energy transfer (QI-FRET), that enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors. Since plasma membranes of cells have complex topologies precluding the acquisition of two-dimensional binding curves, the FRET measurements are performed in plasma membrane derived vesicles that bud off cells as a result of chemical or osmotic stress. The results overviewed here are acquired in vesicles produced with an osmotic vesiculation buffer developed in our laboratory, which does not utilize harsh chemicals. The concentrations of the donor-labeled and the acceptor-labeled proteins are determined, along with the FRET efficiencies, in each vesicle. The experiments utilize transient transfection, such that a wide variety of concentrations is sampled. Then, data from hundreds of vesicles are combined to yield dimerization curves. Here we discuss recent findings about the dimerization of receptor tyrosine kinases (RTKs), membrane proteins that control cell growth and differentiation via lateral dimerization in the plasma membrane. We focus on the dimerization of fibroblast growth factor receptor 3 (FGFR3), a RTK that plays a critically important role in skeletal development. We study the role of different FGFR3 domains in FGFR3 dimerization in the absence of ligand, and we show that FGFR3 extracellular domains inhibit unliganded dimerization, while contacts between the juxtamembrane domains, which connect the transmembrane domains to the kinase domains, stabilize the unliganded FGFR3 dimers. Since FGFR3 has been documented to harbor many pathogenic

Measurement of plasma unbound unconjugated bilirubin.

PubMed

Ahlfors, C E

2000-03-15

A method is described for measuring the unconjugated fraction of the unbound bilirubin concentration in plasma by combining the peroxidase method for determining unbound bilirubin with a diazo method for measuring conjugated and unconjugated bilirubin. The accuracy of the unbound bilirubin determination is improved by decreasing sample dilution, eliminating interference by conjugated bilirubin, monitoring changes in bilirubin concentration using diazo derivatives, and correcting for rate-limiting dissociation of bilirubin from albumin. The unbound unconjugated bilirubin concentration by the combined method in plasma from 20 jaundiced newborns was significantly greater than and poorly correlated with the unbound bilirubin determined by the existing peroxidase method (r = 0.7), possibly due to differences in sample dilution between the methods. The unbound unconjugated bilirubin was an unpredictable fraction of the unbound bilirubin in plasma samples from patients with similar total bilirubin concentrations but varying levels of conjugated bilirubin. A bilirubin-binding competitor was readily detected at a sample dilution typically used for the combined test but not at the dilution used for the existing peroxidase method. The combined method is ideally suited to measuring unbound unconjugated bilirubin in jaundiced human newborns or animal models of kernicterus. Copyright 2000 Academic Press.

Decreased plasma concentrations of brain-derived neurotrophic factor (BDNF) in patients with functional hypothalamic amenorrhea.

PubMed

Podfigurna-Stopa, Agnieszka; Casarosa, Elena; Luisi, Michele; Czyzyk, Adam; Meczekalski, Blazej; Genazzani, Andrea Riccardo

2013-09-01

Functional hypothalamic amenorrhea (FHA) is a non organic, secondary amenorrhea related to gonadotropin-releasing hormone pulsatile secretion impairment. Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family of survival-promoting molecules, plays an important role in the growth, development, maintenance and function of several neuronal systems. The aim of the study was the evaluation of plasma BDNF concentrations in patients with the diagnosis of FHA. We studied 85 subjects diagnosed with FHA who were compared with 10 healthy, eumenorrheic controls with normal body mass index. Plasma BDNF and serum luteinizing hormone, follicle-stimulating hormone and estradiol (E2) concentrations were measured by immunoenzymatic method (enzyme-linked immunosorbent assay). Significantly lower concentration of plasma BDNF was found in FHA patients (196.31 ± 35.26 pg/ml) in comparison to healthy controls (407.20 ± 25.71 pg/ml; p < 0.0001). In the control group, there was a strong positive correlation between plasma BDNF and serum E2 concentrations (r = 0.92, p = 0.0001) but in FHA group it was not found. Role of BDNF in FHA is not yet fully understood. There could be found studies concerning plasma BDNF concentrations in humans and animals in the literature. However, our study is one of the first projects which describes decreased plasma BDNF concentration in patients with diagnosed FHA. Therefore, further studies on BDNF in FHA should clarify the role of this peptide.

Novel hits for acetylcholinesterase inhibition derived by docking-based screening on ZINC database.

PubMed

Doytchinova, Irini; Atanasova, Mariyana; Valkova, Iva; Stavrakov, Georgi; Philipova, Irena; Zhivkova, Zvetanka; Zheleva-Dimitrova, Dimitrina; Konstantinov, Spiro; Dimitrov, Ivan

2018-12-01

The inhibition of the enzyme acetylcholinesterase (AChE) increases the levels of the neurotransmitter acetylcholine and symptomatically improves the affected cognitive function. In the present study, we searched for novel AChE inhibitors by docking-based virtual screening of the standard lead-like set of ZINC database containing more than 6 million small molecules using GOLD software. The top 10 best-scored hits were tested in vitro for AChE affinity, neurotoxicity, GIT and BBB permeability. The main pharmacokinetic parameters like volume of distribution, free fraction in plasma, total clearance, and half-life were predicted by previously derived models. Nine of the compounds bind to the enzyme with affinities from 0.517 to 0.735 µM, eight of them are non-toxic. All hits permeate GIT and BBB and bind extensively to plasma proteins. Most of them are low-clearance compounds. In total, seven of the 10 hits are promising for further lead optimisation. These are structures with ZINC IDs: 00220177, 44455618, 66142300, 71804814, 72065926, 96007907, and 97159977.

Plasma exosomal α-synuclein is likely CNS-derived and increased in Parkinson’s disease

PubMed Central

Cook, Travis J.; Bullock, Kristin M.; Zhao, Yanchun; Ginghina, Carmen; Li, Yanfei; Aro, Patrick; Dator, Romel; He, Chunmei; Hipp, Michael J.; Zabetian, Cyrus P.; Peskind, Elaine R.; Hu, Shu-Ching; Quinn, Joseph F.; Galasko, Douglas R.; Banks, William A.; Zhang, Jing

2014-01-01

Extracellular α-synuclein is important in the pathogenesis of Parkinson disease (PD) and also as a potential biomarker when tested in the cerebrospinal fluid (CSF). The performance of blood plasma or serum α-synuclein as a biomarker has been found to be inconsistent and generally ineffective, largely due to the contribution of peripherally derived α-synuclein. In this study, we discovered, via an intracerebroventricular injection of radiolabeled α-synuclein into mouse brain, that CSF α-synuclein was readily transported to blood, with a small portion being contained in exosomes that are relatively specific to the central nervous system (CNS). Consequently, we developed a technique to evaluate the levels of α-synuclein in these exosomes in individual plasma samples. When applied to a large cohort of clinical samples (267 PD, 215 controls), we found that in contrast to CSF α-synuclein concentrations, which are consistently reported to be lower in PD patients compared to controls, the levels of plasma exosomal α-synuclein were substantially higher in PD patients, suggesting an increased efflux of the protein to the peripheral blood of these patients. Furthermore, although no association was observed between plasma exosomal and CSF α-synuclein, a significant correlation between plasma exosomal α-synuclein and disease severity (r=0.176, p=0.004) was observed, and the diagnostic sensitivity and specificity achieved by plasma exosomal α-synuclein were comparable to those determined by CSF α-synuclein. Further studies are clearly needed to elucidate the mechanism involved in the transport of CNS α-synuclein to the periphery, which may lead to a more convenient and robust assessment of PD clinically. PMID:24997849

Iron-binding antioxidant capacity is impaired in diabetes mellitus.

PubMed

Van Campenhout, Ann; Van Campenhout, Christel; Lagrou, Albert R; Moorkens, Greta; De Block, Christophe; Manuel-y-Keenoy, Begoña

2006-05-15

Increased lipid peroxidation contributes to diabetic complications and redox-active iron is known to play an important role in catalyzing peroxidation reactions. We aimed to investigate if diabetes affects the capacity of plasma to protect against iron-driven lipid peroxidation and to identify underlying factors. Glycemic control, serum iron, proteins involved in iron homeostasis, plasma iron-binding antioxidant capacity in a liposomal model, and non-transferrin-bound iron were measured in 40 type 1 and 67 type 2 diabetic patients compared to 100 nondiabetic healthy control subjects. Iron-binding antioxidant capacity was significantly lower in the plasma of diabetic subjects (83 +/- 6 and 84 +/- 5% in type 1 and type 2 diabetes versus 88 +/- 6% in control subjects, p < 0.0005). The contribution of transferrin, ceruloplasmin, and albumin concentrations to the iron-binding antioxidant capacity was lost in diabetes (explaining only 4.2 and 6.3% of the variance in type 1 and type 2 diabetes versus 13.9% in control subjects). This observation could not be explained by differences in Tf glycation, lipid, or inflammatory status and was not associated with higher non-transferrin-bound iron levels. Iron-binding antioxidant capacity is decreased in diabetes mellitus.

Structural and binding studies of SAP-1 protein with heparin.

PubMed

Yadav, Vikash K; Mandal, Rahul S; Puniya, Bhanwar L; Kumar, Rahul; Dey, Sharmistha; Singh, Sarman; Yadav, Savita

2015-03-01

SAP-1 is a low molecular weight cysteine protease inhibitor (CPI) which belongs to type-2 cystatins family. SAP-1 protein purified from human seminal plasma (HuSP) has been shown to inhibit cysteine and serine proteases and exhibit interesting biological properties, including high temperature and pH stability. Heparin is a naturally occurring glycosaminoglycan (with varied chain length) which interacts with a number of proteins and regulates multiple steps in different biological processes. As an anticoagulant, heparin enhances inhibition of thrombin by the serpin antithrombin III. Therefore, we have employed surface plasmon resonance (SPR) to improve our understanding of the binding interaction between heparin and SAP-1 (protease inhibitor). SPR data suggest that SAP-1 binds to heparin with a significant affinity (KD = 158 nm). SPR solution competition studies using heparin oligosaccharides showed that the binding of SAP-1 to heparin is dependent on chain length. Large oligosaccharides show strong binding affinity for SAP-1. Further to get insight into the structural aspect of interactions between SAP-1 and heparin, we used modelled structure of the SAP-1 and docked with heparin and heparin-derived polysaccharides. The results suggest that a positively charged residue lysine plays important role in these interactions. Such information should improve our understanding of how heparin, present in the reproductive tract, regulates cystatins activity. © 2014 John Wiley & Sons A/S.

Diffusion and binding constants for acetylcholine derived from the falling phase of miniature endplate currents.

PubMed Central

Land, B R; Harris, W V; Salpeter, E E; Salpeter, M M

1984-01-01

In previous papers we studied the rising phase of a miniature endplate current (MEPC) to derive diffusion and forward rate constants controlling acetylcholine (AcCho) in the intact neuromuscular junction. The present study derives similar values (but with smaller error ranges) for these constants by including experimental results from the falling phase of the MEPC. We find diffusion to be 4 X 10(-6) cm2 s-1, slightly slower than free diffusion, forward binding to be 3.3 X 10(7) M-1 s-1, and the distance from an average release site to the nearest exit from the cleft to be 1.6 micron. We also estimate the back reaction rates. From our values we can accurately describe the shape of MEPCs under different conditions of receptor and esterase concentration. Since we suggest that unbinding is slower than isomerization, we further predict that there should be several short "closing flickers" during the total open time for an AcCho-ligated receptor channel. PMID:6584895

Investigations on therapeutic glucocerebrosidases through paired detection with fluorescent activity-based probes

PubMed Central

Kallemeijn, Wouter W.; Scheij, Saskia; Hoogendoorn, Sascha; Witte, Martin D.; Herrera Moro Chao, Daniela; van Roomen, Cindy P. A. A.; Ottenhoff, Roelof; Overkleeft, Herman S.; Boot, Rolf G.; Aerts, Johannes M. F. G.

2017-01-01

Deficiency of glucocerebrosidase (GBA) causes Gaucher disease (GD). In the common non-neuronopathic GD type I variant, glucosylceramide accumulates primarily in the lysosomes of visceral macrophages. Supplementing storage cells with lacking enzyme is accomplished via chronic intravenous administration of recombinant GBA containing mannose-terminated N-linked glycans, mediating the selective uptake by macrophages expressing mannose-binding lectin(s). Two recombinant GBA preparations with distinct N-linked glycans are registered in Europe for treatment of type I GD: imiglucerase (Genzyme), contains predominantly Man(3) glycans, and velaglucerase (Shire PLC) Man(9) glycans. Activity-based probes (ABPs) enable fluorescent labeling of recombinant GBA preparations through their covalent attachment to the catalytic nucleophile E340 of GBA. We comparatively studied binding and uptake of ABP-labeled imiglucerase and velaglucerase in isolated dendritic cells, cultured human macrophages and living mice, through simultaneous detection of different GBAs by paired measurements. Uptake of ABP-labeled rGBAs by dendritic cells was comparable, as well as the bio-distribution following equimolar intravenous administration to mice. ABP-labeled rGBAs were recovered largely in liver, white-blood cells, bone marrow and spleen. Lungs, brain and skin, affected tissues in severe GD types II and III, were only poorly supplemented. Small, but significant differences were noted in binding and uptake of rGBAs in cultured human macrophages, in the absence and presence of mannan. Mannan-competed binding and uptake were largest for velaglucerase, when determined with single enzymes or as equimolar mixtures of both enzymes. Vice versa, imiglucerase showed more prominent binding and uptake not competed by mannan. Uptake of recombinant GBAs by cultured macrophages seems to involve multiple receptors, including several mannose-binding lectins. Differences among cells from different donors (n = 12

Generator of the low-temperature heterogeneous plasma flow

NASA Astrophysics Data System (ADS)

Yusupov, D. I.; Gadzhiev, M. Kh; Tyuftyaev, A. S.; Chinnov, V. F.; Sargsyan, M. A.

2018-01-01

A generator of low-temperature dc plasma with an expanding channel of an output electrode for gas-thermal spraying was designed and constructed. The delivery of the sprayed powder into the cathode and anode arc-binding zones or into the plasma jet below the anode binding was realized. The electrophysical characteristics of both the plasma torch and the heterogeneous plasma flow with Al2O3 powder are studied. It is shown that the current-voltage characteristic (CVC) of a plasma torch depends on the gas flow rate. If the flow rate varies from 1 to 3 g/s, the falling CVC becomes gradually increasing. The speed and temperature of the sprayed powder are determined.

Platelet-Rich Plasma Derived Growth Factors Contribute to Stem Cell Differentiation in Musculoskeletal Regeneration.

PubMed

Qian, Yun; Han, Qixin; Chen, Wei; Song, Jialin; Zhao, Xiaotian; Ouyang, Yuanming; Yuan, Weien; Fan, Cunyi

2017-01-01

Stem cell treatment and platelet-rich plasma (PRP) therapy are two significant issues in regenerative medicine. Stem cells such as bone marrow mesenchymal stem cells, adipose-derived stem cells and periodontal ligament stem cells can be successfully applied in the field of tissue regeneration. PRP, a natural product isolated from whole blood, can secrete multiple growth factors (GFs) for regulating physiological activities. These GFs can stimulate proliferation and differentiation of different stem cells in injury models. Therefore, combination of both agents receives wide expectations in regenerative medicine, especially in bone, cartilage and tendon repair. In this review, we thoroughly discussed the interaction and underlying mechanisms of PRP derived GFs with stem cells, and assessed their functions in cell differentiation for musculoskeletal regeneration.

A novel L-ficolin/mannose-binding lectin chimeric molecule with enhanced activity against Ebola virus.

PubMed

Michelow, Ian C; Dong, Mingdong; Mungall, Bruce A; Yantosca, L Michael; Lear, Calli; Ji, Xin; Karpel, Marshall; Rootes, Christina L; Brudner, Matthew; Houen, Gunnar; Eisen, Damon P; Kinane, T Bernard; Takahashi, Kazue; Stahl, Gregory L; Olinger, Gene G; Spear, Gregory T; Ezekowitz, R Alan B; Schmidt, Emmett V

2010-08-06

Ebola viruses constitute a newly emerging public threat because they cause rapidly fatal hemorrhagic fevers for which no treatment exists, and they can be manipulated as bioweapons. We targeted conserved N-glycosylated carbohydrate ligands on viral envelope surfaces using novel immune therapies. Mannose-binding lectin (MBL) and L-ficolin (L-FCN) were selected because they function as opsonins and activate complement. Given that MBL has a complex quaternary structure unsuitable for large scale cost-effective production, we sought to develop a less complex chimeric fusion protein with similar ligand recognition and enhanced effector functions. We tested recombinant human MBL and three L-FCN/MBL variants that contained the MBL carbohydrate recognition domain and varying lengths of the L-FCN collagenous domain. Non-reduced chimeric proteins formed predominantly nona- and dodecameric oligomers, whereas recombinant human MBL formed octadecameric and larger oligomers. Surface plasmon resonance revealed that L-FCN/MBL76 had the highest binding affinities for N-acetylglucosamine-bovine serum albumin and mannan. The same chimeric protein displayed superior complement C4 cleavage and binding to calreticulin (cC1qR), a putative receptor for MBL. L-FCN/MBL76 reduced infection by wild type Ebola virus Zaire significantly greater than the other molecules. Tapping mode atomic force microscopy revealed that L-FCN/MBL76 was significantly less tall than the other molecules despite similar polypeptide lengths. We propose that alterations in the quaternary structure of L-FCN/MBL76 resulted in greater flexibility in the collagenous or neck region. Similarly, a more pliable molecule might enhance cooperativity between the carbohydrate recognition domains and their cognate ligands, complement activation, and calreticulin binding dynamics. L-FCN/MBL chimeric proteins should be considered as potential novel therapeutics.

Molecular Docking, Synthesis And Biological Evaluation Of Sulphonylureas/Guanidine Derivatives As Promising Antidiabetics Agent.

PubMed

Panchal, Ishan; Sen, Dhrubo Jyoti; Patel, Ashish D; Shah, Umang; Patel, Mehul; Navle, Archana; Bhavsar, Vashisth

2017-10-02

A series of novel sulphonylureas/guanidine derivatives were designed, synthesized, and evaluated for the treatment of diabetes mellitus. In this study, the designed compounds were docked with AKR1C1 complexes by using glide docking program and docking calculations were performed to predict the binding affinity of the designed compounds with the binding pocket of protein 4YVP and QikProp program was used to predict the ADME/T properties of the analogues. All the targeted derivatives were synthesized and purified by recrystallization. Synthesize compounds were characterized by various physicochemical and various spectroscopic techniques like melting point, thin layer chromatography, infrared spectroscopy (KBr pellets), mass spectroscopy(m/z), 1H NMR (DMSO-d6), and 13C NMR. The synthesized compounds were further studied for biological evolution by alloxan (150 mg/dl, intraperitonial) induced diabetic rat model for in-vivo studies. Among all the synthesized derivatives, 5c and 5d were most potent as per binding energy. Compound 5i have shown a better plasma glucose reduction compared to glibenclamide. Hence, it will further use as a lead compound to develop a more such kind of agent. The docking study revealed that in all designed sulphonylureas/guanidine series of compounds 5c and 5d were found to be most potent compounds as per the binding energy compared to glibenclamide. With the help of details study of in vivo biological activity we observed that compound 5i gives better result compared to glibenclamide as standard. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Cytotoxicity, DNA binding and localisation of novel bis-naphthalimidopropyl polyamine derivatives.

PubMed

Pavlov, V; Kong Thoo Lin, P; Rodilla, V

2001-07-31

Bis-naphthalimidopropyl spermidine (BNIPSpd), spermine (BNIPSpm) and oxa-spermine (BNIPOSpm) showed high in vitro cytotoxicity against human breast cancer MCF-7 cells with IC(50) values of 1.38, 2.91 and 8.45 microM, respectively. These compounds were found to effectively displace the intercalating agent ethidium bromide bound to the calf thymus DNA using fluorimetric methods (C(50) 0.08-0.12 microM) and their apparent equilibrium binding constants (K(app)) were calculated to be in the range of 10.5-18 x 10(7) M(-1). Furthermore, strong stabilisation of calf thymus DNA duplex in the presence of bis-naphthalimidopropyl polyamine derivatives (BNIPSpd, BNIPSpm and BNIPOSpm) was observed by UV spectrophotometric analysis (T(m)=93.3-97 degrees C compared with 75 degrees C for calf thymus DNA without drug). Because of their inherent fluorescence, these compounds were localised preferentially inside the nucleus as evidenced by their direct observation under the fluorescence microscope. The results obtained suggest that the cytotoxic activity of the bis-naphthalimidopropyl polyamines may be in part, caused by their effects on DNA.

Mechanism of transfer of LDL-derived free cholesterol to HDL subfractions in human plasma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miida, T.; Fielding, C.J.; Fielding, P.E.

1990-11-01

The transfer of ({sup 3}H)cholesterol in low-density lipoprotein (LDL) to different high-density lipoprotein (HDL) species in native human plasma was determined by using nondenaturing two-dimensional electrophoresis. Transfer from LDL had a t{sub 1/2} at 37{degree}C of 51 {plus minus} 8 min and an activation energy of 18.0 kCal mol{sup {minus}1}. There was unexpected specificity among HDL species as acceptors of LDL-derived labeled cholesterol. The largest fraction of the major {alpha}-migrating class (HDL{sub 2b}) was the major initial acceptor of LDL-derived cholesterol. Kinetic analysis indicated a rapid secondary transfer from HDL{sub 2b} to smaller {alpha}HDL (particularly HDL{sub 3}) driven enzymatically bymore » the lecithin-cholesterol acyltransferase reaction. Rates of transfer among {alpha}HDL were most rapid from the largest {alpha}HDL fraction (HDL{sub 2b}), suggesting possible protein-mediated facilitation. Simultaneous measurements of the transport of LDL-derived and cell-derived isotopic cholesterol indicated that the former preferably utilized the {alpha}HDL pathyway, with little label in pre-{beta}HDL. The same experiments confirmed earlier data that cell-derived cholesterol is preferentially channeled through pre-{beta}HDL. The authors suggest that the functional heterogeneity of HDL demonstrated here includes the ability to independently process cell- and LDL-derived free cholesterol.« less

Plasma-derived exosomes contribute to inflammation via the TLR9-NF-κB pathway in chronic heart failure patients.

PubMed

Ye, Wei; Tang, Xiaojun; Yang, Zhengquan; Liu, Chu; Zhang, Xin; Jin, Jing; Lyu, Jianxin

2017-07-01

Exosomes are small vesicles that contain proteins, DNA and RNA, and play an important role in inflammation; however, the underlying mechanism remains unclear. In the present study, we found increased plasma-derived exosomes in chronic heart failure patients compared with healthy controls. Further, our data demonstrated that plasma-derived exosomes carried mtDNA, and triggered an inflammatory response via the TLR9-NF-κB pathway, as well, the inflammatory effect was closely related to exosomal mtDNA copy number. However, the effect could be blocked by chloroquine (CQ), a TLR9 inhibitor. These findings reveal a new mechanism of exosome-induced inflammation, and provide a new perspective for intervention and treatment of inflammation-related diseases, such as chronic heart failure. Copyright © 2017 Elsevier Ltd. All rights reserved.

Soluble PD-L1 with PD-1-binding capacity exists in the plasma of patients with non-small cell lung cancer.

PubMed

Takeuchi, Masahiro; Doi, Tomomitsu; Obayashi, Kunie; Hirai, Ayako; Yoneda, Kazue; Tanaka, Fumihiro; Iwai, Yoshiko

2018-04-01

PD-L1 is one of the important immune checkpoint molecules that can be targeted by cancer immunotherapies. PD-L1 has a soluble form (sPD-L1) and a membrane-bound form (mPD-L1). Conventional enzyme-linked immunosorbent assay (ELISA) systems can detect sPD-L1 using anti-PD-L1 capture antibody through the antigen-antibody reaction, but cannot evaluate the quality and function of sPD-L1. In this study, we developed a novel ELISA system for the detection and quantification of sPD-L1 with PD-1-binding capacity (bsPD-L1). To capture bsPD-L1 through the ligand-receptor reaction, the anti-PD-L1 capture antibody in the conventional ELISA was replaced with PD-1-Ig fusion protein in the new ELISA. The new ELISA could detect bsPD-L1 in 29 out of 75 plasma samples from patients with non-small cell lung cancer (NSCLC), with higher sensitivity and frequency than the conventional ELISA. The western blot analysis showed that sPD-L1 in the plasma was glycosylated. Treatment of the samples with glycosidase reduced the absorbance determined by the new ELISA but had no effect on the absorbance determined by the conventional ELISA. These results suggest that glycosylation of sPD-L1 is important for its binding to the immobilized PD-1 in the new ELISA. Our new ELISA system may be useful for the evaluation of functional sPD-L1 with PD-1-binding capacity in cancer patients. Copyright © 2018 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Effects of parturition and feed restriction on concentrations and distribution of the insulin-like growth factor-binding proteins in plasma and cerebrospinal fluid of dairy cows.

PubMed

Laeger, T; Wirthgen, E; Piechotta, M; Metzger, F; Metges, C C; Kuhla, B; Hoeflich, A

2014-05-01

Hormones and metabolites act as satiety signals in the brain and play an important role in the control of feed intake (FI). These signals can reach the hypothalamus and brainstem, 2 major centers of FI regulation, via the blood stream or the cerebrospinal fluid (CSF). During the early lactation period of high-yielding dairy cows, the increase of FI is often insufficient. Recently, it has been demonstrated that insulin-like growth factors (IGF) may control FI. Thus, we asked in the present study if IGF-binding proteins (IGFBP) are regulated during the periparturient period and in response to feed restriction and therefore might affect FI as well. In addition, we specifically addressed conditional distribution of IGFBP in plasma and CSF. In one experiment, 10 multiparous German Holstein dairy cows were fed ad libitum and samples of CSF and plasma were obtained before morning feeding on d -20, -10, +1, +10, +20, and +40 relative to calving. In a second experiment, 7 cows in second mid-lactation were sampled for CSF and plasma after ad libitum feeding and again after feeding 50% of the previous ad libitum intake for 4 d. Intact IGFBP-2, IGFBP-3, and IGFBP-4 were detected in plasma by quantitative Western ligand blot analysis. In CSF, we were able to predominantly identify intact IGFBP-2 and a specific IGFBP-2 fragment containing detectable binding affinities for biotinylated IGF-II. Whereas plasma concentrations of IGFBP-2 and IGFBP-4 increased during the periparturient period, IGFBP-3 was unaffected over time. In CSF, concentrations of IGFBP-2, both intact and fragmented, were not affected during the periparturient period. Plasma IGF-I continuously decreased until calving but remained at a lower concentration in early lactation than in late pregnancy. Food restriction did not affect concentrations of IGF components present in plasma or CSF. We could show that the IGFBP profiles in plasma and CSF are clearly distinct and that changes in IGFBP in plasma do not simply

Prediction of Drug-Plasma Protein Binding Using Artificial Intelligence Based Algorithms.

PubMed

Kumar, Rajnish; Sharma, Anju; Siddiqui, Mohammed Haris; Tiwari, Rajesh Kumar

2018-01-01

Plasma protein binding (PPB) has vital importance in the characterization of drug distribution in the systemic circulation. Unfavorable PPB can pose a negative effect on clinical development of promising drug candidates. The drug distribution properties should be considered at the initial phases of the drug design and development. Therefore, PPB prediction models are receiving an increased attention. In the current study, we present a systematic approach using Support vector machine, Artificial neural network, k- nearest neighbor, Probabilistic neural network, Partial least square and Linear discriminant analysis to relate various in vitro and in silico molecular descriptors to a diverse dataset of 736 drugs/drug-like compounds. The overall accuracy of Support vector machine with Radial basis function kernel came out to be comparatively better than the rest of the applied algorithms. The training set accuracy, validation set accuracy, precision, sensitivity, specificity and F1 score for the Suprort vector machine was found to be 89.73%, 89.97%, 92.56%, 87.26%, 91.97% and 0.898, respectively. This model can potentially be useful in screening of relevant drug candidates at the preliminary stages of drug design and development. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Which is safer source plasma for manufacturing in China: apheresis plasma or recovered plasma?

PubMed

Liu, Yu; Li, Changqing; Wang, Ya; Zhang, Yan; Wu, Binting; Ke, Ling; Xu, Min; Liu, Gui; Liu, Zhong

2016-05-01

In most countries, the plasma for derivative production includes two types of plasma, apheresis plasma (AP) and recovered plasma (RP). However, the plasma recovered from whole blood is not permitted for manufacture in China. Because of the lack of source plasma and the surplus of RP, the Chinese government is considering allowing RP as an equivalent source for the production of plasma derivatives. It is known that human blood can be contaminated by various infectious agents. The objective of the study was to evaluate if infectious risk would increase by enacting this policy. The samples from the two types of blood donors from January 1 to December 31, 2013, were collected. Supplementary testing was conducted and the residual risk (RR) of human immunodeficiency virus (HIV), hepatitis B virus, and hepatitis C virus (HCV) in the two types of blood donors and donations were calculated through the incidence-window period model. Prevalence of the markers of hepatitis E virus, hepatitis A virus, severe fever with thrombocytopenia syndrome bunyavirus, cytomegalovirus, B19, and West Nile virus was calculated. No significant difference was found in the RR of the three pathogens in the two types of blood donors. However, after the quarantine period, the RR of HCV and HIV in AP was significantly lower than that in RP. A quarantine period of 2 years will make the infectious risk of RP not significantly different than that of AP. Our data demonstrate that allowing RP to be used for the manufacture of plasma derivatives will not increase its infectious disease risk if coupled with a 2-year inventory hold. © 2016 AABB.

Fatty acids and small organic compounds bind to mineralo-organic nanoparticles derived from human body fluids as revealed by metabolomic analysis.

PubMed

Martel, Jan; Wu, Cheng-Yeu; Hung, Cheng-Yu; Wong, Tsui-Yin; Cheng, Ann-Joy; Cheng, Mei-Ling; Shiao, Ming-Shi; Young, John D

2016-03-14

Nanoparticles entering the human body instantly become coated with a "protein corona" that influences the effects and distribution of the particles in vivo. Yet, whether nanoparticles may bind to other organic compounds remains unclear. Here we use an untargeted metabolomic approach based on ultra-performance liquid chromatography and quadruple time-of-flight mass spectrometry to identify the organic compounds that bind to mineral nanoparticles formed in human body fluids (serum, plasma, saliva, and urine). A wide range of organic compounds is identified, including fatty acids, glycerophospholipids, amino acids, sugars, and amides. Our results reveal that, in addition to the proteins identified previously, nanoparticles harbor an "organic corona" containing several fatty acids which may affect particle-cell interactions in vivo. This study provides a platform to study the organic corona of biological and synthetic nanoparticles found in the human body.

Deglycosylation of serum vitamin D3-binding protein by alpha-N-acetylgalactosaminidase detected in the plasma of patients with systemic lupus erythematosus.

PubMed

Yamamoto, N; Naraparaju, V R; Moore, M; Brent, L H

1997-03-01

A serum glycoprotein, Gc protein (vitamin D3-binding protein), can be converted by beta-galactosidase of B cells and sialidase of T cells to a potent macrophage-activating factor (MAF), a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is the precursor for MAF. Treatment of Gc protein with immobilized beta-galactosidase and sialidase generates a remarkably high titered macrophage-activating factor (GcMAF). When peripheral blood monocytes/ macrophages (designated macrophages) of 33 systemic lupus erythematosus patients were incubated with GcMAF (100 pg/ml), the macrophages of all patients were activated as determined by superoxide generation. However, the precursor activity of patient plasma Gc protein was lost or reduced in these patients. Loss of the precursor activity was the result of deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase activity found in the patient plasma. Levels of plasma alpha-N-acetylgalactosaminidase activity in individual patients had an inverse correlation with the MAF precursor activity of their plasma Gc protein. Deglycosylated Gc protein cannot be converted to macro-phage-activating factor. The resulting defect in macro-phage activation may lead to an inability to clear pathogenic immune complexes. Thus, elevated plasma alpha-N-acetylgalactosaminidase activity resulting in the loss of MAF precursor activity and reduced macro-phage activity may play a role in the pathogenesis of systemic lupus erythematosus.

Further purification of human pituitary-derived chondrocyte growth factor: heparin-binding and cross-reactivity with antiserum to basic FGF.

PubMed

Too, C K; Murphy, P R; Hamel, A M; Friesen, H G

1987-05-14

The previously described human pituitary-derived chondrocyte growth factor (CGF), mitogenic for rabbit fetal chondrocytes, was found to bind to heparin-Sepharose and was eluted with 1.5M NaCl. Further characterization of CGF demonstrated a molecular weight of 18-20 kD and cross-reactivity with antiserum to synthetic bovine basic fibroblast growth factor (FGF1-24). When human pituitaries were homogenized in 0.15 ammonium sulfate (pH 5.5) and the extract chromatographed on heparin-Sepharose, 98% of the mitogenic activity was adsorbed to heparin and eluted with 3M NaCl. These findings indicate that CGF is closely related or identical to basic FGF and that the bulk of mitogenic activity in the human pituitary extracts binds to heparin.

Transthyretin-binding activity of contaminants in blood from polar bear (Ursus maritimus) cubs.

PubMed

Bytingsvik, Jenny; Simon, Eszter; Leonards, Pim E G; Lamoree, Marja; Lie, Elisabeth; Aars, Jon; Derocher, Andrew E; Wiig, Oystein; Jenssen, Bjørn M; Hamers, Timo

2013-05-07

We determined the transthyretin (TTR)-binding activity of blood-accumulating contaminants in blood plasma samples of approximately 4-months-old polar bear (Ursus maritimus) cubs from Svalbard sampled in 1998 and 2008. The TTR-binding activity was measured as thyroxine (T4)-like equivalents (T4-EQMeas). Our findings show that the TTR-binding activity related to contaminant levels was significantly lower (45%) in 2008 than in 1998 (mean ± standard error of mean: 1998, 2265 ± 231 nM; 2008, 1258 ± 170 nM). Although we cannot exclude a potential influence of between-year differences in capture location and cub body mass, our findings most likely reflect reductions of TTR-binding contaminants or their precursors in the arctic environment (e.g., polychlorinated biphenyls [PCBs]). The measured TTR-binding activity correlated positively with the cubs' plasma levels of hydroxylated PCBs (OH-PCBs). No such association was found between TTR-binding activity and the plasma levels of perfluoroalkyl substances (PFASs). The OH-PCBs explained 60 ± 7% and 54 ± 4% of the TTR-binding activity in 1998 and 2008, respectively, and PFASs explained ≤1.2% both years. Still, almost half the TTR-binding activity could not be explained by the contaminants we examined. The considerable levels of TTR-binding contaminants warrant further effect directed analysis (EDA) to identify the contaminants responsible for the unexplained part of the observed TTR-binding activity.

Bilirubin Binding Capacity in the Preterm Neonate

PubMed Central

Amin, Sanjiv B

2016-01-01

SYNOPSIS Total serum/plasma bilirubin (TB), the biochemical measure currently used to evaluate and manage hyperbilirubinemia, is not a useful predictor of bilirubin-induced neurotoxicity in premature infants. Altered bilirubin-albumin binding in premature infants limits the usefulness of TB in premature infants. In this article, bilirubin-albumin binding, a modifying factor for bilirubin-induced neurotoxicity, in premature infants is reviewed. PMID:27235205

Plasma oscillations in spherical Gaussian shaped ultracold neutral plasma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Tianxing; Lu, Ronghua, E-mail: lurh@siom.ac.cn; Guo, Li

2016-04-15

The collective plasma oscillations are investigated in ultracold neutral plasma with a non-uniform density profile. Instead of the plane configuration widely used, we derive the plasma oscillation equations with spherically symmetric distribution and Gaussian density profile. The damping of radial oscillation is found. The Tonks–Dattner resonances of the ultracold neutral plasma with an applied RF field are also calculated.

A 115 kDa calmodulin-binding protein is located in rat liver endosome fractions.

PubMed Central

Enrich, C; Bachs, O; Evans, W H

1988-01-01

The distribution of calmodulin-binding polypeptides in various rat liver subcellular fractions was investigated. Plasma-membrane, endosome, Golgi and lysosome fractions were prepared by established procedures. The calmodulin-binding polypeptides present in the subcellular fractions were identified by using an overlay technique after transfer from gels to nitrocellulose sheets. Distinctive populations of calmodulin-binding polypeptides were present in all the fractions examined except lysosomes. A major 115 kDa calmodulin-binding polypeptide of pI 4.3 was located to the endosome subfractions, and it emerges as a candidate endosome-specific protein. Partitioning of endosome fractions between aqueous and Triton X-114 phases indicated that the calmodulin-binding polypeptide was hydrophobic. Major calmodulin-binding polypeptides of 140 and 240 kDa and minor polypeptides of 40-60 kDa were present in plasma membranes. The distribution of calmodulin in the various endosome and plasma-membrane fractions was also analysed, and the results indicated that the amounts were high compared with those in the cytosol. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:3214436

HIV-Derived ssRNA Binds to TLR8 to Induce Inflammation-Driven Macrophage Foam Cell Formation

PubMed Central

Bernard, Mark A.; Han, Xinbing; Inderbitzin, Sonya; Agbim, Ifunanya; Zhao, Hui; Koziel, Henry; Tachado, Souvenir D.

2014-01-01

Even though combined anti-retroviral therapy (cART) dramatically improves patient survival, they remain at a higher risk of being afflicted with non-infectious complications such as cardiovascular disease (CVD). This increased risk is linked to persistent inflammation and chronic immune activation. In this study, we assessed whether this complication is related to HIV-derived ssRNAs inducing in macrophages increases in TNFα release through TLR8 activation leading to foam cell formation. HIV ssRNAs induced foam cell formation in monocyte-derived macrophages (MDMs) in a dose-dependent manner. This response was reduced when either endocytosis or endosomal acidification was inhibited by dynasore or chloroquine, respectively. Using a flow cytometry FRET assay, we demonstrated that ssRNAs bind to TLR8 in HEK cells. In MDMs, ssRNAs triggered a TLR8-mediated inflammatory response that ultimately lead to foam cell formation. Targeted silencing of the TLR8 and MYD88 genes reduced foam cell formation. Furthermore, foam cell formation induced by these ssRNAs was blocked by an anti-TNFα neutralizing antibody. Taken together in MDMs, HIV ssRNAs are internalized; bind TLR8 in the endosome followed by endosomal acidification. TLR8 signaling then triggers TNFα release and ultimately leads to foam cell formation. As this response was inhibited by a blocking anti-TNFα antibody, drug targeting HIV ssRNA-driven TLR8 activation may serve as a potential therapeutic target to reduce chronic immune activation and inflammation leading to CVD in HIV+ patients. PMID:25090652

Sensitive and accurate identification of protein–DNA binding events in ChIP-chip assays using higher order derivative analysis

PubMed Central

Barrett, Christian L.; Cho, Byung-Kwan

2011-01-01

Immuno-precipitation of protein–DNA complexes followed by microarray hybridization is a powerful and cost-effective technology for discovering protein–DNA binding events at the genome scale. It is still an unresolved challenge to comprehensively, accurately and sensitively extract binding event information from the produced data. We have developed a novel strategy composed of an information-preserving signal-smoothing procedure, higher order derivative analysis and application of the principle of maximum entropy to address this challenge. Importantly, our method does not require any input parameters to be specified by the user. Using genome-scale binding data of two Escherichia coli global transcription regulators for which a relatively large number of experimentally supported sites are known, we show that ∼90% of known sites were resolved to within four probes, or ∼88 bp. Over half of the sites were resolved to within two probes, or ∼38 bp. Furthermore, we demonstrate that our strategy delivers significant quantitative and qualitative performance gains over available methods. Such accurate and sensitive binding site resolution has important consequences for accurately reconstructing transcriptional regulatory networks, for motif discovery, for furthering our understanding of local and non-local factors in protein–DNA interactions and for extending the usefulness horizon of the ChIP-chip platform. PMID:21051353

TnBP⁄Triton X-45 Treatment of Plasma for Transfusion Efficiently Inactivates Hepatitis C Virus

PubMed Central

Chou, Ming-Li; Burnouf, Thierry; Chang, Shun-Pang; Hung, Ting-Chun; Lin, Chun-Ching; Richardson, Christopher D.; Lin, Liang-Tzung

2015-01-01

Risk of transmission of hepatitis C virus (HCV) by clinical plasma remains high in countries with a high prevalence of hepatitis C, justifying the implementation of viral inactivation treatments. In this study, we assessed the extent of inactivation of HCV during minipool solvent/detergent (SD; 1% TnBP / 1% Triton X-45) treatment of human plasma. Luciferase-tagged infectious cell culture-derived HCV (HCVcc) particles were used to spike human plasma prior to treatment by SD at 31 ± 0.5°C for 30 min. Samples were taken before and after SD treatment and filtered on a Sep-Pak Plus C18 cartridge to remove the SD agents. Risk of cytotoxicity was assessed by XTT cell viability assay. Viral infectivity was analyzed based on the luciferase signals, 50% tissue culture infectious dose viral titer, and immunofluorescence staining for HCV NS5A protein. Total protein, cholesterol, and triglyceride contents were determined before and after SD treatment and C18 cartridge filtration. Binding analysis, using patient-derived HCV clinical isolates, was also examined to validate the efficacy of the inactivation by SD. SD treatment effectively inactivated HCVcc within 30 min, as demonstrated by the baseline level of reporter signals, total loss of viral infectivity, and absence of viral protein NS5A. SD specifically targeted HCV particles to render them inactive, with essentially no effect on plasma protein content and hemostatic function. More importantly, the efficacy of the SD inactivation method was confirmed against various genotypes of patient-derived HCV clinical isolates and against HCVcc infection of primary human hepatocytes. Therefore, treatment by 1% TnBP / 1% Triton X-45 at 31°C is highly efficient to inactivate HCV in plasma for transfusion, showing its capacity to enhance the safety of therapeutic plasma products. We propose that the methodology used here to study HCV infectivity can be valuable in the validation of viral inactivation and removal processes of human

Serum and plasma brain-derived neurotrophic factor (BDNF) in abstinent alcoholics and social drinkers

PubMed Central

D’Sa, Carrol; Dileone, Ralph J.; Anderson, George M.; Sinha, Rajita

2013-01-01

Although the effects of alcohol on brain-derived neurotrophic factor (BDNF) have been extensively studied in rodents, BDNF levels have rarely been measured in abstinent, alcohol-dependent (AD) individuals. Interpretation of reported group comparisons of serum BDNF levels is difficult due to limited information regarding analytical variance, biological variability, and the relative contribution of platelet and plasma pools to serum BDNF. Analytical variance (intra- and inter-assay coefficients of variation) of the enzyme-linked immunosorbent assay (ELISA) was characterized. Within- and between-subject variability, and group differences in serum and plasma BDNF, was assessed on three separate days in 16, 4-week abstinent AD individuals (7M/9F) and 16 social drinkers (SDs; 8M/8F). Significantly higher mean (±sd) serum BDNF levels were observed for the AD group compared to the SD (p = 0.003). No significant difference in mean baseline plasma BDNF levels was observed between AD and SD groups. The low analytical variance, high day-to-day within-individual stability and the high degree of individuality demonstrates the potential clinical utility of measuring serum BDNF levels. The low correlations that we observed between plasma and serum levels are congruent with their representing separate pools of BDNF. The observation of higher basal serum BDNF in the AD group without a concomitant elevation in plasma BDNF levels indicates that the elevated serum BDNF in AD patients is not due to greater BDNF exposure. Further research is warranted to fully elucidate mechanisms underlying this alteration and determine the utility of serum BDNF as a predictor or surrogate marker of chronic alcohol abuse. PMID:22364688

Molecular binding of toxic phenothiazinium derivatives, azures to bovine serum albumin: A comparative spectroscopic, calorimetric, and in silico study.

PubMed

Das, Somnath; Islam, Md Maidul; Jana, Gopal Chandra; Patra, Anirudha; Jha, Pradeep K; Hossain, Maidul

2017-07-01

In this paper, the comparative binding behavior of antimalarial drug azure A, azure B and azure C with bovine serum albumin (BSA) has been studied. The interaction has been confirmed by multispectroscopic (UV, fluorescence, Fourier transform infrared (FT-IR), and circular dichroism) and molecular docking techniques. The experimental results show that azure B has the highest BSA binding affinity followed by azure A and azure C. The experimental evidence of binding showed a static quenching mechanism in the interaction azures with BSA. The isothermal titration calorimetry result reveals that the binding was exothermic with positive entropy contribution in each case. The thermodynamic parameters ΔH, ΔG, and ΔS at 25°C were calculated, which indicates that the weak van der Waals forces and hydrogen bonding rather than the hydrophobic effect played an important role in the interaction. According to the theory of Förster nonradiative energy transfer, the distance (r) between the donor (BSA) and acceptor azures found to be <7 nm in all the case. The circular dichroism and FT-IR studies show that the content of α-helix structure has increased for the azures-BSA system. Overall, experimental studies characterize the interaction dynamics and energetics of the binding of three toxic analogs towards the physiologically relevant serum albumins. We hope, the outcome of this work will be most helpful for synthesizing a new type of phenothiazinium derivatives of the better therapeutic application. Copyright © 2017 John Wiley & Sons, Ltd.

gamma. -Preprotachykinin-(72-92)-peptide amide: An endogenous preprotachykinin I gene-derived peptide that preferentially binds to neurokinin-2 receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dam, T.V.; Takeda, Y.; Krause, J.E.

1990-01-01

The presence of N-terminally extended forms of neurokinin A has recently been reported in the mammalian brain. Among them, gamma-preprotachykinin-(72-92)-peptide amide (gamma-PPT-(72-92)-NH2), a peptide derived by posttranslational processing of gamma-preprotachykinin, is most prominent. We report here that this peptide most likely acts on neurokinin-2 receptor sites since neurokinin A (a putative neurokinin-2 agonist) and gamma-PPT-(72-92)-NH2 are potent competitors of 125I-labeled gamma-PPT-(72-92)-NH2 binding whereas selective neurokinin-1 and -3 agonists are not. Moreover, the distribution of 125I-labeled gamma-PPT-(72-92)-NH2 and 125I-labeled neurokinin A binding sites are very similar in rat brain. On the other hand, 125I-labeled Bolton-Hunter-substance P (a neurokinin-1 ligand) and 125I-labeledmore » Bolton-Hunter-eledoisin (a neurokinin-3 ligand) binding sites are differentially located in this tissue. Thus, it appears that gamma-PPT-(72-92)-NH2 binds to neurokinin-2 receptors and should be considered as a putative endogenous ligand for this receptor class.« less

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

PubMed

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

Goodpasture Antigen-binding Protein/Ceramide Transporter Binds to Human Serum Amyloid P-Component and Is Present in Brain Amyloid Plaques*

PubMed Central

Mencarelli, Chiara; Bode, Gerard H.; Losen, Mario; Kulharia, Mahesh; Molenaar, Peter C.; Veerhuis, Robert; Steinbusch, Harry W. M.; De Baets, Marc H.; Nicolaes, Gerry A. F.; Martinez-Martinez, Pilar

2012-01-01

Serum amyloid P component (SAP) is a non-fibrillar glycoprotein belonging to the pentraxin family of the innate immune system. SAP is present in plasma, basement membranes, and amyloid deposits. This study demonstrates, for the first time, that the Goodpasture antigen-binding protein (GPBP) binds to human SAP. GPBP is a nonconventional Ser/Thr kinase for basement membrane type IV collagen. Also GPBP is found in plasma and in the extracellular matrix. In the present study, we demonstrate that GPBP specifically binds SAP in its physiological conformations, pentamers and decamers. The START domain in GPBP is important for this interaction. SAP and GPBP form complexes in blood and partly colocalize in amyloid plaques from Alzheimer disease patients. These data suggest the existence of complexes of SAP and GPBP under physiological and pathological conditions. These complexes are important for understanding basement membrane, blood physiology, and plaque formation in Alzheimer disease. PMID:22396542

Glutamine-derived 2-hydroxyglutarate is associated with disease progression in plasma cell malignancies

PubMed Central

Gonsalves, Wilson I.; Hitosugi, Taro; Ghosh, Toshi; Jevremovic, Dragan; Petterson, Xuan-Mai; Wellik, Linda; Kumar, Shaji K.; Nair, K. Sreekumaran

2018-01-01

The production of the oncometabolite 2-hydroxyglutarate (2-HG) has been associated with c-MYC overexpression. c-MYC also regulates glutamine metabolism and drives progression of asymptomatic precursor plasma cell (PC) malignancies to symptomatic multiple myeloma (MM). However, the presence of 2-HG and its clinical significance in PC malignancies is unknown. By performing 13C stable isotope resolved metabolomics (SIRM) using U[13C6]Glucose and U[13C5]Glutamine in human myeloma cell lines (HMCLs), we show that 2-HG is produced in clonal PCs and is derived predominantly from glutamine anaplerosis into the TCA cycle. Furthermore, the 13C SIRM studies in HMCLs also demonstrate that glutamine is preferentially utilized by the TCA cycle compared with glucose. Finally, measuring the levels of 2-HG in the BM supernatant and peripheral blood plasma from patients with precursor PC malignancies such as smoldering MM (SMM) demonstrates that relatively elevated levels of 2-HG are associated with higher levels of c-MYC expression in the BM clonal PCs and with a subsequent shorter time to progression (TTP) to MM. Thus, measuring 2-HG levels in BM supernatant or peripheral blood plasma of SMM patients offers potential early identification of those patients at high risk of progression to MM, who could benefit from early therapeutic intervention. PMID:29321378

Determination of osteogenic or adipogenic lineages in muscle-derived stem cells (MDSCs) by a collagen-binding peptide (CBP) derived from bone sialoprotein (BSP).

PubMed

Choi, Yoon Jung; Lee, Jue Yeon; Lee, Seung Jin; Chung, Chong-Pyoung; Park, Yoon Jeong

2012-03-09

Bone sialoprotein (BSP) is a mineralized, tissue-specific, non-collagenous protein that is normally expressed only in mineralized tissues such as bone, dentin, cementum, and calcified cartilage, and at sites of new mineral formation. The binding of BSP to collagen is thought to be important for initiating bone mineralization and bone cell adhesion to the mineralized matrix. Several recent studies have isolated stem cells from muscle tissue, but their functional properties are still unclear. In this study, we examined the effects of a synthetic collagen-binding peptide (CBP) on the differentiation efficiency of muscle-derived stem cells (MDSCs). The CBP sequence (NGVFKYRPRYYLYKHAYFYPHLKRFPVQ) corresponds to residues 35-62 of bone sialoprotein (BSP), which are located within the collagen-binding domain in BSP. Interestingly, this synthetic CBP inhibited adipogenic differentiation but increased osteogenic differentiation in MDSCs. The CBP also induced expression of osteoblastic marker proteins, including alkaline phosphatase (ALP), type I collagen, Runt-related transcription factor 2 (Runx2), and osteocalcin; prevented adipogenic differentiation in MDSCs; and down-regulated adipose-specific mRNAs, such as adipocyte protein 2 (aP2) and peroxisome proliferator-activated receptor γ. The CBP increased Extracellular signal-regulated kinases (ERK) 1/2 protein phosphorylation, which is important in lineage determination. These observations suggest that this CBP determines the osteogenic or adipogenic lineage in MDSCs by activating ERK1/2. Taken together, a novel CBP could be a useful candidate for regenerating bone and treating osteoporosis, which result from an imbalance in osteogenesis and adipogenesis differentiation. Copyright © 2012 Elsevier Inc. All rights reserved.

Effect of platelet-derived β-thromboglobulins on coagulation.

PubMed

Egan, Karl; van Geffen, Johanna P; Ma, Hui; Kevane, Barry; Lennon, Aine; Allen, Seamus; Neary, Elaine; Parsons, Martin; Maguire, Patricia; Wynne, Kieran; O' Kennedy, Richard; Heemskerk, Johan W M; Áinle, Fionnuala Ní

2017-06-01

β-thromboglobulins are derived from the cleavage of the CXC chemokine platelet basic protein and are released in high concentrations by activated platelets. Platelet-derived β-thromboglobulins (βTG) share 70% homology with platelet factor 4 (PF4), another CXC chemokine released by activated platelets. PF4 modulates coagulation by inhibiting heparin-antithrombin interactions, promoting protein C activation, and attenuating the activity of activated protein C. In contrast, the effect of βTG on coagulation is unknown. Clotting times, thrombin generation, chromogenic clotting factor assays, and surface plasmon resonance (SPR) were used to assess the effect of purified βTG on coagulation. In normal pooled plasma, βTG shortened the lagtime and time to peak thrombin generation of tissue factor (TF)-dependent and TF-independent thrombin generation. In factor VIII and factor IX-deficient plasmas, βTG induced thrombin generation in the absence of a TF stimulus and in the presence of anti-TF and factor VIIa inhibitory antibodies. The procoagulant effect was not observed when thrombin generation was independent of factor X activation (supplementation of factor X-deficient plasma with factor Xa). Cleavage of a factor Xa-specific chromogenic substrate was observed when βTG was incubated with factor X, suggesting a direct interaction between βTG and factor X. Using SPR, βTG were found to bind to immobilised factor X in a dose dependent manner. βTG modulate coagulation in vitro via an interaction with factor X. Copyright © 2017 Elsevier Ltd. All rights reserved.

Plasma enhancement of in vitro attachment of rat bone-marrow-derived stem cells on cross-linked gelatin films.

PubMed

Prasertsung, I; Kanokpanont, S; Mongkolnavin, R; Wong, C S; Panpranot, J; Damrongsakkul, S

2012-01-01

In this work, nitrogen, oxygen and air glow discharges powered by 50 Hz AC power supply are used for the treatment of type-A gelatin film cross-linked by a dehydrothermal (DHT) process. The properties of cross-linked gelatin were characterized by contact angle measurement, atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) analysis. The results showed that the water contact angle of gelatin films decrease with increasing plasma treatment time. The treatment of nitrogen, oxygen and air plasma up to 30 s had no effects on the surface roughness of the gelatin film as revealed by AFM results. The XPS analysis showed that the N-containing functional groups generated by nitrogen and air plasma, and O-containing functional groups generated by oxygen and air plasmas were incorporated onto the film surface, the functional groups were found to increase with increasing treatment time. An in vitro test using rat bone-marrow-mesenchym-derived stem cells (MSCs) revealed that the number of cells attached on plasma-treated gelatin films was significantly increased compared to untreated samples. The best enhancement of cell attachment was noticed when the film was treated with nitrogen plasma for 15-30 s, oxygen plasma for 3 s, and air plasma for 9 s. In addition, among the three types of plasmas used, nitrogen plasma treatment gave the best MSCs attachment on the gelatin surface. The results suggest that a type-A gelatin film with water contact angle of 27-28° and an O/N ratio of 1.4 is most suitable for MSCs attachment.

Binding of alkylphenols and alkylated non-phenolics to the rainbow trout (Oncorhynchus mykiss) plasma sex steroid-binding protein.

PubMed

Tollefsen, K-E

2007-09-01

Alkylphenols are well-known endocrine disrupters, mediating effects through the estrogen receptor (ER). Although the estrogenic properties of the alkylphenols are well documented, alternative mechanisms of action are poorly described. In the present work, the interaction of a range of alkyl-substituted phenols and alkyl-substituted non-phenolics with the rainbow trout (Oncorhynchus mykiss) sex steroid-binding protein (rtSBP) were determined by competitive ligand-binding studies. The role of alkyl chain length and branching, substituent position, number of alkylated groups, and the requirement of a phenolic ring structure were assessed. The results showed that the rtSBP binds to most chemical structures tested, although the highest affinity was obtained for mono-substituted alkylphenols with a chain length of four to eight methyl groups. Interestingly, rtSBP binding was also observed for non-phenolic compounds such as 4-t-butylcyclohexanol and 4-t-butylnitrobenzene suggesting that the rtSBP has a broad binding specificity for alkylphenols and alkylated non-phenolics.

Non-activated plasma-derived PC improves amputation rate of children undergoing sepsis.

PubMed

Piccin, Andrea; O' Marcaigh, Aengus; Mc Mahon, Corrina; Murphy, Ciaran; Okafor, Ikechukwu; Marcheselli, Luigi; Casey, William; Claffey, Liam; Smith, Owen Patrick

2014-07-01

Low circulating protein C (PC) levels have been observed in sepsis, especially in patients with Neisseriae Meningitides infections. Poor clinical outcome and high limb amputation rates have been associated in infected patients with low circulating PC levels. Published studies using activated PC replacement therapy patients with sepsis have shown reduced mortality rates, however, its use has been associated with severe bleeding events. Paediatric sepsis studies using non-activated plasma-derived PC (Ceprotin®) are lacking. We present a retrospective study in children with sepsis who were treated with Ceprotin® focusing on amputation rate post treatment. Thirty subjects were identified. Median age at diagnosis was 2 years. Twenty-one (70%) were treated for Nesseria Meningitides and one (3%) for Streptococcus-A β-haemolyticus, another 8 (26%) patients with malignancies were treated for neutropenic sepsis. Following Ceprotin® administration, a significant increase in leukocyte count (p=0.004), neutrophil count (p=0.001) and PC (pretreatment=13%, posttreatment=88.5%; p=0.0001) was seen. Prothrombin time (pretreatment =30.3 seconds, posttreatment =16.5; p=0.000) and activated partial thromboplastin time (pretreatment =61.8 sec, postreatment =42.6 sec; p=0.000) were significantly reduced, while fibrinogen levels were significantly elevated (pretreatment =1.9 g/dL, posttreatment =4.4 g/dL; p=0.000). The median time between admission to intensive care and Ceprotin® administration was 10 hrs. Limb amputation rate was reduced (16-23% versus 30-50% from previous studies) and there were no haemorrhagic events observed. This study demonstrates the safe administration of non-activated plasma-derived PC concentrate in patients with sepsis who are coagulopathic and it associated with a reduction in amputation rates. Copyright © 2014 Elsevier Ltd. All rights reserved.

Plasma Modes

NASA Astrophysics Data System (ADS)

Dubin, D. H. E.

This chapter explores several aspects of the linear electrostatic normal modes of oscillation for a single-species non-neutral plasma in a Penning trap. Linearized fluid equations of motion are developed, assuming the plasma is cold but collisionless, which allow derivation of the cold plasma dielectric tensor and the electrostatic wave equation. Upper hybrid and magnetized plasma waves in an infinite uniform plasma are described. The effect of the plasma surface in a bounded plasma system is considered, and the properties of surface plasma waves are characterized. The normal modes of a cylindrical plasma column are discussed, and finally, modes of spheroidal plasmas, and finite temperature effects on the modes, are briefly described.

Arabidopsis-derived shrimp viral-binding protein, PmRab7 can protect white spot syndrome virus infection in shrimp.

PubMed

Thagun, Chonprakun; Srisala, Jiraporn; Sritunyalucksana, Kallaya; Narangajavana, Jarunya; Sojikul, Punchapat

2012-09-15

White spot syndrome virus is currently the leading cause of production losses in the shrimp industry. Penaeus monodon Rab7 protein has been recognized as a viral-binding protein with an efficient protective effect against white spot syndrome infection. Plant-derived recombinant PmRab7 might serve as an alternative source for in-feed vaccination, considering the remarkable abilities of plant expression systems. PmRab7 was introduced into the Arabidopsis thaliana T87 genome. Arabidopsis-derived recombinant PmRab7 showed high binding activity against white spot syndrome virus and a viral envelope, VP28. The growth profile of Arabidopsis suspension culture expressing PmRab7 (ECR21# 35) resembled that of its counterpart. PmRab7 expression in ECR21# 35 reached its maximum level at 5 mg g(-1) dry weight in 12 days, which was higher than those previously reported in Escherichia coli and in Pichia. Co-injection of white spot syndrome virus and Arabidopsis crude extract containing PmRab7 in Litopenaeus vannamei showed an 87% increase in shrimp survival rate at 5 day after injection. In this study, we propose an alternative PmRab7 source with higher production yield, and cheaper culture media costs, that might serve the industry's need for an in-feed supplement against white spot syndrome infection. Copyright © 2012 Elsevier B.V. All rights reserved.

Hupresin Retains Binding Capacity for Butyrylcholinesterase and Acetylcholinesterase after Sanitation with Sodium Hydroxide.

PubMed

Onder, Seda; David, Emilie; Tacal, Ozden; Schopfer, Lawrence M; Lockridge, Oksana

2017-01-01

Hupresin is a new affinity resin that binds butyrylcholinesterase (BChE) in human plasma and acetylcholinesterase (AChE) solubilized from red blood cells (RBC). Hupresin is available from the CHEMFORASE company. BChE in human plasma binds to Hupresin and is released with 0.1 M trimethylammonium bromide (TMA) with full activity and 10-15% purity. BChE immunopurified from plasma by binding to immobilized monoclonal beads has fewer contaminating proteins than the one-step Hupresin-purified BChE. However, when affinity chromatography on Hupresin follows ion exchange chromatography at pH 4.5, BChE is 99% pure. The membrane bound AChE, solubilized from human RBC with 0.6% Triton X-100, binds to Hupresin and remains bound during washing with sodium chloride. Human AChE is not released in significant quantities with non-denaturing solvents, but is recovered in 1% trifluoroacetic acid. The denatured, partially purified AChE is useful for detecting exposure to nerve agents by mass spectrometry. Our goal was to determine whether Hupresin retains binding capacity for BChE and AChE after Hupresin is washed with 0.1 M NaOH. A 2 mL column of Hupresin equilibrated in 20 mM TrisCl pH 7.5 was used in seven consecutive trials to measure binding and recovery of BChE from 100 mL human plasma. Between each trial the Hupresin was washed with 10 column volumes of 0.1 M sodium hydroxide. A similar trial was conducted with red blood cell AChE in 0.6% Triton X-100. It was found that the binding capacity for BChE and AChE was unaffected by washing Hupresin with 0.1 M sodium hydroxide. Hupresin could be washed with sodium hydroxide at least seven times without losing binding capacity.

Determination of osteogenic or adipogenic lineages in muscle-derived stem cells (MDSCs) by a collagen-binding peptide (CBP) derived from bone sialoprotein (BSP)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Yoon Jung; Lee, Jue Yeon; Lee, Seung Jin

Highlights: Black-Right-Pointing-Pointer CBP sequence is identified from BSP and has collagen binding activity. Black-Right-Pointing-Pointer CBP directly activates the MAPK signaling, especially ERK1/2. Black-Right-Pointing-Pointer CBP increase osteoblastic differentiation by the activation of Runx2. Black-Right-Pointing-Pointer CBP decrease adipogenic differentiation by the inhibition of PPAR{gamma}. -- Abstract: Bone sialoprotein (BSP) is a mineralized, tissue-specific, non-collagenous protein that is normally expressed only in mineralized tissues such as bone, dentin, cementum, and calcified cartilage, and at sites of new mineral formation. The binding of BSP to collagen is thought to be important for initiating bone mineralization and bone cell adhesion to the mineralized matrix. Severalmore » recent studies have isolated stem cells from muscle tissue, but their functional properties are still unclear. In this study, we examined the effects of a synthetic collagen-binding peptide (CBP) on the differentiation efficiency of muscle-derived stem cells (MDSCs). The CBP sequence (NGVFKYRPRYYLYKHAYFYPHLKRFPVQ) corresponds to residues 35-62 of bone sialoprotein (BSP), which are located within the collagen-binding domain in BSP. Interestingly, this synthetic CBP inhibited adipogenic differentiation but increased osteogenic differentiation in MDSCs. The CBP also induced expression of osteoblastic marker proteins, including alkaline phosphatase (ALP), type I collagen, Runt-related transcription factor 2 (Runx2), and osteocalcin; prevented adipogenic differentiation in MDSCs; and down-regulated adipose-specific mRNAs, such as adipocyte protein 2 (aP2) and peroxisome proliferator-activated receptor {gamma}. The CBP increased Extracellular signal-regulated kinases (ERK) 1/2 protein phosphorylation, which is important in lineage determination. These observations suggest that this CBP determines the osteogenic or adipogenic lineage in MDSCs by activating ERK1/2. Taken

A specific l-tri-iodothyronine-binding protein in the cytosol fraction of human breast adipose tissue

PubMed Central

Rao, Marie Luise; Rao, Govind S.

1982-01-01

1. Binding of l-tri-[125I]iodothyronine to the cytosol fraction of normal human female breast adipose tissue was investigated by the charcoal adsorption method. Equilibrium of binding was reached after 120s at 25°C. 2. The l-tri-[125I]iodothyronine-binding component is a protein; this was confirmed by experiments in which binding was totally lost after heating the cytosol fraction for 10min at 100°C and in which binding was diminished after treatment with proteolytic enzymes and with thiol-group-blocking reagents. The binding protein was stable at −38°C for several months. 3. It displayed saturability, high affinity (apparent Kd 3.28nm) and a single class of binding sites. 4. High specificity for l-tri-iodothyronine and l-3,5-di-iodo-3′-isopropylthyronine was observed, whereas other iodothyronines were less effective in displacing l-tri-[125I]-iodothyronine from its binding site. 5. The binding of the hormone by the cytosol fraction did not show a pH optimum. 6. When cytosol fractions of adipose tissue from different females were subjected to radioimmunoassay for the determination of thyroxine-binding globulin a value of 0.304±0.11μg/mg of cytosol protein (mean±s.d., n=4) was obtained; the mean concentration in plasma was 0.309±0.07μg/mg of plasma protein (mean±s.d., n=3). 7. The Ka value of 6.3×108m−1 of l-tri-[125I]iodothyronine for binding to plasma, the similar thermalinactivation profiles of binding and the reactivity to thiol-group-blocking reagents were some properties common between the binding components from the cytosol fraction and plasma. 8. These results suggest that the cytosol fraction of human female breast adipose tissue contains thyroxine-binding globulin; the protein that binds l-tri-[125I]iodothyronine with high affinity and specificity appears to be similar to thyroxine-binding globulin. PMID:6289813

Prion removal effect of a specific affinity ligand introduced into the manufacturing process of the pharmaceutical quality solvent/detergent (S/D)-treated plasma OctaplasLG.

PubMed

Neisser-Svae, A; Bailey, A; Gregori, L; Heger, A; Jordan, S; Behizad, M; Reichl, H; Römisch, J; Svae, T-E

2009-10-01

A new chromatographic step for the selective binding of abnormal prion protein (PrP(Sc)) was developed, and optimization for PrP(Sc) capture was achieved by binding to an affinity ligand attached to synthetic resin particles. This step was implemented into the manufacturing process of the solvent/detergent (S/D)-treated biopharmaceutical quality plasma Octaplas to further improve the safety margin in terms of risk for variant Creutzfeldt-Jakob disease (vCJD) transmission. Intermediates and Octaplas final container material, spiked with hamster brain-derived PrP(Sc)-containing fractions, were used for experiments to establish the feasibility of introducing this novel chromatography step. The binding capacity per millilitre of ligand gel was determined under the selected manufacturing conditions. In addition, the specificity of the ligand gel to bind PrP(Sc) from human sources was investigated. A validated Western blot test was used for the identification and quantification of PrP(Sc). A reduction factor of > or = 3.0 log(10) could be demonstrated by Western blotting, utilizing the relevant Octaplas matrix from manufacturing. In this particular cell-free plasma solution, the PrP(Sc) binding capacity of the selected gel was very high (> or = 6 log(10) ID(50)/ml, equivalent to roughly 10 log(10) ID(50)/column at manufacturing scale). The gel binds specifically PrP(Sc) from both animal (hamster and mouse) and human (sporadic and variant CJD) sources. This new single-use, disposable PrP(Sc)-harvesting gel ensures a very high capacity in terms of removing the pathogenic agent causing vCJD from the new generation OctaplasLG, in the event that prions can be found in plasma from donors incubating the disease and thereby contaminating the raw material plasma used for manufacturing.

A validated inductively coupled plasma mass spectrometry (ICP-MS) method for the quantification of total platinum content in plasma, plasma ultrafiltrate, urine and peritoneal fluid.

PubMed

Lemoine, Lieselotte; Thijssen, Elsy; Noben, Jean-Paul; Adriaensens, Peter; Carleer, Robert; Speeten, Kurt Van der

2018-04-15

Oxaliplatin is a platinum (Pt) 1 containing antineoplastic agent that is applied in current clinical practice for the treatment of colon and appendiceal neoplasms. A fully validated, highly sensitive, high throughput inductively coupled plasma mass spectrometry (ICP-MS) method is provided to quantify the total Pt content in plasma, plasma ultrafiltrate, urine and peritoneal fluid. In this ICP-MS approach, the only step of sample preparation is a 1000-fold dilution in 0.5% nitric acid, allowing the analysis of 17 samples per hour. Detection of Pt was achieved over a linear range of 0.01-100 ng/mL. The limit of quantification was 18.0 ng/mL Pt in plasma, 8.0 ng/mL in ultrafiltrate and 6.1 ng/mL in urine and peritoneal fluid. The ICP-MS method was further validated for inter-and intraday precision and accuracy (≤15%), recovery, robustness and stability. Short-term storage of the biofluids, for 14 days, can be performed at -4 °C, -24 °C and -80 °C. As to long-term stability, up to 5 months, storage at -80 °C is encouraged. Furthermore, a timeline assessing the total and unbound Pt fraction in plasma and ultrafiltrate over a period of 45 h is provided. Following an incubation period of 5 h at 37 °C, 19-21% of Pt was recovered in the ultrafiltrate, emphasizing the extensive and rapid binding of oxaliplatin-derived Pt to plasma proteins. The described method can easily be implemented in a routine setting for pharmacokinetic studies in patients treated with oxaliplatin-based hyperthermic intraperitoneal perioperative chemotherapy. Copyright © 2018 Elsevier B.V. All rights reserved.

Systematic identification of yeast proteins extracted into model wine during aging on the yeast lees.

PubMed

Rowe, Jeffrey D; Harbertson, James F; Osborne, James P; Freitag, Michael; Lim, Juyun; Bakalinsky, Alan T

2010-02-24

Total protein and protein-associated mannan concentrations were measured, and individual proteins were identified during extraction into model wines over 9 months of aging on the yeast lees following completion of fermentations by seven wine strains of Saccharomyces cerevisiae. In aged wines, protein-associated mannan increased about 6-fold (+/-66%), while total protein only increased 2-fold (+/-20%), which resulted in a significantly greater protein-associated mannan/total protein ratio for three strains. A total of 219 proteins were identified among all wine samples taken over the entire time course. Of the 17 "long-lived" proteins detected in all 9 month samples, 13 were cell wall mannoproteins, and four were glycolytic enzymes. Most cytosolic proteins were not detected after 6 months. Native mannosylated yeast invertase was assayed for binding to wine tannin and was found to have a 10-fold lower affinity than nonglycosylated bovine serum albumin. Enrichment of mannoproteins in the aged model wines implies greater solution stability than other yeast proteins and the possibility that their contributions to wine quality may persist long after bottling.