On the general concept of buoyancy in sedimentation and ultracentrifugation.

PubMed

Piazza, Roberto; Buzzaccaro, Stefano; Secchi, Eleonora; Parola, Alberto

2013-08-02

Gravity or ultracentrifuge settling of colloidal particles and macromolecules usually involves several disperse species, either because natural and industrial colloids display a large size polydispersity, or because additives are put in on purpose to allow for density-based fractionation of the suspension. Such 'macromolecular crowding', however, may have surprising effects on sedimentation, for it strongly affects the buoyant force felt by a settling particle. Here we show that, as a matter of fact, the standard Archimedes' principle is just a limiting law, valid only for mesoscopic particles settling in a molecular fluid, and we obtain a fully general expression for the actual buoyancy force providing a microscopic basis to the general thermodynamic analysis of sedimentation in multi-component mixtures. The effective buoyancy also depends on the particle shape, being much more pronounced for thin rods and discs. Our model is successfully tested on simple colloidal mixtures, and used to predict rather unexpected effects, such as denser particles floating on top of a lighter fluid, which we actually observe in targeted experiments. This 'generalized Archimedes principle' may provide a tool to devise novel separation methods sensitive to particle size and shape.

On the general concept of buoyancy in sedimentation and ultracentrifugation

NASA Astrophysics Data System (ADS)

Piazza, Roberto; Buzzaccaro, Stefano; Secchi, Eleonora; Parola, Alberto

2013-08-01

Gravity or ultracentrifuge settling of colloidal particles and macromolecules usually involves several disperse species, either because natural and industrial colloids display a large size polydispersity, or because additives are put in on purpose to allow for density-based fractionation of the suspension. Such ‘macromolecular crowding’, however, may have surprising effects on sedimentation, for it strongly affects the buoyant force felt by a settling particle. Here we show that, as a matter of fact, the standard Archimedes' principle is just a limiting law, valid only for mesoscopic particles settling in a molecular fluid, and we obtain a fully general expression for the actual buoyancy force providing a microscopic basis to the general thermodynamic analysis of sedimentation in multi-component mixtures. The effective buoyancy also depends on the particle shape, being much more pronounced for thin rods and discs. Our model is successfully tested on simple colloidal mixtures, and used to predict rather unexpected effects, such as denser particles floating on top of a lighter fluid, which we actually observe in targeted experiments. This ‘generalized Archimedes principle’ may provide a tool to devise novel separation methods sensitive to particle size and shape.

Volume of reaction by the Archibald ultracentrifuge method (lobster hemocyanin).

PubMed

Saxena, V P; Kegeles, G; Kikas, R

1976-07-01

Samples of lobster hemocyanin (Homarus americanus) under conditions of reversible reaction between whole (25 S) and half (17 S) molecules have been subjected to accurately known nitrogen pressures in analytical ultracentrifuge cells. A modified pressurization chamber of the type developed by Schumaker and colleagues has been constructed for this purpose. The molecular weight was then determined at the top (liquid-gas) meniscus, by means of the Archibald method. The logarithmic dependence upon pressure of the derived equilibrium constant then gave directly the volume of reaction. Experiments were performed in veronal-citrate buffers at pH 8, where the molar volume of formation of whole (dodecameric) molecules from half molecules appears to be negative, and at pH 8.46 in veronal-citrate buffer in the presence of 0.003 molar free calcium ion, where the molar volume of formation was estimated to be + 390 cm3/mole. In glycine-sodium hydroxide buffer at pH 9.6 containing 0.0047 molar free calcium, the molar volume of formation of whole molecules was estimated to be +120 +/- 70 cm3, corresponding to an estimated difference in partial specific volume between whole molecules and half molecules of only 1.3 (10)-4cm3/gram. The correctness of the sign of this value in glycine buffer has been verified by pressure-jump light-scattering experiments.

Residual urinary extracellular vesicles in ultracentrifugation supernatants after hydrostatic filtration dialysis enrichment.

PubMed

Musante, Luca; Tataruch-Weinert, Dorota; Kerjaschki, Dontscho; Henry, Michael; Meleady, Paula; Holthofer, Harry

2017-01-01

Urinary extracellular vesicles (UEVs) appear an ideal source of biomarkers for kidney and urogenital diseases. The majority of protocols designed for their isolation are based on differential centrifugation steps. However, little is still known of the type and amount of vesicles left in the supernatant. Here we used an isolation protocol for UEVs which uses hydrostatic filtration dialysis as first pre-enrichment step, followed by differential centrifugation. Transmission electron microscopy (TEM), mass spectrometry (MS), western blot, ELISA assays and tuneable resistive pulse sensing (TRPS) were used to characterise and quantify UEVs in the ultracentrifugation supernatant. TEM showed the presence of a variety of small size vesicles in the supernatant while protein identification by MS matched accurately with the protein list available in Vesiclepedia. Screening and relative quantification for specific vesicle markers showed that the supernatant was preferentially positive for CD9 and TSG101. ELISA tests for quantification of exosome revealed that 14%, was left in the supernatant with a particle diameter of 110 nm and concentration of 1.54 × 10 10 /ml. Here we show a comprehensive characterisation of exosomes and other small size urinary vesicles which the conventional differential centrifugation protocol may lose.

Residual urinary extracellular vesicles in ultracentrifugation supernatants after hydrostatic filtration dialysis enrichment

PubMed Central

Musante, Luca; Tataruch-Weinert, Dorota; Kerjaschki, Dontscho; Henry, Michael; Meleady, Paula; Holthofer, Harry

2017-01-01

ABSTRACT Urinary extracellular vesicles (UEVs) appear an ideal source of biomarkers for kidney and urogenital diseases. The majority of protocols designed for their isolation are based on differential centrifugation steps. However, little is still known of the type and amount of vesicles left in the supernatant. Here we used an isolation protocol for UEVs which uses hydrostatic filtration dialysis as first pre-enrichment step, followed by differential centrifugation. Transmission electron microscopy (TEM), mass spectrometry (MS), western blot, ELISA assays and tuneable resistive pulse sensing (TRPS) were used to characterise and quantify UEVs in the ultracentrifugation supernatant. TEM showed the presence of a variety of small size vesicles in the supernatant while protein identification by MS matched accurately with the protein list available in Vesiclepedia. Screening and relative quantification for specific vesicle markers showed that the supernatant was preferentially positive for CD9 and TSG101. ELISA tests for quantification of exosome revealed that 14%, was left in the supernatant with a particle diameter of 110 nm and concentration of 1.54 × 1010/ml. Here we show a comprehensive characterisation of exosomes and other small size urinary vesicles which the conventional differential centrifugation protocol may lose. PMID:28326167

Purification of infectious adenovirus in two hours by ultracentrifugation and tangential flow filtration

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

Ugai, Hideyo; Yamasaki, Takahito; Hirose, Megumi

2005-06-17

Adenoviruses are excellent vectors for gene transfer and are used extensively for high-level expression of the products of transgenes in living cells. The development of simple and rapid methods for the purification of stable infectious recombinant adenoviruses (rAds) remains a challenge. We report here a method for the purification of infectious adenovirus type 5 (Ad5) that involves ultracentrifugation on a cesium chloride gradient at 604,000g for 15 min at 4 deg C and tangential flow filtration. The entire procedure requires less than two hours and infectious Ad5 can be recovered at levels higher than 64% of the number of plaque-formingmore » units (pfu) in the initial crude preparation of viruses. We have obtained titers of infectious purified Ad5 of 1.35 x 10{sup 10} pfu/ml and a ratio of particle titer to infectious titer of seven. The method described here allows the rapid purification of rAds for studies of gene function in vivo and in vitro, as well as the rapid purification of Ad5.« less

Variable-Field Analytical Ultracentrifugation: I. Time-Optimized Sedimentation Equilibrium

PubMed Central

Ma, Jia; Metrick, Michael; Ghirlando, Rodolfo; Zhao, Huaying; Schuck, Peter

2015-01-01

Sedimentation equilibrium (SE) analytical ultracentrifugation (AUC) is a gold standard for the rigorous determination of macromolecular buoyant molar masses and the thermodynamic study of reversible interactions in solution. A significant experimental drawback is the long time required to attain SE, which is usually on the order of days. We have developed a method for time-optimized SE (toSE) with defined time-varying centrifugal fields that allow SE to be attained in a significantly (up to 10-fold) shorter time than is usually required. To achieve this, numerical Lamm equation solutions for sedimentation in time-varying fields are computed based on initial estimates of macromolecular transport properties. A parameterized rotor-speed schedule is optimized with the goal of achieving a minimal time to equilibrium while limiting transient sample preconcentration at the base of the solution column. The resulting rotor-speed schedule may include multiple over- and underspeeding phases, balancing the formation of gradients from strong sedimentation fluxes with periods of high diffusional transport. The computation is carried out in a new software program called TOSE, which also facilitates convenient experimental implementation. Further, we extend AUC data analysis to sedimentation processes in such time-varying centrifugal fields. Due to the initially high centrifugal fields in toSE and the resulting strong migration, it is possible to extract sedimentation coefficient distributions from the early data. This can provide better estimates of the size of macromolecular complexes and report on sample homogeneity early on, which may be used to further refine the prediction of the rotor-speed schedule. In this manner, the toSE experiment can be adapted in real time to the system under study, maximizing both the information content and the time efficiency of SE experiments. PMID:26287634

On the use of ultracentrifugal devices for routine sample preparation in biomolecular magic-angle-spinning NMR

PubMed Central

Mandal, Abhishek; Boatz, Jennifer C.; Wheeler, Travis; van der Wel, Patrick C. A.

2017-01-01

A number of recent advances in the field of magic-angle-spinning (MAS) solid-state NMR have enabled its application to a range of biological systems of ever increasing complexity. To retain biological relevance, these samples are increasingly studied in a hydrated state. At the same time, experimental feasibility requires the sample preparation process to attain a high sample concentration within the final MAS rotor. We discuss these considerations, and how they have led to a number of different approaches to MAS NMR sample preparation. We describe our experience of how custom-made (or commercially available) ultracentrifugal devices can facilitate a simple, fast and reliable sample preparation process. A number of groups have since adopted such tools, in some cases to prepare samples for sedimentation-style MAS NMR experiments. Here we argue for a more widespread adoption of their use for routine MAS NMR sample preparation. PMID:28229262

Analysis of the complex formation of heparin with protamine by light scattering and analytical ultracentrifugation: implications for blood coagulation management.

PubMed

Maurer, Jürgen; Haselbach, Stephanie; Klein, Oliver; Baykut, Doan; Vogel, Vitali; Mäntele, Werner

2011-02-02

Heparin, a linear glycosaminoglycan, is used in different forms in anticoagulation treatment. Protamine, a highly positive charged peptide containing about 32 amino acids, acts as an antagonist for heparin to restore normal blood coagulation. The complex formation of protamine with heparin was analyzed by a combination of analytical ultracentrifugation and light scattering. Titration of heparin with protamine in blood plasma preparations results in a drastic increase of turbidity, indicating the formation of nanoscale particles. A similar increase of turbidity was observed in physiological saline solution with or without human serum albumin (HSA). Particle size analysis by analytical ultracentrifugation revealed a particle radius of approximately 30 nm for unfractionated heparin and of approximately 60 nm for low molecular weight heparin upon complexation with excess protamine, in agreement with atomic force microscopy data. In the absence of HSA, larger and more heterogeneous particles were observed. The particles obtained were found to be stable for hours. The particle formation kinetics was analyzed by light scattering at different scattering angles and was found to be complete within several minutes. The time course of particle formation suggests a condensation reaction, with sigmoidal traces for low heparin concentrations and quasi-first-order reaction for high heparin concentrations. Under all conditions, the final scattering intensity reached after several minutes was found to be proportional to the amount of heparin in the blood plasma or buffer solution, provided that excess protamine was available and no multiple scattering occurred. On the basis of a direct relation between particle concentration and the heparin concentration present before protaminization, a light scattering assay was developed which permits the quantitative analysis of the heparin concentration in blood plasma and which could complement or even replace the activated clotting time test

Determination of nanoparticle size distribution together with density or molecular weight by 2D analytical ultracentrifugation

PubMed Central

Carney, Randy P.; Kim, Jin Young; Qian, Huifeng; Jin, Rongchao; Mehenni, Hakim; Stellacci, Francesco; Bakr, Osman M.

2011-01-01

Nanoparticles are finding many research and industrial applications, yet their characterization remains a challenge. Their cores are often polydisperse and coated by a stabilizing shell that varies in size and composition. No single technique can characterize both the size distribution and the nature of the shell. Advances in analytical ultracentrifugation allow for the extraction of the sedimentation (s) and diffusion coefficients (D). Here we report an approach to transform the s and D distributions of nanoparticles in solution into precise molecular weight (M), density (ρP) and particle diameter (dp) distributions. M for mixtures of discrete nanocrystals is found within 4% of the known quantities. The accuracy and the density information we achieve on nanoparticles are unparalleled. A single experimental run is sufficient for full nanoparticle characterization, without the need for standards or other auxiliary measurements. We believe that our method is of general applicability and we discuss its limitations. PMID:21654635

Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling.

PubMed

Schuck, P

2000-03-01

A new method for the size-distribution analysis of polymers by sedimentation velocity analytical ultracentrifugation is described. It exploits the ability of Lamm equation modeling to discriminate between the spreading of the sedimentation boundary arising from sample heterogeneity and from diffusion. Finite element solutions of the Lamm equation for a large number of discrete noninteracting species are combined with maximum entropy regularization to represent a continuous size-distribution. As in the program CONTIN, the parameter governing the regularization constraint is adjusted by variance analysis to a predefined confidence level. Estimates of the partial specific volume and the frictional ratio of the macromolecules are used to calculate the diffusion coefficients, resulting in relatively high-resolution sedimentation coefficient distributions c(s) or molar mass distributions c(M). It can be applied to interference optical data that exhibit systematic noise components, and it does not require solution or solvent plateaus to be established. More details on the size-distribution can be obtained than from van Holde-Weischet analysis. The sensitivity to the values of the regularization parameter and to the shape parameters is explored with the help of simulated sedimentation data of discrete and continuous model size distributions, and by applications to experimental data of continuous and discrete protein mixtures.

AN IMPROVED AIR-DRIVEN TYPE OF ULTRACENTRIFUGE FOR MOLECULAR SEDIMENTATION.

PubMed

Bauer, J H; Pickels, E G

1937-03-31

1. A description is given of the construction details and operation characteristics of an improved type of air-driven ultracentrifuge operating in vacuum and suitable for the determination of sedimentation constants of protein molecules. 2. The rotor of the centrifuge is made of a forged aluminum alloy; it is oval in shape, measures 185 mm. at its greatest diameter, and weighs 3,430 gm. It carries a transparent cell located at a distance of 65 mm. from the axis of rotation and designed to accommodate a fluid column 15 mm. high. 3. The rotor has been run repeatedly over long periods at a speed of 60,000 R.P.M., which corresponds to a centrifugal force of 260,000 times gravity in the center of the cell. At this speed no deformation of the rotor nor leakage of the cell has been observed. 4. The sharp definition of sedimentation photographs taken at high speed serves to indicate the absence of detectable vibrations in the centrifuge. 5. When a vacuum of less than 1 micron of mercury is maintained in the centrifuge chamber, the rise in the rotor temperature amounts to only 1 or 2 degrees C. after several hours' run at high speed. 6. There has been no evidence of convection currents interfering with normal sedimentation of protein molecules in the centrifugal field. 7. A driving air pressure of about 18 pounds per square inch is sufficient to maintain the centrifuge at a steady speed of 60,000 R.P.M. With a driving pressure of 80 pounds per square inch, it can be accelerated to this speed in less than 20 minutes, and also brought to rest in about the same length of time by the application of the braking system. 8. The adaptation of Svedberg's optical systems to this centrifuge for photographically recording the movement of sedimentation boundaries is described.

AN IMPROVED AIR-DRIVEN TYPE OF ULTRACENTRIFUGE FOR MOLECULAR SEDIMENTATION

PubMed Central

Bauer, Johannes H.; Pickels, Edward G.

1937-01-01

1. A description is given of the construction details and operation characteristics of an improved type of air-driven ultracentrifuge operating in vacuum and suitable for the determination of sedimentation constants of protein molecules. 2. The rotor of the centrifuge is made of a forged aluminum alloy; it is oval in shape, measures 185 mm. at its greatest diameter, and weighs 3,430 gm. It carries a transparent cell located at a distance of 65 mm. from the axis of rotation and designed to accommodate a fluid column 15 mm. high. 3. The rotor has been run repeatedly over long periods at a speed of 60,000 R.P.M., which corresponds to a centrifugal force of 260,000 times gravity in the center of the cell. At this speed no deformation of the rotor nor leakage of the cell has been observed. 4. The sharp definition of sedimentation photographs taken at high speed serves to indicate the absence of detectable vibrations in the centrifuge. 5. When a vacuum of less than 1 micron of mercury is maintained in the centrifuge chamber, the rise in the rotor temperature amounts to only 1 or 2°C. after several hours' run at high speed. 6. There has been no evidence of convection currents interfering with normal sedimentation of protein molecules in the centrifugal field. 7. A driving air pressure of about 18 pounds per square inch is sufficient to maintain the centrifuge at a steady speed of 60,000 R.P.M. With a driving pressure of 80 pounds per square inch, it can be accelerated to this speed in less than 20 minutes, and also brought to rest in about the same length of time by the application of the braking system. 8. The adaptation of Svedberg's optical systems to this centrifuge for photographically recording the movement of sedimentation boundaries is described. PMID:19870619

A television scanner for the ultracentrifuge. II. Multiple cell operation.

PubMed

Rockholt, D L; Royce, C R; Richards, E G

1976-07-01

The "Optical Multichannel Analyzer" (OMA) is a commercially available instrument that with the absorption optical system of the ultracentrifuge, provides an entire 500 channel intensity profile of a cell in real time. With its own analog-todigital converter, the OMA integrates a selectable number of 32.8 msec scans to provide a time-averaged image in digital form. This paper describes an interface-controller for operation of the OMA with single- and double-sector cells in multi-cell rotors, simulating double-beam measurement required for absorbance determinations. The desired sector is selected by "gating" the intensifier stage of a "Silicon Intensified Target" vidicon (SIT) used as the light detector. The cell location in the rotor and the position of the gate relative to the cell centerline is obtained from a phase-locked loop circuit which divides each rotation of the rotor into 3600 parts independent of rotor speed. (This circuit employed with photo-multiplier scanners would select the gate position for integration of photomultiplier pulses.) From examination of appropriate signals with an oscilloscope, it was verified that gate positions and widths are located with an accuracy of 0.1degree or better and with a precision of +/- 0.1 mus. The light intensity profile for any desired cell can be examined in "real time", even during acceleration of the rotor. Additional circuits employing a 10 MHz crystal clock 1) control the automatic collection of data for all sectors in multicell rotors at digitally selected time intervals, 2) display the rotor speed, and 3) indicate the elapsed time of the experiment. Constructed but not tested are additional circuits for pulsing a laser into the absorption or Rayleigh optical system. The accuracy of the pulsed SIT has been demonstrated by measurement of absorbances of solutions and also by sedimentation equilibrium experiments with myoglobin. The estimated error is 0.003 for absorbances ranging from 0 to 1. The interface

Investigating the early stages of mineral precipitation by potentiometric titration and analytical ultracentrifugation.

PubMed

Kellermeier, Matthias; Cölfen, Helmut; Gebauer, Denis

2013-01-01

Despite the importance of crystallization for various areas of research, our understanding of the early stages of the mineral precipitation from solution and of the actual mechanism of nucleation is still rather limited. Indeed, detailed insights into the processes underlying nucleation may enable a systematic development of novel strategies for controlling mineralization, which is highly relevant for fields ranging from materials chemistry to medicine. In this work, we describe experimental aspects of a quantitative assay, which relies on pH titrations combined with in situ metal ion potentiometry and conductivity measurements. The assay has originally been designed to study the crystallization of calcium carbonate, one of the most abundant biominerals. However, the developed procedures can also be readily applied to any compound containing cations for which ion-selective electrodes are available. Besides the possibility to quantitatively assess ion association prior to nucleation and to directly determine thermodynamic solubility products of precipitated phases, the main advantage of the crystallization assay is the unambiguous identification of the different stages of precipitation (i.e., prenucleation, nucleation, and early postnucleation) and the characterization of the multiple effects of additives. Furthermore, the experiments permit targeted access to distinct precursor species and intermediate stages, which thus can be analyzed by additional methods such as cryo-electron microscopy or analytical ultracentrifugation (AUC). Regarding ion association in solution, AUC detects entities significantly larger than simple ion pairs, so-called prenucleation clusters. Sedimentation coefficient values and distributions obtained for the calcium carbonate system are discussed in light of recent insights into the structural nature of prenucleation clusters. © 2013 Elsevier Inc. All rights reserved.

Enhanced Electrochemical Performance of Ultracentrifugation-Derived nc-Li3VO4/MWCNT Composites for Hybrid Supercapacitors.

PubMed

Iwama, Etsuro; Kawabata, Nozomi; Nishio, Nagare; Kisu, Kazuaki; Miyamoto, Junichi; Naoi, Wako; Rozier, Patrick; Simon, Patrice; Naoi, Katsuhiko

2016-05-24

Nanocrystalline Li3VO4 dispersed within multiwalled carbon nanotubes (MWCNTs) was prepared using an ultracentrifugation (uc) process and electrochemically characterized in Li-containing electrolyte. When charged and discharged down to 0.1 V vs Li, the material reached 330 mAh g(-1) (per composite) at an average voltage of about 1.0 V vs Li, with more than 50% capacity retention at a high current density of 20 A g(-1). This current corresponds to a nearly 500C rate (7.2 s) for a porous carbon electrode normally used in electric double-layer capacitor devices (1C = 40 mA g(-1) per activated carbon). The irreversible structure transformation during the first lithiation, assimilated as an activation process, was elucidated by careful investigation of in operando X-ray diffraction and X-ray absorption fine structure measurements. The activation process switches the reaction mechanism from a slow "two-phase" to a fast "solid-solution" in a limited voltage range (2.5-0.76 V vs Li), still keeping the capacity as high as 115 mAh g(-1) (per composite). The uc-Li3VO4 composite operated in this potential range after the activation process allows fast Li(+) intercalation/deintercalation with a small voltage hysteresis, leading to higher energy efficiency. It offers a promising alternative to replace high-rate Li4Ti5O12 electrodes in hybrid supercapacitor applications.

Quantifying Trace Amounts of Aggregates in Biopharmaceuticals Using Analytical Ultracentrifugation Sedimentation Velocity: Bayesian Analyses and F Statistics.

PubMed

Wafer, Lucas; Kloczewiak, Marek; Luo, Yin

2016-07-01

Analytical ultracentrifugation-sedimentation velocity (AUC-SV) is often used to quantify high molar mass species (HMMS) present in biopharmaceuticals. Although these species are often present in trace quantities, they have received significant attention due to their potential immunogenicity. Commonly, AUC-SV data is analyzed as a diffusion-corrected, sedimentation coefficient distribution, or c(s), using SEDFIT to numerically solve Lamm-type equations. SEDFIT also utilizes maximum entropy or Tikhonov-Phillips regularization to further allow the user to determine relevant sample information, including the number of species present, their sedimentation coefficients, and their relative abundance. However, this methodology has several, often unstated, limitations, which may impact the final analysis of protein therapeutics. These include regularization-specific effects, artificial "ripple peaks," and spurious shifts in the sedimentation coefficients. In this investigation, we experimentally verified that an explicit Bayesian approach, as implemented in SEDFIT, can largely correct for these effects. Clear guidelines on how to implement this technique and interpret the resulting data, especially for samples containing micro-heterogeneity (e.g., differential glycosylation), are also provided. In addition, we demonstrated how the Bayesian approach can be combined with F statistics to draw more accurate conclusions and rigorously exclude artifactual peaks. Numerous examples with an antibody and an antibody-drug conjugate were used to illustrate the strengths and drawbacks of each technique.

A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.

PubMed

Zhao, Huaying; Ghirlando, Rodolfo; Alfonso, Carlos; Arisaka, Fumio; Attali, Ilan; Bain, David L; Bakhtina, Marina M; Becker, Donald F; Bedwell, Gregory J; Bekdemir, Ahmet; Besong, Tabot M D; Birck, Catherine; Brautigam, Chad A; Brennerman, William; Byron, Olwyn; Bzowska, Agnieszka; Chaires, Jonathan B; Chaton, Catherine T; Cölfen, Helmut; Connaghan, Keith D; Crowley, Kimberly A; Curth, Ute; Daviter, Tina; Dean, William L; Díez, Ana I; Ebel, Christine; Eckert, Debra M; Eisele, Leslie E; Eisenstein, Edward; England, Patrick; Escalante, Carlos; Fagan, Jeffrey A; Fairman, Robert; Finn, Ron M; Fischle, Wolfgang; de la Torre, José García; Gor, Jayesh; Gustafsson, Henning; Hall, Damien; Harding, Stephen E; Cifre, José G Hernández; Herr, Andrew B; Howell, Elizabeth E; Isaac, Richard S; Jao, Shu-Chuan; Jose, Davis; Kim, Soon-Jong; Kokona, Bashkim; Kornblatt, Jack A; Kosek, Dalibor; Krayukhina, Elena; Krzizike, Daniel; Kusznir, Eric A; Kwon, Hyewon; Larson, Adam; Laue, Thomas M; Le Roy, Aline; Leech, Andrew P; Lilie, Hauke; Luger, Karolin; Luque-Ortega, Juan R; Ma, Jia; May, Carrie A; Maynard, Ernest L; Modrak-Wojcik, Anna; Mok, Yee-Foong; Mücke, Norbert; Nagel-Steger, Luitgard; Narlikar, Geeta J; Noda, Masanori; Nourse, Amanda; Obsil, Tomas; Park, Chad K; Park, Jin-Ku; Pawelek, Peter D; Perdue, Erby E; Perkins, Stephen J; Perugini, Matthew A; Peterson, Craig L; Peverelli, Martin G; Piszczek, Grzegorz; Prag, Gali; Prevelige, Peter E; Raynal, Bertrand D E; Rezabkova, Lenka; Richter, Klaus; Ringel, Alison E; Rosenberg, Rose; Rowe, Arthur J; Rufer, Arne C; Scott, David J; Seravalli, Javier G; Solovyova, Alexandra S; Song, Renjie; Staunton, David; Stoddard, Caitlin; Stott, Katherine; Strauss, Holger M; Streicher, Werner W; Sumida, John P; Swygert, Sarah G; Szczepanowski, Roman H; Tessmer, Ingrid; Toth, Ronald T; Tripathy, Ashutosh; Uchiyama, Susumu; Uebel, Stephan F W; Unzai, Satoru; Gruber, Anna Vitlin; von Hippel, Peter H; Wandrey, Christine; Wang, Szu-Huan; Weitzel, Steven E; Wielgus-Kutrowska, Beata; Wolberger, Cynthia; Wolff, Martin; Wright, Edward; Wu, Yu-Sung; Wubben, Jacinta M; Schuck, Peter

2015-01-01

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation

PubMed Central

Zhao, Huaying; Ghirlando, Rodolfo; Alfonso, Carlos; Arisaka, Fumio; Attali, Ilan; Bain, David L.; Bakhtina, Marina M.; Becker, Donald F.; Bedwell, Gregory J.; Bekdemir, Ahmet; Besong, Tabot M. D.; Birck, Catherine; Brautigam, Chad A.; Brennerman, William; Byron, Olwyn; Bzowska, Agnieszka; Chaires, Jonathan B.; Chaton, Catherine T.; Cölfen, Helmut; Connaghan, Keith D.; Crowley, Kimberly A.; Curth, Ute; Daviter, Tina; Dean, William L.; Díez, Ana I.; Ebel, Christine; Eckert, Debra M.; Eisele, Leslie E.; Eisenstein, Edward; England, Patrick; Escalante, Carlos; Fagan, Jeffrey A.; Fairman, Robert; Finn, Ron M.; Fischle, Wolfgang; de la Torre, José García; Gor, Jayesh; Gustafsson, Henning; Hall, Damien; Harding, Stephen E.; Cifre, José G. Hernández; Herr, Andrew B.; Howell, Elizabeth E.; Isaac, Richard S.; Jao, Shu-Chuan; Jose, Davis; Kim, Soon-Jong; Kokona, Bashkim; Kornblatt, Jack A.; Kosek, Dalibor; Krayukhina, Elena; Krzizike, Daniel; Kusznir, Eric A.; Kwon, Hyewon; Larson, Adam; Laue, Thomas M.; Le Roy, Aline; Leech, Andrew P.; Lilie, Hauke; Luger, Karolin; Luque-Ortega, Juan R.; Ma, Jia; May, Carrie A.; Maynard, Ernest L.; Modrak-Wojcik, Anna; Mok, Yee-Foong; Mücke, Norbert; Nagel-Steger, Luitgard; Narlikar, Geeta J.; Noda, Masanori; Nourse, Amanda; Obsil, Tomas; Park, Chad K.; Park, Jin-Ku; Pawelek, Peter D.; Perdue, Erby E.; Perkins, Stephen J.; Perugini, Matthew A.; Peterson, Craig L.; Peverelli, Martin G.; Piszczek, Grzegorz; Prag, Gali; Prevelige, Peter E.; Raynal, Bertrand D. E.; Rezabkova, Lenka; Richter, Klaus; Ringel, Alison E.; Rosenberg, Rose; Rowe, Arthur J.; Rufer, Arne C.; Scott, David J.; Seravalli, Javier G.; Solovyova, Alexandra S.; Song, Renjie; Staunton, David; Stoddard, Caitlin; Stott, Katherine; Strauss, Holger M.; Streicher, Werner W.; Sumida, John P.; Swygert, Sarah G.; Szczepanowski, Roman H.; Tessmer, Ingrid; Toth, Ronald T.; Tripathy, Ashutosh; Uchiyama, Susumu; Uebel, Stephan F. W.; Unzai, Satoru; Gruber, Anna Vitlin; von Hippel, Peter H.; Wandrey, Christine; Wang, Szu-Huan; Weitzel, Steven E.; Wielgus-Kutrowska, Beata; Wolberger, Cynthia; Wolff, Martin; Wright, Edward; Wu, Yu-Sung; Wubben, Jacinta M.; Schuck, Peter

2015-01-01

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies. PMID:25997164

Analytical Ultracentrifugation Sedimentation Velocity for the Characterization of Detergent-Solubilized Membrane Proteins Ca++-ATPase and ExbB

PubMed Central

Salvay, Andrés G.; Santamaria, Monica; le Maire, Marc

2008-01-01

We have investigated the potential of new methods of analysis of sedimentation velocity (SV) analytical ultracentrifugation (AUC) for the characterization of detergent-solubilized membrane proteins. We analyze the membrane proteins Ca++-ATPase and ExbB solubilized with DDM (dodecyl-β-d-maltoside). SV is extremely well suited for characterizing sample heterogeneity. DDM micelles (s20w = 3.1 S) and complexes (Ca++-ATPase: s20w = 7.3 S; ExbB: s20w = 4 S) are easily distinguished. Using different detergent and protein concentrations, SV does not detect any evidence of self-association for the two proteins. An estimate of bound detergent of 0.9 g/g for Ca++-ATPase and 1.5 g/g for ExbB is obtained from the combined analysis of SV profiles obtained using absorbance and interference optics. Combining s20w with values of the hydrodynamic radius, Rs = 5.5 nm for Ca++-ATPase or Rs = 3.4 nm for ExbB, allows the determination of buoyant molar masses, Mb. In view of their Mb and composition, Ca++-ATPase and ExbB are monomers in our experimental conditions. We conclude that one of the main advantages of SV versus other techniques is the possibility to ascertain the homogeneity of the samples and to focus on a given complex even in the presence of other impurities or aggregates. The relative rapidity of SV measurements also allows experiments on unstable samples. PMID:19669527

Low density lipoprotein for oxidation and metabolic studies. Isolation from small volumes of plasma using a tabletop ultracentrifuge.

PubMed

Himber, J; Bühler, E; Moll, D; Moser, U K

1995-01-01

A rapid method is described for the isolation of small volumes of plasma low density lipoprotein (LDL) free of plasma protein contaminants using the TL-100 Tabletop Ultracentrifuge (Beckman). The isolation of LDL was achieved by a 25 min discontinuous gradient density centrifugation between the density range of 1.006 and 1.21 g/ml, recovery of LDL by tube slicing followed by a 90 min flotation step (d = 1.12 g/ml). The purity of LDL and apolipoprotein B100 (apo B100) were monitored by agarose electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), radial immunodiffusion and micropreparative fast protein liquid chromatography (FPLC). The ability of LDL oxidation was assessed by following absorbance at 234 nm after addition of copper ions. The functional integrity of the isolated LDL was checked by clearance kinetics after injection of [125I]-labelled LDL in estrogen-treated rats. The additional purification step led to LDL fractions free of protein contamination and left apo B100, alpha-tocopherol and beta-carotene intact. The LDL prepared in this way was free of albumin, as evident from analytic tests and from its enhanced oxidative modification by copper ions. Used for analytical purposes, this method allows LDL preparations from plasma volumes up to 570 microliters. This method is also convenient for metabolic studies in small animals, especially those relating to the determination of kinetic parameters of LDL in which LDL-apo B100 has to be specifically radiolabelled.

A protocol for exosome isolation and characterization: evaluation of ultracentrifugation, density-gradient separation, and immunoaffinity capture methods.

PubMed

Greening, David W; Xu, Rong; Ji, Hong; Tauro, Bow J; Simpson, Richard J

2015-01-01

Exosomes are 40-150 nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of tumorigenic proteins, mRNA and miRNA. Exosomes are important regulators of the cellular niche, and their altered characteristics in many diseases, such as cancer, suggest their importance for diagnostic and therapeutic applications, and as drug delivery vehicles. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. In this chapter, we reveal the protocol and key insights into the isolation, purification and characterization of exosomes, distinct from shed microvesicles and apoptotic blebs. Using the colorectal cancer cell line LIM1863 as a cell model, a comprehensive evaluation of exosome isolation methods including ultracentrifugation (UC-Exos), OptiPrep™ density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM-coated magnetic beads (IAC-Exos) were examined. All exosome isolation methodologies contained 40-150 nm vesicles based on electron microscopy, and positive for exosome markers (Alix, TSG101, HSP70) based on immunoblotting. This protocol employed a proteomic profiling approach to characterize the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method in exosome isolation. Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, IAC-Exos was shown to be the most effective method to isolate exosomes. However, the use of density-based separation (DG-Exos) provides significant advantages for exosome isolation when the use of immunoaffinity capture is limited (due to antibody availability and suitability of exosome markers).

A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents.

PubMed

Helwa, Inas; Cai, Jingwen; Drewry, Michelle D; Zimmerman, Arthur; Dinkins, Michael B; Khaled, Mariam Lotfy; Seremwe, Mutsa; Dismuke, W Michael; Bieberich, Erhard; Stamer, W Daniel; Hamrick, Mark W; Liu, Yutao

2017-01-01

Exosomes play a role in cell-to-cell signaling and serve as possible biomarkers. Isolating exosomes with reliable quality and substantial concentration is a major challenge. Our purpose is to compare the exosomes extracted by three different exosome isolation kits (miRCURY, ExoQuick, and Invitrogen Total Exosome Isolation Reagent) and differential ultracentrifugation (UC) using six different volumes of a non-cancerous human serum (5 ml, 1 ml, 500 μl, 250 μl, 100 μl, and 50 μl) and three different volumes (1 ml, 500 μl and 100 μl) of six individual commercial serum samples collected from human donors. The smaller starting volumes (100 μl and 50 μl) are used to mimic conditions of limited availability of heterogeneous biological samples. The isolated exosomes were characterized based upon size, quantity, zeta potential, CD63 and CD9 protein expression, and exosomal RNA (exRNA) quality and quantity using several complementary methods: nanoparticle tracking analysis (NTA) with ZetaView, western blot, transmission electron microscopy (TEM), the Agilent Bioanalyzer system, and droplet digital PCR (ddPCR). Our NTA results showed that all isolation techniques produced exosomes within the expected size range (40-150 nm). The three kits, though, produced a significantly higher yield (80-300 fold) of exosomes as compared to UC for all serum volumes, except 5 mL. We also found that exosomes isolated by the different techniques and serum volumes had similar zeta potentials to previous studies. Western blot analysis and TEM immunogold labelling confirmed the expression of two common exosomal protein markers, CD63 and CD9, in samples isolated by all techniques. All exosome isolations yielded high quality exRNA, containing mostly small RNA with a peak between 25 and 200 nucleotides in size. ddPCR results indicated that exosomes isolated from similar serum volumes but different isolation techniques rendered similar concentrations of two selected exRNA: hsa-miR-16 and hsa

Assessment of prion reduction filters in decreasing infectivity of ultracentrifuged 263K scrapie-infected brain homogenates in "spiked" human blood and red blood cells.

PubMed

Cardone, Franco; Sowemimo-Coker, Samuel; Abdel-Haq, Hanin; Sbriccoli, Marco; Graziano, Silvia; Valanzano, Angelina; Berardi, Vito Angelo; Galeno, Roberta; Puopolo, Maria; Pocchiari, Maurizio

2014-04-01

The safety of red blood cells (RBCs) is of concern because of the occurrence of four transfusion-transmitted variant Creutzfeldt-Jakob disease (vCJD) cases in the United Kingdom. The absence of validated screening tests requires the use of procedures to remove prions from blood to minimize the risk of transmission. These procedures must be validated using infectious prions in a form that is as close as possible to one in blood. Units of human whole blood (WB) and RBCs were spiked with high-speed supernatants of 263K scrapie-infected hamster brain homogenates. Spiked samples were leukoreduced and then passed through prion-removing filters (Pall Corporation). In another experiment, RBCs from 263K scrapie-infected hamsters were treated as above, and residual infectivity was measured by bioassay. The overall removal of infectivity by the filters from prion-spiked WB and RBCs was approximately two orders of magnitude. No infectivity was detected in filtered hamster RBCs endogenously infected with scrapie. The use of prion-removing filters may help to reduce the risk of transfusion-transmitted vCJD. To avoid overestimation of prion removal efficiency in validation studies, it may be more appropriate to use supernates from ultracentrifugation of scrapie-infected hamster brain homogenate rather than the current standard brain homogenates. © 2013 American Association of Blood Banks.

Analytical ultracentrifugation with fluorescence detection system reveals differences in complex formation between recombinant human TNF and different biological TNF antagonists in various environments

PubMed Central

Krayukhina, Elena; Noda, Masanori; Ishii, Kentaro; Maruno, Takahiro; Wakabayashi, Hirotsugu; Tada, Minoru; Suzuki, Takuo; Ishii-Watabe, Akiko; Kato, Masahiko; Uchiyama, Susumu

2017-01-01

ABSTRACT A number of studies have attempted to elucidate the binding mechanism between tumor necrosis factor (TNF) and clinically relevant antagonists. None of these studies, however, have been conducted as close as possible to physiologic conditions, and so the relationship between the size distribution of TNF-antagonist complexes and the antagonists' biological activity or adverse effects remains elusive. Here, we characterized the binding stoichiometry and sizes of soluble TNF-antagonist complexes for adalimumab, infliximab, and etanercept that were formed in human serum and in phosphate-buffered saline (PBS). Fluorescence-detected sedimentation velocity analytical ultracentrifugation analyses revealed that adalimumab and infliximab formed a range of complexes with TNF, with the major complexes consisting of 3 molcules of the respective antagonist and one or 2 molcules of TNF. Considerably greater amounts of high-molecular-weight complexes were detected for infliximab in human serum. The emergence of peaks with higher sedimentation coefficients than the adalimumab monomer as a function of added human serum albumin (HSA) concentration in PBS suggested weak reversible interactions between HSA and immunoglobulins. Etanerept exclusively formed 1:1 complexes with TNF in PBS, and a small amount of complexes with higher stoichiometry was detected in human serum. Consistent with these biophysical characterizations, a reporter assay showed that adalimumab and infliximab, but not etanercept, exerted FcγRIIa- and FcγRIIIa-mediated cell signaling in the presence of TNF and that infliximab exhibited higher potency than adalimumab. This study shows that assessing distribution profiles in serum will contribute to a more comprehensive understanding of the in vivo behavior of therapeutic proteins. PMID:28387583

Complexes prepared from protein A and human serum, IgG, or Fc gamma fragments: characterization by immunochemical analysis of ultracentrifugation fractions and studies on their interconversion.

PubMed

Langone, J J; Das, C; Mainwaring, R; Shearer, W T

1985-01-01

Protein A of Staphylococcus aureus is an Fc receptor for IgG that has been used as a therapeutic reagent to treat cancer in humans and experimental animals. We used ultracentrifugation combined with analysis of isolated fractions by radioimmunoprecipitation and competitive radioimmunoassay with chicken antibodies that bind free protein A or protein A in complexes but do bind free immunoglobulin reagents to localize and characterize the types of complexes formed with different molar ratios of 125I-protein A and human 131I-IgG alone or in serum, and 131I-Fc gamma fragments. This approach offers a distinct advantage over direct counting of radioactivity in the fractions because resolution of complexes and free reagents is much improved. With excess 131I-IgG or 131I-Fc, all the 125I-protein A is present only in complexes that contained 4 molecules of immunoglobulin reagent and 2 molecules of protein A (4:2 complexes), whereas with excess 125I-protein A the stoichiometry of the complexes was 1:1. We have also shown the preformed 4:2 and 1:1 complexes will interconvert in the presence of added excess protein A or IgG, respectively, and that fresh IgG will exchange with IgG or Fc gamma in preformed complexes. Because protein A has been found to elute from an immobilized reagent used in serotherapy of human cancer and is present in a large excess of IgG, the 4:2 complexes may play an active role in the tumoricidal or toxic reactions observed.

Capitalizing Resolving Power of Density Gradient Ultracentrifugation by Freezing and Precisely Slicing Centrifuged Solution: Enabling Identification of Complex Proteins from Mitochondria by Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

PubMed Central

Yu, Haiqing; Lu, Joann J.; Rao, Wei

2016-01-01

Density gradient centrifugation is widely utilized for various high purity sample preparations, and density gradient ultracentrifugation (DGU) is often used for more resolution-demanding purification of organelles and protein complexes. Accurately locating different isopycnic layers and precisely extracting solutions from these layers play a critical role in achieving high-resolution DGU separations. In this technique note, we develop a DGU procedure by freezing the solution rapidly (but gently) after centrifugation to fix the resolved layers and by slicing the frozen solution to fractionate the sample. Because the thickness of each slice can be controlled to be as thin as 10 micrometers, we retain virtually all the resolution produced by DGU. To demonstrate the effectiveness of this method, we fractionate complex V from HeLa mitochondria using a conventional technique and this freezing-slicing (F-S) method. The comparison indicates that our F-S method can reduce complex V layer thicknesses by ~40%. After fractionation, we analyze complex V proteins directly on a matrix assisted laser desorption/ionization, time-of-flight mass spectrometer. Twelve out of fifteen subunits of complex V are positively identified. Our method provides a practical protocol to identify proteins from complexes, which is useful to investigate biomolecular complexes and pathways in various conditions and cell types. PMID:27668122

Distribution of thiobarbituric acid-reactive substances in lipoproteins and proteins in serum.

PubMed

Bonnefont, D; Legrand, A; Peynet, J; Emerit, J; Delattre, J; Galli, A

1989-10-01

We assessed the distribution of malondialdehyde (MDA) in lipoproteins and proteins in serum after using two procedures to separate the lipoproteins: sequential ultracentrifugation or selective precipitation with a sodium phosphotungstate and magnesium chloride reagent followed by ultracentrifugation of the supernate. MDA concentrations were determined by the thiobarbituric acid reaction and quantified by fluorometry. We found that 43% of the thiobarbituric acid-reactive substances (TBARS) was bound to the lipoproteins--27% to very-low- and low-density lipoproteins (VLDL-LDL) and 16% to high-density lipoproteins (HDL)--and from 11.5% to 15.8% to proteins, depending on the separation procedure. Residual unbound TBARS were located in the ultracentrifugation layers that contained no lipoproteins or proteins. The TBARS concentration in serum lipoproteins containing apolipoprotein B (i.e., VLDL-LDL) was the same after ultracentrifugation or selective precipitation. We therefore consider the precipitation method more suitable for routine TBARS determination in these lipoproteins, because it is easier to handle and faster. However, for determination of TBARS in HDL, selective precipitation requires subsequent ultracentrifugation at a density of 1.21 kg/L.

AXOPLASMIC PROTEINS OF THE SQUID GIANT NERVE FIBER WITH PARTICULAR REFERENCE TO THE FIBROUS PROTEIN

PubMed Central

Maxfield, Myles

1953-01-01

1. Axoplasm of squid giant nerve fibers is examined with the ultracentrifuge and electrophoresis apparatus and several distinct components demonstrated. 2. One of these components, a protein called axon filaments, is isolated by fractional extraction followed by differential ultracentrifugation and redissolving in glycine solution. Axon filaments are monodisperse by ultracentrifugation. Their physical chemical properties have been studied. 3. The existence of a reversible transformation of axon filaments into a particle of lower molecular weight and lower asymmetry has been demonstrated. PMID:13109156

Ultracentrifuge for separating fluid mixtures

DOEpatents

Lowry, Ralph A.

1976-01-01

1. A centrifuge for the separation of fluid mixtures having light and heavy fractions comprising a cylindrical rotor, disc type end-plugs closing the ends of the rotor, means for mounting said rotor for rotation about its cylindrical axis, a housing member enclosing the rotor, a vacuum chamber in said housing about the central portion of the rotor, a collection chamber at each end of the housing, the innermost side of which is substantially formed by the outer face of the end-plug, means for preventing flow of the fluid from the collection chambers to said vacuum chamber, at least one of said end-plugs having a plurality of holes therethrough communicating between the collection chamber adjacent thereto and the inside of the rotor to induce countercurrent flow of the fluid in the centrifuge, means for feeding fluid to be processed into the centrifuge, means communicating with the collection chambers to extract the light and heavy separated fractions of the fluid, and means for rotating the rotor.

Simple method provides resolution of albumin, lipoprotein, free fraction, and chylomicron to enhance the utility of protein binding assays.

PubMed

Brockman, Adam H; Oller, Haley R; Moreau, Benoît; Kriksciukaite, Kristina; Bilodeau, Mark T

2015-02-12

Medicinal chemists have been encouraged in recent years to embrace high speed protein binding assays. These methods employ dialysis membranes in 96-well format or spin filters. Membrane-based methods do not separate lipoprotein binding from albumin binding and introduce interference despite membrane binding controls. Ultracentrifugation methods, in contrast, do not introduce interference if density gradients can be avoided and they resolve lipoprotein from albumin. A new generation of compact, fast ultracentrifuges facilitates the rapid and fully informative separation of plasma into albumin, albumin/fatty acid complex, lipoprotein, protein-free, and chylomicron fractions with no need of salt or sugar density gradients. We present a simple and fast ultracentrifuge method here for two platinum compounds and a taxane that otherwise bound irreversibly to dialysis membranes and which exhibited distinctive lipoprotein binding behaviors. This new generation of ultracentrifugation methods underscores a need to further discuss protein binding assessments as they relate to medicinal chemistry efforts.

Lipemia interferences in routine clinical biochemical tests.

PubMed

Calmarza, Pilar; Cordero, José

2011-01-01

Lipemic specimens are a common and frequent, but yet unresolved problem in clinical chemistry, and may produce significant interferences in the analytical results of different biochemical parameters. The aim of this study was to examine the effect of lipid removal using ultracentrifugation of lipemic samples, on some routine biochemistry parameters. Among all the samples obtained daily in our laboratory, the ones which were visibly muddy were selected and underwent to a process of ultracentrifugation, being determined a variety of biochemical tests before and after ultracentrifugation. A total of 110 samples were studied. We found significant differences in all the parameters studied except for total bilirubin, glucose, gamma-glutamyl transferase (GGT) and aspartate aminotransferase (AST). The greatest differences in the parameters analyzed were found in the concentration of alanine aminotransferase (ALT) (7.36%) and the smallest ones in the concentration of glucose (0.014%). Clinically significant interferences were found for phosphorus, creatinine, total protein and calcium. Lipemia causes clinically significant interferences for phosphorus, creatinine, total protein and calcium measurement and those interferences could be effectively removed by ultracentrifugation.

Professor Jesse W. Beams and the first practical magnetic suspension

NASA Technical Reports Server (NTRS)

Allaire, P. E.; Humphris, R. R.; Lewis, D. W.

1992-01-01

Dr. Jesse W. Beams developed the first practical magnetic suspension for high speed rotating devices. The devices included high speed rotating mirrors, ultracentrifuges, and high speed centrifugal field rotors. A brief biography of Dr. Beams is presented, and the following topics are discussed: (1) early axial magnetic suspension for ultracentrifuges; and (2) magnetic suspension for high centrifugal fields.

Virus purification by CsCl density gradient using general centrifugation.

PubMed

Nasukawa, Tadahiro; Uchiyama, Jumpei; Taharaguchi, Satoshi; Ota, Sumire; Ujihara, Takako; Matsuzaki, Shigenobu; Murakami, Hironobu; Mizukami, Keijirou; Sakaguchi, Masahiro

2017-11-01

Virus purification by cesium chloride (CsCl) density gradient, which generally requires an expensive ultracentrifuge, is an essential technique in virology. Here, we optimized virus purification by CsCl density gradient using general centrifugation (40,000 × g, 2 h, 4 °C), which showed almost the same purification ability as conventional CsCl density gradient ultracentrifugation (100,000 × g, 1 h, 4 °C) using phages S13' and φEF24C. Moreover, adenovirus strain JM1/1 was also successfully purified by this method. We suggest that general centrifugation can become a less costly alternative to ultracentrifugation for virus purification by CsCl densiy gradient and will thus encourage research in virology.

Virioplankton 'pegylation': use of PEG (polyethylene glycol) to concentrate and purify viruses in pelagic ecosystems.

PubMed

Colombet, J; Robin, A; Lavie, L; Bettarel, Y; Cauchie, H M; Sime-Ngando, T

2007-12-01

We have described the use of Polyethylene glycol (PEG) for the precipitation of natural communities of aquatic viruses, and its comparison with the usual concentration method based on ultracentrifugation. Experimental samples were obtained from different freshwater ecosystems whose trophic status varied. Based on transmission electron microscope observations and counting of phage-shaped particles, our results showed that the greatest recovery efficiency for all ecosystems was obtained when we used the PEG protocol. On average, this protocol allowed the recovery of >2-fold more viruses, compared to ultracentrifugation. In addition, the diversity of virioplankton, based on genomic size profiling using pulsed field gel electrophoresis, was higher and better discriminated when we used the PEG method. We conclude that pegylation offers a valid, simple and cheaper alternative method to ultracentrifugation, for the concentration and the purification of pelagic viruses.

Reversible dimer formation and stability of the anti-tumour single-chain Fv antibody MFE-23 by neutron scattering, analytical ultracentrifugation, and NMR and FT-IR spectroscopy.

PubMed

Lee, Yie Chia; Boehm, Mark K; Chester, Kerry A; Begent, Richard H J; Perkins, Stephen J

2002-06-28

MFE-23 is a single chain Fv (scFv) antibody molecule used to target colorectal cancer through its high affinity for the tumour marker carcinoembryonic antigen (CEA). ScFv molecules are formed from peptide-linked antibody V(H) and V(L) domains, and many of these form dimers. Our recent crystal structure for MFE-23 showed that this formed an unusual symmetric back-to-back association of two monomers that is consistent with a domain-swapped diabody structure. Neutron scattering and modelling fits showed that MFE-23 existed as compact V(H)-V(L)-linked monomers at therapeutically relevant concentrations below 1 mg/ml. Size-exclusion gel chromatography showed that the monomeric and dimeric forms of MFE-23 could be separated, and that the proportions of these two forms depended on the starting MFE-23 concentration. Sedimentation equilibrium experiments by analytical ultracentrifugation at nine concentrations of MFE-23 indicated a reversible monomer-dimer self-association equilibrium with an association constant of 1.9x10(3)-2.2x10(3) M(-1). Sedimentation velocity experiments using the time derivative g(s(*)) method showed that MFE-23-His has a concentration-dependent weight average sedimentation coefficient that increased from 1.8 S for the monomer to about 3-6 S for the dimer. Both values agreed with those calculated from the MFE-23 crystal structure. In relation to the thermal stability of MFE-23, denaturation experiments by (1)H NMR and FT-IR spectroscopy showed that the molecule is stable up to 47 degrees C, after which denaturation was irreversible. MFE-23 dimerisation is discussed in terms of a new model for diabody structures, in which the V(H) and V(L) domains in the monomer are able to dissociate and reassociate to form a dimer, or diabody, but in which symmetric back-to-back contacts between the two monomers are formed. This dimerisation in solution is attributed to the complementary nature of the C-terminal surface of the MFE-23 monomer. Crystal structures for

Urine Exosome Isolation and Characterization.

PubMed

Street, Jonathan M; Koritzinsky, Erik H; Glispie, Deonna M; Yuen, Peter S T

2017-01-01

Exosomes are nanometer-scale, membrane-enclosed vesicles that can potentially be used to detect nephrotoxicity, and reveal the subsequent response of the kidney. Epithelial cells of every nephron segment can contribute to the urinary exosome population, which is rich in potential biomarkers, including membrane proteins such as transporters and receptors, transcription factors, and microRNAs. These exosomal biomarkers may be up- or downregulated upon nephrotoxicant exposure. Exosome isolation is an area of ongoing research. Although faster and simpler methods have been developed, ultracentrifugation remains a mainstay for purification. A single ultracentrifugation step provides an enriched preparation suitable for biomarker discovery, and a second ultracentrifugation on a sucrose/D 2 O cushion provides the purest exosome preparation currently available and may be preferred for bioactivity assays. The concentration of exosomes can be determined using Nanosight Nanoparticle Tracking Analysis and their contents studied with a variety of approaches including western blots for proteins and RT-qPCR for microRNAs.

The Susceptibilities of Respiratory Syncytial Virus to Nucleolin Receptor Blocking and Antibody Neutralization Are Dependent upon the Method of Virus Purification

PubMed Central

Bilawchuk, Leanne M.; Jensen, Lionel D.; Marchant, David J.

2017-01-01

Respiratory Syncytial Virus (RSV) that is propagated in cell culture is purified from cellular contaminants that can confound experimental results. A number of different purification methods have been described, including methods that utilize fast protein liquid chromatography (FPLC) and gradient ultracentrifugation. Thus, the constituents and experimental responses of RSV stocks purified by ultracentrifugation in sucrose and by FPLC were analyzed and compared by infectivity assay, Coomassie stain, Western blot, mass spectrometry, immuno-transmission electron microscopy (TEM), and ImageStream flow cytometry. The FPLC-purified RSV had more albumin contamination, but there was less evidence of host-derived exosomes when compared to ultracentrifugation-purified RSV as detected by Western blot and mass spectrometry for the exosome markers superoxide dismutase [Cu-Zn] (SOD1) and the tetraspanin CD63. Although the purified virus stocks were equally susceptible to nucleolin-receptor blocking by the DNA aptamer AS1411, the FPLC-purified RSV was significantly less susceptible to anti-RSV polyclonal antibody neutralization; there was 69% inhibition (p = 0.02) of the sucrose ultracentrifugation-purified RSV, 38% inhibition (p = 0.03) of the unpurified RSV, but statistically ineffective neutralization in the FPLC-purified RSV (22% inhibition; p = 0.30). The amount of RSV neutralization of the purified RSV stocks was correlated with anti-RSV antibody occupancy on RSV particles observed by immuno-TEM. RSV purified by different methods alters the stock composition and morphological characteristics of virions that can lead to different experimental responses. PMID:28771197

Effect of exosome isolation methods on physicochemical properties of exosomes and clearance of exosomes from the blood circulation.

PubMed

Yamashita, Takuma; Takahashi, Yuki; Nishikawa, Makiya; Takakura, Yoshinobu

2016-01-01

Exosomes, which are expected to be delivery systems for biomolecules such as nucleic acids, are collected by several methods. However, the effect of exosome isolation methods on the characteristics of exosomes as drug carriers, such as recovery efficiency after sterile filtration and pharmacokinetics, has not been investigated despite the importance of these characteristics for the development of exosome-based delivery systems. In the present study, exosomes collected from murine melanoma B16-BL6 cells by several methods were compared with respect to dispersibility, recovery rate after filtering, and clearance from the blood circulation in mice. The exosomes were collected by three ultracentrifugation-based methods: simple ultracentrifugation/pelleting (pelleting method), ultracentrifugation with an iodixanol cushion (cushion method), and ultracentrifugation on an iodixanol density gradient (gradient method). The isolation methods had little effect on the particle number of exosomes. In contrast, transmission electron microscopy observation and size distribution measurement using tunable resistive pulse sensing indicated that the exosomes of the gradient method were more dispersed than the others. The exosomes were labeled with Gaussia luciferase and intravenously injected into mice. Clearance of injected exosomes from the blood circulation did not significantly change with isolation methods. When the exosomes were filtered using a 0.2-μm filter, the recovery rate was 82% for the exosomes of the gradient method, whereas it was less than 50% for the others. These results indicate that the exosome isolation method markedly affects the dispersibility and filtration efficiency of the exosomes. Copyright © 2015 Elsevier B.V. All rights reserved.

Purification of bacteriophage M13 by anion exchange chromatography.

PubMed

Monjezi, Razieh; Tey, Beng Ti; Sieo, Chin Chin; Tan, Wen Siang

2010-07-01

M13 is a non-lytic filamentous bacteriophage (phage). It has been used widely in phage display technology for displaying foreign peptides, and also for studying macromolecule structures and interactions. Traditionally, this phage has been purified by cesium chloride (CsCl) density gradient ultracentrifugation which is highly laborious and time consuming. In the present study, a simple, rapid and efficient method for the purification of M13 based on anion exchange chromatography was established. A pre-packed SepFast Super Q column connected to a fast protein liquid chromatography (FPLC) system was employed to capture released phages in clarified Escherichia coli fermented broth. An average yield of 74% was obtained from a packed bed mode elution using citrate buffer (pH 4), containing 1.5 M NaCl at 1 ml/min flow rate. The purification process was shortened substantially to less than 2 h from 18 h in the conventional ultracentrifugation method. SDS-PAGE revealed that the purity of particles was comparable to that of CsCl gradient density ultracentrifugation method. Plaque forming assay showed that the purified phages were still infectious. Copyright 2010 Elsevier B.V. All rights reserved.

Comparison of internal process control viruses for detection of food and waterborne viruses.

PubMed

Blanco Fernández, María Dolores; Barrios, Melina Elizabeth; Cammarata, Robertina Viviana; Torres, Carolina; Taboga, Oscar Alberto; Mbayed, Viviana Andrea

2017-05-01

Enteric viruses are pathogens associated with food- and waterborne outbreaks. The recovery of viruses from food or water samples is affected by the procedures applied to detect and concentrate them. The incorporation of an internal process control virus to the analyses allows monitoring the performance of the methodology. The aim of this study was to produce a recombinant adenovirus (rAdV) and apply it together with bacteriophage PP7 as process controls. The rAdV carries a DNA construction in its genome to differentiate it from wild-type adenovirus by qPCR. The stability of both control viruses was evaluated at different pH conditions. The rAdV was stable at pH 3, 7, and 10 for 18 h. PP7 infectious particles were stable at pH 7 and showed a 2.14 log reduction at pH 10 and total decay at pH 3 after 18 h. Three virus concentration methods were evaluated: hollow-fiber tap water ultrafiltration, wastewater ultracentrifugation, and elution-PEG precipitation from lettuce. Total and infectious viruses were quantified and their recoveries were calculated. Virus recovery for rAdV and PP7 by ultrafiltration showed a wide range (2.10-84.42 and 13.54-84.62%, respectively), whereas that by ultracentrifugation was 5.05-13.71 and 6.98-13.27%, respectively. The performance of ultracentrifugation to concentrate norovirus and enteroviruses present in sewage was not significantly different to the recovery of control viruses. For detection of viruses from lettuce, genomic copies of PP7 were significantly more highly recovered than adenovirus (14.74-18.82 and 0.00-3.44%, respectively). The recovery of infectious virus particles was significantly affected during sewage ultracentrifugation and concentration from lettuce. The simultaneous use of virus controls with dissimilar characteristics and behaviors might resemble different enteric viruses.

Enrichment and characterization of ferritin for nanomaterial applications

NASA Astrophysics Data System (ADS)

Ghirlando, Rodolfo; Mutskova, Radina; Schwartz, Chad

2016-01-01

Ferritin is a ubiquitous iron storage protein utilized as a nanomaterial for labeling biomolecules and nanoparticle construction. Commercially available preparations of horse spleen ferritin, widely used as a starting material, contain a distribution of ferritins with different iron loads. We describe a detailed approach to the enrichment of differentially loaded ferritin molecules by common biophysical techniques such as size exclusion chromatography and preparative ultracentrifugation, and characterize these preparations by dynamic light scattering, and analytical ultracentrifugation. We demonstrate a combination of methods to standardize an approach for determining the chemical load of nearly any particle, including nanoparticles and metal colloids. Purification and characterization of iron content in monodisperse ferritin species is particularly critical for several applications in nanomaterial science.

Chromatographic Separation, and Characteristics of Nucleic Acids from HeLa Cells

PubMed Central

Philipson, Lennart

1961-01-01

The application of the phenol-duponol method to extraction of nucleic acids from HeLa cells is described. Chromatography of the phenol extract on an esterified bovine serum albumin column with a salt gradient of sodium chloride gives separation of soluble RNA, DNA, and two different high molecular RNA fractions. Ultracentrifugation of the DNA eluted from the column gives a sedimentation coefficient (s 20 o,w) of 38, which agrees with ultracentrifugation data on the phenol extract. The eluted RNA appears polydisperse at low ionic strength, but at high ionic strength and after alcohol precipitation two fractions with the sedimentation coefficients of 16 and 25 to 29, respectively, were obtained. PMID:13735276

Methods for pore water extraction from unsaturated zone tuff, Yucca Mountain, Nevada

USGS Publications Warehouse

Scofield, K.M.

2006-01-01

Assessing the performance of the proposed high-level radioactive waste repository at Yucca Mountain, Nevada, requires an understanding of the chemistry of the water that moves through the host rock. The uniaxial compression method used to extract pore water from samples of tuffaceous borehole core was successful only for nonwelded tuff. An ultracentrifugation method was adopted to extract pore water from samples of the densely welded tuff of the proposed repository horizon. Tests were performed using both methods to determine the efficiency of pore water extraction and the potential effects on pore water chemistry. Test results indicate that uniaxial compression is most efficient for extracting pore water from nonwelded tuff, while ultracentrifugation is more successful in extracting pore water from densely welded tuff. Pore water splits collected from a single nonwelded tuff core during uniaxial compression tests have shown changes in pore water chemistry with increasing pressure for calcium, chloride, sulfate, and nitrate. Pore water samples collected from the intermediate pressure ranges should prevent the influence of re-dissolved, evaporative salts and the addition of ion-deficient water from clays and zeolites. Chemistry of pore water splits from welded and nonwelded tuffs using ultracentrifugation indicates that there is no substantial fractionation of solutes.

Semi-purification procedures of prions from a prion-infected brain using sucrose has no influence on the nonenzymatic glycation of the disease-associated prion isoform.

PubMed

Choi, Yeong-Gon; Kim, Jae-Il; Choi, Eun-Kyoung; Carp, Richard I; Kim, Yong-Sun

2016-01-01

Previous studies have shown that the Nε-carboxymethyl group is linked to not only one or more N-terminal Lys residues but also to one or more Lys residues of the protease-resistant core region of the pathogenic prion isoform (PrPSc) in prion-infected brains. Using an anti-advanced glycation end product (AGE) antibody, we detected nonenzymatically glycated PrPSc (AGE-PrPSc) in prion-infected brains following concentration by a series of ultracentrifugation steps with a sucrose cushion. In the present study, the levels of in vitro nonenzymatic glycation of PrPSc using sucrose were investigated to determine whether sucrose cushion can artificially and nonenzymatically induce in vitro glycation during ultracentrifugation. The first insoluble pellet fraction following the first ultracentrifugation (PU1st) collected from 263K scrapie-infected brains was incubated with sucrose, glucose or colloidal silica coated with polyvinylpyrrolidone (percoll). None of the compounds in vitro resulted in AGE-PrPSc. Nonetheless, glucose and percoll produced AGEs in vitro from other proteins within PU1st of the infected brains. This reaction could lead to the AGE-modified polymer(s) of nonenzymatic glycation-prone protein(s). This study showed that PrPSc is not nonenzymatically glycated in vitro with sucrose, glucose or percoll and that AGE-modified PrPSc can be isolated and enriched from prion-infected brains.

The Open AUC Project.

PubMed

Cölfen, Helmut; Laue, Thomas M; Wohlleben, Wendel; Schilling, Kristian; Karabudak, Engin; Langhorst, Bradley W; Brookes, Emre; Dubbs, Bruce; Zollars, Dan; Rocco, Mattia; Demeler, Borries

2010-02-01

Progress in analytical ultracentrifugation (AUC) has been hindered by obstructions to hardware innovation and by software incompatibility. In this paper, we announce and outline the Open AUC Project. The goals of the Open AUC Project are to stimulate AUC innovation by improving instrumentation, detectors, acquisition and analysis software, and collaborative tools. These improvements are needed for the next generation of AUC-based research. The Open AUC Project combines on-going work from several different groups. A new base instrument is described, one that is designed from the ground up to be an analytical ultracentrifuge. This machine offers an open architecture, hardware standards, and application programming interfaces for detector developers. All software will use the GNU Public License to assure that intellectual property is available in open source format. The Open AUC strategy facilitates collaborations, encourages sharing, and eliminates the chronic impediments that have plagued AUC innovation for the last 20 years. This ultracentrifuge will be equipped with multiple and interchangeable optical tracks so that state-of-the-art electronics and improved detectors will be available for a variety of optical systems. The instrument will be complemented by a new rotor, enhanced data acquisition and analysis software, as well as collaboration software. Described here are the instrument, the modular software components, and a standardized database that will encourage and ease integration of data analysis and interpretation software.

Low density lipoprotein fraction assay for cardiac disease risk

DOEpatents

Krauss, Ronald M.; Blanche, Patricia J.; Orr, Joseph

1999-01-01

A variable rate density gradient electrophoric gel is described which separate LDL subfractions with the precision of ultracentrifugation techniques. Also, an innovative bottom inlet mixing chamber particularly useful for producing these gels is described.

Low density lipoprotein fraction assay for cardiac disease risk

DOEpatents

Krauss, R.M.; Blanche, P.J.; Orr, J.

1999-07-20

A variable rate density gradient electrophoric gel is described which separates LDL subfractions with the precision of ultracentrifugation techniques. Also, an innovative bottom inlet mixing chamber particularly useful for producing these gels is described. 8 figs.

IDENTIFICATION, ISOLATION AND CHARACTERIZATION OF THE INFECTIOUS HEPATITIS (HEPATITIS A) AGENT

EPA Science Inventory

The research program has the overall objective of combining the techniques of electron microscopy, ultracentrifugation, column chromatography, tissue culture and serology to identify, isolate and characterize the etiologic agent of infectious hepatitis, to propagate it in cell cu...

Physical, Chemical, Bibological, and Biotechnological sciences are incomplete without each other

USDA-ARS?s Scientific Manuscript database

By coupling of mechanics, optics, and mathematics, Theodor Svedberg invented the ultracentrifuge, which allowed separation of important biological materials by high centrifugal force, resulting in physical chemical separation and characterization of atherogenic low density lipoproteins and other bio...

Comparison of ELISA antigen preparations alone or in combination for serodiagnosing Helicobacter pylori infections.

PubMed Central

Hirschl, A M; Rathbone, B J; Wyatt, J I; Berger, J; Rotter, M L

1990-01-01

The immunoglobulin G antibody response to Helicobacter pylori was assessed in 78 patients with non-ulcer dyspepsia using five different antigen preparations. All patients were endoscoped and biopsied. The H pylori state was determined histologically on at least two endoscopic biopsy specimens using a modified Giemsa stain. The ultracentrifuged cell sonicate, acid glycine extract, and 120 kilodalton protein antigens were specific in diagnosing infection (95-98%), but had only moderate sensitivity (70-84%). By mixing either of the two complex antigens with the 120 kilodalton protein, the sensitivity of the test was increased to 97% without affecting the high specificity. The combination of ultracentrifuged sonicate or acid glycine extract with the 120 kilodalton protein therefore seems to be superior to the individual antigen preparations and is particularly suitable for the serodiagnosis of H pylori infection. PMID:2380396

Retrovirus purification: method that conserves envelope glycoprotein and maximizes infectivity.

PubMed Central

McGrath, M; Witte, O; Pincus, T; Weissman, I L

1978-01-01

A Sepharose 4B chromatographic method for purification of retroviruses is described which was less time consuming, increased purified virus yields, conserved viral glycoprotein, and increased recovery of biological infectivity in comparison with conventional sucrose gradient ultracentrifugation techniques. Images PMID:205680

Association of a Model Transmembrane Peptide Containing Gly in a Heptad Sequence Motif

PubMed Central

Lear, James D.; Stouffer, Amanda L.; Gratkowski, Holly; Nanda, Vikas; DeGrado, William F.

2004-01-01

A peptide containing glycine at a and d positions of a heptad motif was synthesized to investigate the possibility that membrane-soluble peptides with a Gly-based, left-handed helical packing motif would associate. Based on analytical ultracentrifugation in C14-betaine detergent micelles, the peptide did associate in a monomer-dimer equilibrium, although the association constant was significantly less than that reported for the right-handed dimer of the glycophorin A transmembrane peptide in similar detergents. Fluorescence resonance energy transfer (FRET) experiments conducted on peptides labeled at their N-termini with either tetramethylrhodamine (TMR) or 7-nitrobenz-2-oxa-1,3-diazole (NBD) also indicated association. However, analysis of the FRET data using the usual assumption of complete quenching for NBD-TMR pairs in the dimer could not be quantitatively reconciled with the analytical ultracentrifugation-measured dimerization constant. This led us to develop a general treatment for the association of helices to either parallel or antiparallel structures of any aggregation state. Applying this treatment to the FRET data, constraining the dimerization constant to be within experimental uncertainty of that measured by analytical ultracentrifugation, we found the data could be well described by a monomer-dimer equilibrium with only partial quenching of the dimer, suggesting that the helices are most probably antiparallel. These results also suggest that a left-handed Gly heptad repeat motif can drive membrane helix association, but the affinity is likely to be less strong than the previously reported right-handed motif described for glycophorin A. PMID:15315956

Solubilization of single-walled carbon nanotubes using a peptide aptamer in water below the critical micelle concentration.

PubMed

Li, Zha; Kameda, Tomoshi; Isoshima, Takashi; Kobatake, Eiry; Tanaka, Takeshi; Ito, Yoshihiro; Kawamoto, Masuki

2015-03-24

The solubilizing ability of single-walled carbon nanotubes (SWCNTs) in water with several dispersants was investigated. Among the dispersants, including low-molecular-weight surfactants, peptides, DNA, and a water-soluble polymer, the peptide aptamer, A2 (IFRLSWGTYFS), exhibited the highest dispersion capability below the critical micelle concentration at a concentration of 0.02 w/v%. The dispersion of supernatant aqueous solution of SWCNTs containing aptamer A2 was essentially unchanged for several months after high-speed ultracentrifugation and gave rise to an efficient and stable dispersion of the SWCNTs in water. From the results of isothermal titration calorimetry and molecular dynamics simulations, the effective binding capability of A2 was due to π-π interaction between aromatic groups in the peptide aptamer and the side walls of SWCNTs. Interestingly, the peptide aptamer showed the possibility of diameter separation of semiconducting SWCNTs using a uniform density gradient ultracentrifuge. These phenomena are encouraging results toward an effective approach to the dispersion and separation of SWCNTs.

Characterization of blood lipoproteins and validation of cholesterol and triacylglycerol assays for free-ranging polar bears (Ursus maritimus).

PubMed

Whiteman, John P; Frank, Nicholas; Greller, Katie A; Harlow, Henry J; Ben-David, Merav

2013-05-01

Blood triacylglycerol (TG) and lipoproteins are important variables for evaluating nutritional status of wildlife, but measurements are often expensive and difficult. Performance of a small, portable blood analyzer intended for human medical diagnostics was evaluated in measuring these variables in plasma and serum from free-ranging polar bears (Ursus maritimus), which are experiencing nutritional stress related to sea ice loss. The analyzer accurately tracked changes in concentration of total cholesterol (Ctotal), cholesterol associated with high-density lipoprotein (CHDL), and TG during a validation protocol of diluting samples and spiking them with exogenous cholesterol and glycerol. Values of Ctotal and TG agreed well with values obtained by other methods (ultracentrifugation followed by colorimetric assays); agreement was variable for values of cholesterol associated with specific lipoproteins. Similar to a study of captive polar bears, ultracentrifugation methods revealed greater TG in very low-density lipoproteins than in low-density lipoprotein, which is unusual and merits additional study.

Crystallization Caught in the Act with Terahertz Spectroscopy: Non-Classical Pathway for l-(+)-Tartaric Acid.

PubMed

Soltani, Amin; Gebauer, Denis; Duschek, Lennart; Fischer, Bernd M; Cölfen, Helmut; Koch, Martin

2017-10-12

Crystal formation is a highly debated problem. This report shows that the crystallization of l-(+)-tartaric acid from water follows a non-classical path involving intermediate hydrated states. Analytical ultracentrifugation indicates solution clusters of the initial stages aggregate to form an early intermediate. Terahertz spectroscopy performed during water evaporation highlights a transient increase in the absorption during nucleation; this indicates the recurrence of water molecules that are expelled from the intermediate phase. Besides, a transient resonance at 750 GHz, which can be assigned to a natural vibration of large hydrated aggregates, vanishes after the final crystal has formed. Furthermore, THz data reveal the vibration of nanosized clusters in the dilute solution indicated by analytical ultracentrifugation. Infrared spectroscopy and wide-angle X-ray scattering highlight that the intermediate is not a crystalline hydrate. These results demonstrate that nanoscopic intermediate units assemble to form the first solvent-free crystalline nuclei upon dehydration. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

DNA stable-isotope probing (DNA-SIP).

PubMed

Dunford, Eric A; Neufeld, Josh D

2010-08-02

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

Excessive centrifugal fields damage high density lipoprotein[S

PubMed Central

Munroe, William H.; Phillips, Martin L.; Schumaker, Verne N.

2015-01-01

HDL is typically isolated ultracentrifugally at 40,000 rpm or greater, however, such high centrifugal forces are responsible for altering the recovered HDL particle. We demonstrate that this damage to HDL begins at approximately 30,000 rpm and the magnitude of loss increases in a rotor speed-dependent manner. The HDL is affected by elevated ultracentrifugal fields resulting in a lower particle density due to the shedding of associated proteins. To circumvent the alteration of the recovered HDL, we utilize a KBr-containing density gradient and a lowered rotor speed of 15,000 rpm to separate the lipoproteins using a single 96 h centrifugation step. This recovers the HDL at two density ranges; the bulk of the material has a density of about 1.115 g/ml, while lessor amounts of material are recovered at >1.2 g/ml. Thus, demonstrating the isolation of intact HDL is possible utilizing lower centrifuge rotor speeds. PMID:25910941

Direct evidence on the existence of [Mo132]Keplerate-type species in aqueous solution.

PubMed

Roy, Soumyajit; Planken, Karel L; Kim, Robbert; Mandele, Dexx v d; Kegel, Willem K

2007-10-15

We demonstrate the existence of discrete single molecular [Mo(132)] Keplerate-type clusters in aqueous solution. Starting from a discrete spherical [Mo(132)] cluster, the formation of an open-basket-type [Mo(116)] defect structure is shown for the first time in solution using analytical ultracentrifugation sedimentation velocity experiments.

Surface Glycosylation Profiles of Urine Extracellular Vesicles

PubMed Central

Gerlach, Jared Q.; Krüger, Anja; Gallogly, Susan; Hanley, Shirley A.; Hogan, Marie C.; Ward, Christopher J.

2013-01-01

Urinary extracellular vesicles (uEVs) are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications. PMID:24069349

Laboratory Aspects of Blood Lipids

NASA Technical Reports Server (NTRS)

Townsend, F. M.

1970-01-01

Classification of blood hyperlipemias by electrophoresis or ultracentrifugation according to density fraction is described and therapeutic measures for humans with hyperlipoproteinemia are outlined. The statistically significant relationship between high serum cholesterol levels and incidence of coronary disease prescribes restricted caloric intake or physical exercise to burn excess calories as preventive measures.

NMR spectroscopic and analytical ultracentrifuge analysis of membrane protein detergent complexes.

PubMed

Maslennikov, Innokentiy; Kefala, Georgia; Johnson, Casey; Riek, Roland; Choe, Senyon; Kwiatkowski, Witek

2007-11-08

Structural studies of integral membrane proteins (IMPs) are hampered by inherent difficulties in their heterologous expression and in the purification of solubilized protein-detergent complexes (PDCs). The choice and concentrations of detergents used in an IMP preparation play a critical role in protein homogeneity and are thus important for successful crystallization. Seeking an effective and standardized means applicable to genomic approaches for the characterization of PDCs, we chose 1D-NMR spectroscopic analysis to monitor the detergent content throughout their purification: protein extraction, detergent exchange, and sample concentration. We demonstrate that a single NMR measurement combined with a SDS-PAGE of a detergent extracted sample provides a useful gauge of the detergent's extraction potential for a given protein. Furthermore, careful monitoring of the detergent content during the process of IMP production allows for a high level of reproducibility. We also show that in many cases a simple sedimentation velocity measurement provides sufficient data to estimate both the oligomeric state and the detergent-to-protein ratio in PDCs, as well as to evaluate the homogeneity of the samples prior to crystallization screening. The techniques presented here facilitate the screening and selection of the extraction detergent, as well as help to maintain reproducibility in the detergent exchange and PDC concentration procedures. Such reproducibility is particularly important for the optimization of initial crystallization conditions, for which multiple purifications are routinely required.

NMR spectroscopic and analytical ultracentrifuge analysis of membrane protein detergent complexes

PubMed Central

Maslennikov, Innokentiy; Kefala, Georgia; Johnson, Casey; Riek, Roland; Choe, Senyon; Kwiatkowski, Witek

2007-01-01

Background Structural studies of integral membrane proteins (IMPs) are hampered by inherent difficulties in their heterologous expression and in the purification of solubilized protein-detergent complexes (PDCs). The choice and concentrations of detergents used in an IMP preparation play a critical role in protein homogeneity and are thus important for successful crystallization. Results Seeking an effective and standardized means applicable to genomic approaches for the characterization of PDCs, we chose 1D-NMR spectroscopic analysis to monitor the detergent content throughout their purification: protein extraction, detergent exchange, and sample concentration. We demonstrate that a single NMR measurement combined with a SDS-PAGE of a detergent extracted sample provides a useful gauge of the detergent's extraction potential for a given protein. Furthermore, careful monitoring of the detergent content during the process of IMP production allows for a high level of reproducibility. We also show that in many cases a simple sedimentation velocity measurement provides sufficient data to estimate both the oligomeric state and the detergent-to-protein ratio in PDCs, as well as to evaluate the homogeneity of the samples prior to crystallization screening. Conclusion The techniques presented here facilitate the screening and selection of the extraction detergent, as well as help to maintain reproducibility in the detergent exchange and PDC concentration procedures. Such reproducibility is particularly important for the optimization of initial crystallization conditions, for which multiple purifications are routinely required. PMID:17988403

Arsenic Mobilization Through Microbial Bioreduction of Ferrihydrite Nanoparticles

NASA Astrophysics Data System (ADS)

Tadanier, C. J.; Roller, J.; Schreiber, M. E.

2004-12-01

Under anaerobic conditions Fe(III)-reducing microorganisms can couple the reduction of solid phase Fe(III) (hydr)oxides with the oxidation of organic carbon. Nutrients and trace metals, such as arsenic, associated with Fe(III) hydroxides may be mobilized through microbially-mediated surface reduction. Although arsenic mobilization has been attributed to mineral surface reduction in a variety of pristine and contaminated environments, minimal information exists on the mechanisms causing this arsenic mobilization. Understanding of the fundamental biochemical and physicochemical processes involved in these mobilization mechanisms is still limited, and has been complicated by the often contradictory and interchangeable terminology used in the literature to describe them. We studied arsenic mobilization mechanisms using a series of controlled microcosm experiments containing aggregated arsenic-bearing ferrihydrite nanoparticles and an Fe(III)-reducing microorganism, Geobacter metallireducens. The phase distribution of iron and arsenic was determined through filtration and ultracentrifugation techniques. Experimental results showed that in the biotic trials, approximately 10 percent of the Fe(III) was reduced to Fe(II) by microbial activity, which remained associated with ferrihydrite surfaces. Biotic activity resulted in changes in nanoparticle surface potential and caused deflocculation of nanoparticle aggregates. Deflocculated nanoparticles were able to pass through a 0.2 micron filter and could only be removed from solution by ultracentrifugation. Arsenic mobilized over time in the biotic trials was found to be exclusively associated with the nanoparticles; 98 percent of arsenic that passed through a 0.2 micron filter was removed from solution by ultracentrifugation. None of these changes were observed in abiotic controls. Because arsenic contamination of natural waters due to mobilization from mineral surfaces is a significant route of human arsenic exposure

Efficient ultrafiltration-based protocol to deplete extracellular vesicles from fetal bovine serum

PubMed Central

Kornilov, Roman; Puhka, Maija; Mannerström, Bettina; Hiidenmaa, Hanna; Peltoniemi, Hilkka; Siljander, Pia; Seppänen-Kaijansinkko, Riitta; Kaur, Sippy

2018-01-01

ABSTRACT Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and, thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell-culture applications. We investigated different EV-depleted FBS prepared by our novel ultrafiltration-based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose-tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 h. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell-culture applications helping to increase comparability of EV research results between laboratories. PMID:29410778

Comparison of guinea-pig serum lipoproteins after iodination by two different methods.

PubMed Central

Weech, P K; McTaggart, F; Mills, G L

1978-01-01

1. Guinea-pig low-density lipoproteins were isolated by ultracentrifugation and iodinated either by the IC1 method or by the chloramine-T procedure. 2. The efficiency of labelling by both methods was essentially the same. 3. When the two products were compared by ultracentrifugation, gel chromatography and immunodiffusion analysis, no significant difference in their properties was detected. 4. When they were compared by gradient-gel electrophoresis, aggregates were found in the product of the IC1 method, but not in the lipoprotein iodinated by the chloramine-T process. 5. Both products were metabolized by the guinea pig with essentially the same fractional catabolic rate. 6. The fractional catabolic rate of lipoprotein iodinated by the chloramine-T method was not significantly different from that of lipoprotein biologically labelled in the protein moiety with [75Se]selenomethionine. 7. It is concluded that the products of both methods of iodination are almost equally acceptable, provided that the optimum conditions for the chloramine-T reaction are carefully established. Images Fig. 4. Fig. 5. PMID:206260

NUTS and BOLTS: Applications of Fluorescence Detected Sedimentation

PubMed Central

Kroe, Rachel R.; Laue, Thomas M.

2008-01-01

Analytical ultracentrifugation is a widely used method for characterizing the solution behavior of macromolecules. However, the two commonly used detectors (absorbance and interference) impose some fundamental restrictions on the concentrations and complexity of the solutions that can be analyzed. The recent addition of a fluorescence detector for the XL-I analytical ultracentrifuge (AU-FDS) enables two different types of sedimentation experiments. First, the AU-FDS can detect picomolar concentrations of labeled solutes allowing the characterization of very dilute solutions of macromolecules, applications we call Normal Use Tracer Sedimentation (NUTS). The great sensitivity of NUTS analysis allows the characterization of small quantities of materials and high affinity interactions. Second, AU-FDS allows characterization of trace quantities of labeled molecules in solutions containing high concentrations and complex mixtures of unlabeled molecules, applications we call Biological On Line Tracer Sedimentation (BOLTS). The discrimination of BOLTS enables the size distribution of a labeled macromolecule to be determined in biological milieu such as cell lysates and serum. Examples are presented that embody features of both NUTS and BOLTS applications, along with our observations on these applications. PMID:19103145

Analysis of Protein Interactions with Picomolar Binding Affinity by Fluorescence-Detected Sedimentation Velocity

PubMed Central

2014-01-01

The study of high-affinity protein interactions with equilibrium dissociation constants (KD) in the picomolar range is of significant interest in many fields, but the characterization of stoichiometry and free energy of such high-affinity binding can be far from trivial. Analytical ultracentrifugation has long been considered a gold standard in the study of protein interactions but is typically applied to systems with micromolar KD. Here we present a new approach for the study of high-affinity interactions using fluorescence detected sedimentation velocity analytical ultracentrifugation (FDS-SV). Taking full advantage of the large data sets in FDS-SV by direct boundary modeling with sedimentation coefficient distributions c(s), we demonstrate detection and hydrodynamic resolution of protein complexes at low picomolar concentrations. We show how this permits the characterization of the antibody–antigen interactions with low picomolar binding constants, 2 orders of magnitude lower than previously achieved. The strongly size-dependent separation and quantitation by concentration, size, and shape of free and complex species in free solution by FDS-SV has significant potential for studying high-affinity multistep and multicomponent protein assemblies. PMID:24552356

ACTIVITY OF DISSOCIATED AND REASSOCIATED 19S ANTI-γ-GLOBULINS

PubMed Central

Schrohenloher, Ralph E.; Kunkel, Henry G.; Tomasi, Thomas B.

1964-01-01

19S anti-γ-globulins were isolated in a high state of purity from the sera of two patients with rheumatoid arthritis. Following reduction with ethyl mercaptan and alkylation by iodoacetamide, fragments were produced which retained the capacity to combine with 7S γ-globulin. The fragments from one of the 19S anti-γ-globulins agglutinated red cells coated with incomplete anti-Rh antibodies. This activity was shown by density gradient ultracentrifugation to be associated with low molecular weight fractions. The agglutination of the coated red cells by the fragments was strongly inhibited by normal and myeloma 7S γ-globulins and showed a greater specificity than the parent 19S material. Analytical ultracentrifuge experiments demonstrated that the fragments from either of the 19S anti-γ-globulins formed complexes with 7S γ-globulin. Reassociation of the dissociated fragments through reformation of disulfide bonds resulted in the formation of fast sedimenting molecules having properties similar to those of the untreated 19S material in respect to precipitation with aggregated γ-globulin and agglutination of coated red cells. PMID:14238936

Nitrogenase of Klebsiella pneumoniae. Interaction of the component proteins studied by ultracentrifugation

PubMed Central

Eady, Robert R.

1973-01-01

Sedimentation-velocity analyses of mixtures of the component proteins of nitrogenase of Klebsiella pneumoniae at a 1:1 molar ratio, showed a single peak of sedimentation coefficient (12.4S) considerably greater than that obtained for the larger (Fe+Mo-containing) protein centrifuged alone (10.4S). When the ratio exceeded 1:1 (the smaller Fe-containing protein in excess) an additional peak corresponding in sedimentation coefficient (about 4.5S) to free Fe-containing protein appeared. When proteins, which had been inactivated by exposure to air were used, no interaction occurred. Na2S2O4 at 2mm both reversed and prevented interaction between the two proteins; sedimentation coefficients corresponded to those of the proteins when centrifuged alone. These results demonstrate the formation of a complex between the nitrogenase proteins, and, together with data of activity titration curves, are consistent with the formulation of the nitrogenase complex of K. pneumoniae as (Fe-containing protein)–(Fe+Mo-containing protein). ImagesFig. 1. PMID:4589392

Characterization of Protein Detergent Complexes by NMR, Light Scattering, and Analytical Ultracentrifugation

PubMed Central

Maslennikov, Innokentiy; Krupa, Martin; Dickson, Christopher; Esquivies, Luis; Blain, Katherine; Kefala, Georgia; Choe, Senyon; Kwiatkowski, Witek

2009-01-01

Bottlenecks in expression, solubilization, purification and crystallization hamper the structural study of integral membrane proteins (IMPs). Successful crystallization is critically dependent on the purity, stability and oligomeric homogeneity of an IMP sample. These characteristics are in turn strongly influenced by the type and concentration of the detergents used in IMP preparation. By utilizing the techniques and analytical tools we earlier developed for the characterization of protein-detergent complexes (PDCs) (Maslennikov et al., 2007), we demonstrate that for successful protein extraction from E. coli membrane fractions, the solubilizing detergent associates preferentially to IMPs rather than to membrane lipids. Notably, this result is contrary to the generally accepted mechanism of detergent-mediated IMP solubilization. We find that for one particular member of the family of proteins studied (E. coli receptor kinases, which is purified in mixed multimeric states and oligomerizes through its transmembrane region), the protein oligomeric composition is largely unaffected by a 10-fold increase in protein concentration, by alteration of micelle properties through addition of other detergents to the PDC sample, or by a 20-fold variation in the detergent concentration used for solubilization of the IMP from the membrane. We observed that the conditions used for expression of the IMP, which impact protein density in the membrane, has the greatest influence on the IMP oligomeric structure. Finally, we argue that for concentrating PDCs smaller than 30 kDa, stirred concentration cells are less prone to over-concentration of detergent and are therefore more effective than centrifugal ultrafiltration devices. PMID:19214777

Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors.

PubMed

Kutner, Robert H; Zhang, Xian-Yang; Reiser, Jakob

2009-01-01

Over the past decade, lentiviral vectors have emerged as powerful tools for transgene delivery. The use of lentiviral vectors has become commonplace and applications in the fields of neuroscience, hematology, developmental biology, stem cell biology and transgenesis are rapidly emerging. Also, lentiviral vectors are at present being explored in the context of human clinical trials. Here we describe improved protocols to generate highly concentrated lentiviral vector pseudotypes involving different envelope glycoproteins. In this protocol, vector stocks are prepared by transient transfection using standard cell culture media or serum-free media. Such stocks are then concentrated by ultracentrifugation and/or ion exchange chromatography, or by precipitation using polyethylene glycol 6000, resulting in vector titers of up to 10(10) transducing units per milliliter and above. We also provide reliable real-time PCR protocols to titrate lentiviral vectors based on proviral DNA copies present in genomic DNA extracted from transduced cells or on vector RNA. These production/concentration methods result in high-titer vector preparations that show reduced toxicity compared with lentiviral vectors produced using standard protocols involving ultracentrifugation-based methods. The vector production and titration protocol described here can be completed within 8 d.

Evidence for involvement of the C-terminal domain in the dimerization of the CopY repressor protein from Enterococcus hirae

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

Pazehoski, Kristina O., E-mail: pazehosk@pitt.edu; Cobine, Paul A., E-mail: pac0006@auburn.edu; Winzor, Donald J.

2011-03-11

Research highlights: {yields} A metal-binding protein domain is directly involved in protein dimerization. {yields} Fusing the metal-binding domain to a monomeric protein induces dimerization. {yields} Frontal size-exclusion chromatography measures the strength of dimer interaction. {yields} Ultracentrifugation studies confirm the influence of metal binding on dimerization. -- Abstract: Metal binding to the C-terminal region of the copper-responsive repressor protein CopY is responsible for homodimerization and the regulation of the copper homeostasis pathway in Enterococcus hirae. Specific involvement of the 38 C-terminal residues of CopY in dimerization is indicated by zonal and frontal (large zone) size-exclusion chromatography studies. The studies demonstrate thatmore » the attachment of these CopY residues to the immunoglobulin-binding domain of streptococcal protein G (GB1) promotes dimerization of the monomeric protein. Although sensitivity of dimerization to removal of metal from the fusion protein is smaller than that found for CopY (as measured by ultracentrifugation studies), the demonstration that an unrelated protein (GB1) can be induced to dimerize by extending its sequence with the C-terminal portion of CopY confirms the involvement of this region in CopY homodimerization.« less

Studies on the "Aerobic" Acetyl-Coenzyme A Synthetase of Saccharomyces Cerevisiae: Purification, Crystallization, and Physical Properties of the Enzyme

NASA Technical Reports Server (NTRS)

Satyanarayana, T.; Klein, Harold P.

1976-01-01

A procedure for the purification of a stable acetyl-coenzyme A synthetase (ACS) from aerobic cells of Saccharomyces cerevisiae is presented. The steps include differential centrifugation, solubilization of the bound enzyme from the crude mitochondrial fraction, ammonium sulfate fractionation, crystallization to constant specific activity from ammonium sulfate solutions followed by Bio-Gel A-1.5 m column chromatography. The resulting enzyme preparation is homogeneous as judged by chromatography on Bio-Gel columns, QAE-Sephadex A-50 anion exchange columns, analytical ultracentrifugal studies, and polyacrylamide gel electrophoresis. Sedimentation velocity runs revealed a single symmetric peak with an s(sub (20,w)) value of 10.6. The molecular weight of the native enzyme, as determined by gel filtration and analytical ultracentrifugation, is 250,000 +/- 500. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the molecular weight of the single polypeptide chain is 83,000 +/- 500. The purified enzyme is inhibited by palmityl-coenzyme A with a Hill interaction coefficient, n, of 2.88. These studies indicate that the ACS of aerobic Saccharomyces cerevisiae is composed of three subunits of identical or nearly identical size.

Acquisition of Scientific Equipment

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

Noland, Lynn

2014-05-16

Whitworth University constructed a 63,00 sq. ft. biology and chemistry building which opened in the Fall of 2011. This project provided for new state-of-the-art science instrumentation enabling Whitworth students to develop skills and knowledge that are directly transferable to practical applications thus enhancing Whitworth student's ability to compete and perform in the scientific workforce. Additionally, STEM faculty undertake outreach programs in the area schools, bringing students to our campus to engage in activities with our science students. The ability to work with insturmentation that is current helps to make science exciting for middle school and high school students and getsmore » them thinking about careers in science. 14 items were purchased following the university's purchasing policy, that benefit instruction and research in the departments of biology, chemistry, and health sciences. They are: Cadaver Dissection Tables with Exhaust Chamber and accessories, Research Microscope with DF DIC, Phase and Fluorescence illumination with DP72 Camera, Microscope with Fluorescence, Microcomputer controlled ultracentrifuge, Ultracentrifuge rotor, Variable Temperature steam pressure sterilizer, Alliance APLC System, DNA Speedvac, Gel Cocumentation System, BioPac MP150, Glovebox personal workstation,Lyophilizer, Nano Drop 2000/2000c Spectrophotometer, C02 Incubator.« less

Protein Biosynthesis and Maturation in the ER.

PubMed

Pedrazzini, Emanuela; Vitale, Alessandro

2018-01-01

The endoplasmic reticulum takes care of the folding, assembly, and quality control of thousands of proteins destined to the different compartments of the endomembrane system, or to be secreted in the apoplast. Here we describe how these early events in the life of all these proteins can be followed biochemically by using velocity or isopycnic ultracentrifugation, metabolic labeling with radioactive amino acids, and immunoprecipitation in various conditions.

Search for Hepatitis A Viruses by New Methods

DTIC Science & Technology

1981-09-01

VII. Detection of HAV and Rotavirus In a Community Water Supply Following an Outbreak of Gastroenteritls and Infectious Hepatitis . . . 1* VUL...22). An "aliquot of each concentrate was further concentrated by ultracentrifugation to assay for HAV antigen and rotavirus . Samples were assayed for... rotavirus using an Indirect "immunofluorescence test (23) and for HAV antigen using a radloirmunoassay (24, 25). Ř. Table 9 shows the concentrations

Progress Report for the First Year (1 January-31 December 1992) on Contract N00014-93-C-0019 (Hawaii Biotechnology Group, Inc.)

DTIC Science & Technology

1994-02-10

resulting from ultracentrifugation reduced the hydrophobicity of the solution, thus allowing improved precipitation of the casein which exists as micelles ...clarification of the whole ferret milk (supplied by Dr. Olgerts Pavlovskis of the Naval Medical Research Institute) for the removal of lipids and casein ...acetic acid until a final pH of 4.6 was achieved. Acidification of he solution precipitated casein (pi=4.6) which was then removed by centrifugation

Model function to calculate the refractive index of native hemoglobin in the wavelength range of 250-1100 nm dependent on concentration.

PubMed

Friebel, Moritz; Meinke, Martina

2006-04-20

The real part of the complex refractive index of oxygenated native hemoglobin solutions dependent on concentration was determined in the wavelength range 250 to 1100 nm by Fresnel reflectance measurements. The hemoglobin solution was produced by physical hemolysis of human erythrocytes followed by ultracentrifugation and filtration. A model function is presented for calculating the refractive index of hemoglobin solutions depending on concentration in the wavelength range 250 to 1100 nm.

Characterisation and Next-generation Sequencing Analysis of Unknown Arboviruses

DTIC Science & Technology

2012-09-01

on the development of real- time PCR detection assays for Vibrio cholerae, a water-borne bacterium responsible for severe enteric disease. From...specific sequence [22]. The length of time from harvesting virus to generating samples that are ready for sequencing takes about two weeks, which is a...two viruses, and on day 4 post infection significant and widespread cytopathic effect was observed. The viruses were harvested by ultracentrifugation

A simplified method to recover urinary vesicles for clinical applications, and sample banking.

PubMed

Musante, Luca; Tataruch, Dorota; Gu, Dongfeng; Benito-Martin, Alberto; Calzaferri, Giulio; Aherne, Sinead; Holthofer, Harry

2014-12-23

Urinary extracellular vesicles provide a novel source for valuable biomarkers for kidney and urogenital diseases: Current isolation protocols include laborious, sequential centrifugation steps which hampers their widespread research and clinical use. Furthermore, large individual urine sample volumes or sizable target cohorts are to be processed (e.g. for biobanking), the storage capacity is an additional problem. Thus, alternative methods are necessary to overcome such limitations. We have developed a practical vesicle isolation technique to yield easily manageable sample volumes in an exceptionally cost efficient way to facilitate their full utilization in less privileged environments and maximize the benefit of biobanking. Urinary vesicles were isolated by hydrostatic dialysis with minimal interference of soluble proteins or vesicle loss. Large volumes of urine were concentrated up to 1/100 of original volume and the dialysis step allowed equalization of urine physico-chemical characteristics. Vesicle fractions were found suitable to any applications, including RNA analysis. In the yield, our hydrostatic filtration dialysis system outperforms the conventional ultracentrifugation-based methods and the labour intensive and potentially hazardous step of ultracentrifugations are eliminated. Likewise, the need for trained laboratory personnel and heavy initial investment is avoided. Thus, our method qualifies as a method for laboratories working with urinary vesicles and biobanking.

Comparison of FTIR-ATR and Raman spectroscopy in determination of VLDL triglycerides in blood serum with PLS regression

NASA Astrophysics Data System (ADS)

Oleszko, Adam; Hartwich, Jadwiga; Wójtowicz, Anna; Gąsior-Głogowska, Marlena; Huras, Hubert; Komorowska, Małgorzata

2017-08-01

Hypertriglyceridemia, related with triglyceride (TG) in plasma above 1.7 mmol/L is one of the cardiovascular risk factors. Very low density lipoproteins (VLDL) are the main TG carriers. Despite being time consuming, demanding well-qualified staff and expensive instrumentation, ultracentrifugation technique still remains the gold standard for the VLDL isolation. Therefore faster and simpler method of VLDL-TG determination is needed. Vibrational spectroscopy, including FT-IR and Raman, is widely used technique in lipid and protein research. The aim of this study was assessment of Raman and FT-IR spectroscopy in determination of VLDL-TG directly in serum with the isolation step omitted. TG concentration in serum and in ultracentrifugated VLDL fractions from 32 patients were measured with reference colorimetric method. FT-IR and Raman spectra of VLDL and serum samples were acquired. Partial least square (PLS) regression was used for calibration and leave-one-out cross validation. Our results confirmed possibility of reagent-free determination of VLDL-TG directly in serum with both Raman and FT-IR spectroscopy. Quantitative VLDL testing by FT-IR and/or Raman spectroscopy applied directly to maternal serum seems to be promising screening test to identify women with increased risk of adverse pregnancy outcomes and patient friendly method of choice based on ease of performance, accuracy and efficiency.

A Simplified Method to Recover Urinary Vesicles for Clinical Applications, and Sample Banking

PubMed Central

Musante, Luca; Tataruch, Dorota; Gu, Dongfeng; Benito-Martin, Alberto; Calzaferri, Giulio; Aherne, Sinead; Holthofer, Harry

2014-01-01

Urinary extracellular vesicles provide a novel source for valuable biomarkers for kidney and urogenital diseases: Current isolation protocols include laborious, sequential centrifugation steps which hampers their widespread research and clinical use. Furthermore, large individual urine sample volumes or sizable target cohorts are to be processed (e.g. for biobanking), the storage capacity is an additional problem. Thus, alternative methods are necessary to overcome such limitations. We have developed a practical vesicle isolation technique to yield easily manageable sample volumes in an exceptionally cost efficient way to facilitate their full utilization in less privileged environments and maximize the benefit of biobanking. Urinary vesicles were isolated by hydrostatic dialysis with minimal interference of soluble proteins or vesicle loss. Large volumes of urine were concentrated up to 1/100 of original volume and the dialysis step allowed equalization of urine physico-chemical characteristics. Vesicle fractions were found suitable to any applications, including RNA analysis. In the yield, our hydrostatic filtration dialysis system outperforms the conventional ultracentrifugation-based methods and the labour intensive and potentially hazardous step of ultracentrifugations are eliminated. Likewise, the need for trained laboratory personnel and heavy initial investment is avoided. Thus, our method qualifies as a method for laboratories working with urinary vesicles and biobanking. PMID:25532487

Halophilic viruses with varying biochemical and biophysical properties are amenable to purification with asymmetrical flow field-flow fractionation.

PubMed

Eskelin, Katri; Lampi, Mirka; Meier, Florian; Moldenhauer, Evelin; Bamford, Dennis H; Oksanen, Hanna M

2017-11-01

Viruses come in various shapes and sizes, and a number of viruses originate from extremities, e.g. high salinity or elevated temperature. One challenge for studying extreme viruses is to find efficient purification conditions where viruses maintain their infectivity. Asymmetrical flow field-flow fractionation (AF4) is a gentle native chromatography-like technique for size-based separation. It does not have solid stationary phase and the mobile phase composition is readily adjustable according to the sample needs. Due to the high separation power of specimens up to 50 µm, AF4 is suitable for virus purification. Here, we applied AF4 for extremophilic viruses representing four morphotypes: lemon-shaped, tailed and tailless icosahedral, as well as pleomorphic enveloped. AF4 was applied to input samples of different purity: crude supernatants of infected cultures, polyethylene glycol-precipitated viruses and viruses purified by ultracentrifugation. All four virus morphotypes were successfully purified by AF4. AF4 purification of culture supernatants or polyethylene glycol-precipitated viruses yielded high recoveries, and the purities were comparable to those obtained by the multistep ultracentrifugation purification methods. In addition, we also demonstrate that AF4 is a rapid monitoring tool for virus production in slowly growing host cells living in extreme conditions.

Quaternary re-arrangement analysed by spectral enhancement: the interaction of a sporulation repressor with its antagonist.

PubMed

Scott, D J; Leejeerajumnean, S; Brannigan, J A; Lewis, R J; Wilkinson, A J; Hoggett, J G

1999-11-12

The protein/protein interaction between SinI and SinR has been studied by analytical ultracentrifugation and gel electrophoresis in an attempt to understand how these proteins contribute to developmental control of sporulation in Bacillus subtilis. SinR was found to be tetrameric, while SinI was found to exist as monomers and dimers in a rapidly reversible equilibrium. Labelling of SinR by incorporating the tryptophan analogue 7-azatryptophan (7AW) into the protein in place of tryptophan shifts the UV absorbance spectrum, thus allowing selective monitoring of 7AWSinR at 315 nm using the UV absorption optics of the analytical ultracentrifuge. Selective monitoring of SinR in mixtures of SinR and SinI enables the binding and stoichiometry of the interaction to be investigated quantitatively and unambiguously. We demonstrate that the oligomeric forms of SinR and SinI re-arrange to form a tight 1:1 SinR:SinI complex, with no stable intermediate species. A fragment of SinR, SinR(1-69), which contains only the DNA-binding domain, was found to be monomeric, showing that the protein appears not to oligomerise in a similar manner to the Cro repressor, a protein with which it shares a marked structural similarity. Copyright 1999 Academic Press.

The in vitro interactions between serum lipoproteins and proteoglycans of the neointima of rabbit aorta after a single balloon catheter injury.

PubMed

Alavi, M Z; Richardson, M; Moore, S

1989-02-01

The effect of injury-induced alterations in the aortic neointimal proteoglycans on their binding with homologous serum lipoproteins was examined. Proteoglycans of the aortic intimal-medial tissues of rabbits that had undergone denudation with a balloon catheter 12 weeks earlier were isolated after homogenization of the tissues in 0.33 M sucrose, ultracentrifugation and subsequently by gel-exclusion chromatography. Lipoproteins from the plasma of healthy donors were prepared by sequential, ultracentrifugal floatation after density adjustment with KBr. To study the interactions, aliquots of electrophoretically pure very low-density lipoproteins (VLDL, d less than 1.006 g/ml), low-density lipoproteins (LDL, d = 1.019-1.063 g/ml), or high-density lipoproteins (HDL, d = 1.210 g/ml) were incubated with proteoglycans in the presence of Ca++ and Mg++ at 4 C. The amount of cholesterol found in the resulting pellet was measured as a marker of the binding capacity of the proteoglycans. Among lipoprotein fractions both VLDL and LDL showed strong binding with proteoglycans, whereas no appreciable binding was observed when incubation experiments were done with HDL. There were significant differences in the lipoprotein binding capacity of proteoglycan of control and injured animals, indicating that injury induced changes in proteoglycan composition exert profound influences on their ionic interactions.

Exosome-like vesicles in uterine aspirates: a comparison of ultracentrifugation-based isolation protocols.

PubMed

Campoy, Irene; Lanau, Lucia; Altadill, Tatiana; Sequeiros, Tamara; Cabrera, Silvia; Cubo-Abert, Montserrat; Pérez-Benavente, Assumpción; Garcia, Angel; Borrós, Salvador; Santamaria, Anna; Ponce, Jordi; Matias-Guiu, Xavier; Reventós, Jaume; Gil-Moreno, Antonio; Rigau, Marina; Colas, Eva

2016-06-18

Uterine aspirates are used in the diagnostic process of endometrial disorders, yet further applications could emerge if its complex milieu was simplified. Exosome-like vesicles isolated from uterine aspirates could become an attractive source of biomarkers, but there is a need to standardize isolation protocols. The objective of the study was to determine whether exosome-like vesicles exist in the fluid fraction of uterine aspirates and to compare protocols for their isolation, characterization, and analysis. We collected uterine aspirates from 39 pre-menopausal women suffering from benign gynecological diseases. The fluid fraction of 27 of those aspirates were pooled and split into equal volumes to evaluate three differential centrifugation-based procedures: (1) a standard protocol, (2) a filtration protocol, and (3) a sucrose cushion protocol. Characterization of isolated vesicles was assessed by electron microscopy, nanoparticle tracking analysis and immunoblot. Specifically for RNA material, we evaluate the effect of sonication and RNase A treatment at different steps of the protocol. We finally confirmed the efficiency of the selected methods in non-pooled samples. All protocols were useful to isolate exosome-like vesicles. However, the Standard procedure was the best performing protocol to isolate exosome-like vesicles from uterine aspirates: nanoparticle tracking analysis revealed a higher concentration of vesicles with a mode of 135 ± 5 nm, and immunoblot showed a higher expression of exosome-related markers (CD9, CD63, and CD81) thus verifying an enrichment in this type of vesicles. RNA contained in exosome-like vesicles was successfully extracted with no sonication treatment and exogenous nucleic acids digestion with RNaseA, allowing the analysis of the specific inner cargo by Real-Time qPCR. We confirmed the existence of exosome-like vesicles in the fluid fraction of uterine aspirates. They were successfully isolated by differential centrifugation giving sufficient proteomic and transcriptomic material for further analyses. The Standard protocol was the best performing procedure since the other two tested protocols did not ameliorate neither yield nor purity of exosome-like vesicles. This study contributes to establishing the basis for future comparative studies to foster the field of biomarker research in gynecology.

Surface plasmon resonance biosensing of the monomer and the linked dimer of the variants of protein G under mass transport limitation.

PubMed

Imamura, Hiroshi; Honda, Shinya

2016-12-01

This article presented the data related to the research article entitled "Calibration-free concentration analysis for an analyte prone to self-association" (H. Imamura, S. Honda, 2017) [1]. The data included surface plasmon resonance (SPR) responses of the variants of protein G with different masses under mass transport limitation. The friction factors of the proteins analyzed by an ultracentrifugation were recorded. Calculation of the SPR response of the proteins was also described.

Genetic Analysis of a Novel Human Adenovirus With a Serologically Unique Hexon and a Recombinant Fiber Gene

DTIC Science & Technology

2011-09-07

diarrhea and was subsequently hospitalized. Cryptosporidium parvum and Giardia lamblia were also found in the fecal matter of the patient; therefore, the... bovine serum (FBS), penicillin (200 U/ml), L-glutamine (2 mM), Fungizone (1 mg/ml), and streptomycin (200 mg/ml). HAdV-D58 was investigated serologi... particles were separated from Hep-2 cells by ultracentrifugation. Genomic DNA was acquired from viral particles using AccuPrep Genomic DNA Extraction Kit

Radiolabeled Exosomes for the Early Detection of Metastases and to Predict Breast Cancer Premetastatic Niche

DTIC Science & Technology

2014-08-01

purification methods such as ultra-centrifugation at 100,000g for 2-3 hours and/or gel filtration on PD-10 columns. Specific Aim 2.2. Iodine - 131 ...with I- 131 was performed 6 5 using the Iodogen method and PIERCE® pre-coated iodination tubes. Na131I of high radiochemical purity was supplied by...hydroxyphenylpropionic acid that serves as free I- 131 scavenger. Unbound I- 131 was removed by size exclusion chromatography on PD-10 columns pre

Induction of cell-mediated cytotoxicity by lipoprotein containing histocompatibility antigens.

PubMed Central

Dennert, G

1979-01-01

Lipoprotein was isolated from tumour cells by sonication and ultracentrifugal flotation on KBr gradients. It contained H-2 antigen detectable by antibody binding and induced a primary or secondary cell-mediated cytotoxic response in vitro which was H-2 specific. In a syngeneic model only a secondary cell-mediated response was stimulated and no competitive inhibition of the effector step of cell-mediated lysis could be demonstrated. The implications of these findings are discussed. PMID:521060

[A preparative method for isolating the synaptonemal complexes from mammalian spermatocytes].

PubMed

Dadashev, S Ia; Bogdanov, Iu F; Gorach, G G; Kolomiets, O L; Karpova, O I

1993-01-01

A method of isolation of synaptonemal complexes (SC) from mouse, rat and Syrian hamster spermatocytes is described. A fraction of pachytene spermatocyte nuclei was obtained by centrifugation of the testis homogenate in stepwise sucrose gradient and then lysed. The resulting chromatine was hydrolysed with DNAse II, and a fraction of isolated SCs was obtained by ultracentrifugation of the hydrolysate. The method can be applied for obtaining the SC fraction from spermatocytes sufficient for cytological, biochemical and molecular biology studies.

Isolation of mitochondria from Saccharomyces cerevisiae using magnetic bead affinity purification

PubMed Central

Liao, Pin-Chao; Boldogh, Istvan R.; Siegmund, Stephanie E.

2018-01-01

Isolated mitochondria are widely used to study the function of the organelle. Typically, mitochondria are prepared using differential centrifugation alone or in conjunction with density gradient ultracentrifugation. However, mitochondria isolated using differential centrifugation contain membrane or organelle contaminants, and further purification of crude mitochondria by density gradient ultracentrifugation requires large amounts of starting material, and is time-consuming. Mitochondria have also been isolated by irreversible binding to antibody-coated magnetic beads. We developed a method to prepare mitochondria from budding yeast that overcomes many of the limitations of other methods. Mitochondria are tagged by insertion of 6 histidines (6xHis) into the TOM70 (Translocase of outer membrane 70) gene at its chromosomal locus, isolated using Ni-NTA (nickel (II) nitrilotriacetic acid) paramagnetic beads and released from the magnetic beads by washing with imidazole. Mitochondria prepared using this method contain fewer contaminants, and are similar in ultrastructure as well as protein import and cytochrome c oxidase complex activity compared to mitochondria isolated by differential centrifugation. Moreover, this isolation method is amenable to small samples, faster than purification by differential and density gradient centrifugation, and more cost-effective than purification using antibody-coated magnetic beads. Importantly, this method can be applied to any cell type where the genetic modification can be introduced by CRISPR or other methods. PMID:29698455

Hydrodynamics of the VanA-type VanS histidine kinase: an extended solution conformation and first evidence for interactions with vancomycin

PubMed Central

Phillips-Jones, Mary K.; Channell, Guy; Kelsall, Claire J.; Hughes, Charlotte S.; Ashcroft, Alison E.; Patching, Simon G.; Dinu, Vlad; Gillis, Richard B.; Adams, Gary G.; Harding, Stephen E.

2017-01-01

VanA-type resistance to glycopeptide antibiotics in clinical enterococci is regulated by the VanSARA two-component signal transduction system. The nature of the molecular ligand that is recognised by the VanSA sensory component has not hitherto been identified. Here we employ purified, intact and active VanSA membrane protein (henceforth referred to as VanS) in analytical ultracentrifugation experiments to study VanS oligomeric state and conformation in the absence and presence of vancomycin. A combination of sedimentation velocity and sedimentation equilibrium in the analytical ultracentrifuge (SEDFIT, SEDFIT-MSTAR and MULTISIG analysis) showed that VanS in the absence of the ligand is almost entirely monomeric (molar mass M = 45.7 kDa) in dilute aqueous solution with a trace amount of high molar mass material (M ~ 200 kDa). The sedimentation coefficient s suggests the monomer adopts an extended conformation in aqueous solution with an equivalent aspect ratio of ~(12 ± 2). In the presence of vancomycin over a 33% increase in the sedimentation coefficient is observed with the appearance of additional higher s components, demonstrating an interaction, an observation consistent with our circular dichroism measurements. The two possible causes of this increase in s – either a ligand induced dimerization and/or compaction of the monomer are considered. PMID:28397853

Determination of bioaccessibility of beta-carotene in vegetables by in vitro methods.

PubMed

Veda, Supriya; Kamath, Akshaya; Platel, Kalpana; Begum, Khyrunnisa; Srinivasan, Krishnapura

2006-11-01

The in vitro method in use for the determination of beta-carotene bioaccessibility involves simulated gastrointestinal digestion followed by ultracentrifugation to separate the micellar fraction containing bioaccessible beta-carotene and its quantitation. In this study, the suitability of two alternatives viz., membrane filtration and equilibrium dialysis were examined to separate the micellar fraction. Values of beta-carotene bioaccessibility obtained with the membrane filtration method were similar to those obtained by the ultracentrifugation method. Equilibrium dialysis was found not suitable for this purpose. Among the vegetables analyzed, fenugreek leaves had the highest content of beta-carotene (9.15 mg/100 g), followed by amaranth (8.17 mg/100 g), carrot (8.14 mg/100 g) and pumpkin (1.90 mg/100 g). Percent bioaccessibility of beta-carotene ranged from 6.7 in fenugreek leaves to 20.3 in carrot. Heat treatment of these vegetables by pressure cooking and stir-frying had a beneficial influence on the bioaccessibility of beta-carotene from these vegetables. The increase in the percent bioaccessibility of beta-carotene as a result of pressure-cooking was 100, 48 and 19% for fenugreek leaves, amaranth and carrot, respectively. Stir-frying in presence of a small quantity of oil led to an enormous increase in the bioaccessibility of beta-carotene from these vegetables, the increase being 263% (fenugreek leaves), 192% (amaranth leaves), 63% (carrot) and 53% (pumpkin).

Direct isolation of labeled low density lipoproteins for the determination of cholesteryl ester transfer protein activity.

PubMed

Sich, D; Saïdi, Y; Egloff, M; Giral, P; Gautier, V; Federspiel, M C; Turpin, G; Beucler, I

1997-10-31

The measurement of the activity of cholesteryl ester transfer protein (CETP), is of high clinical interest and this study reports the use of a direct LDL isolation (d-LDL) technique to determine in one step the amount of radiolabeled cholesteryls esters ([3H]-CE) transferred from exogenous HDL3 to LDL, avoiding the conveniences of the usually used ultracentrifugation or precipitation of apo-B containing lipoproteins in the CETP methodologies. The d-LDL technique providing a specific immunoprecipitation of VLDL, IDL and HDL allowed to directly determine the [3H]-CE transferred on LDL (d-[3H]-CE-LDL). Two methodologies were assayed for the CETP activity using either exogenous or endogenous lipoproteins, and the results with the d-LDL technique were compared with those obtained using the ultracentrifugation (u-[3H]-CE-LDL) considered as the reference method. The intra- and inter-assays were similar in both techniques for the two CETP activity assays. Strong positive correlations were established between values obtained with d-[3H]-CE-LDL and u-[3H]-CE-LDL isolation procedures for CETP activities with exogenous or endogenous lipoproteins (r = 0.972; p = 0.0001 and r = 0.965; p = 0.0001 respectively). In conclusion, the d-LDL technique represents an easy and accurate procedure to measure directly, in normotriglyceridemic plasmas, the amount of [3H]-CE transferred from HDL to LDL by the CETP.

NP-PAH Interaction Dataset

EPA Pesticide Factsheets

Dataset presents concentrations of organic pollutants, such as polyaromatic hydrocarbon compounds, in water samples. Water samples of known volume and concentration were allowed to equilibrate with known mass of nanoparticles. The mixture was then ultracentrifuged and sampled for analysis. This dataset is associated with the following publication:Sahle-Demessie, E., A. Zhao, C. Han, B. Hann, and H. Grecsek. Interaction of engineered nanomaterials with hydrophobic organic pollutants.. Journal of Nanotechnology. Hindawi Publishing Corporation, New York, NY, USA, 27(28): 284003, (2016).

Elution of macrophage-bound immunoglobulins by temperature changes in vitro

PubMed Central

Tizard, I. R.

1972-01-01

Some mouse anti-sheep-erythrocyte macrophage cytophilic antibodies are more readily adsorbed to cells at 4° than at 37°. By treating cells with serum at 4°, washing them well and eluting bound material at 30°, it was found possible to isolate cytophilic immunoglobulins. These were found to have fast γ-mobility, but were heterogeneous upon preparative ultracentrifugation and gel-filtration. The macrophage eluate contained relatively high titres of anti-sheep-erythrocyte cytophilic antibodies. ImagesFIG. 1 PMID:5013911

Sub-micron particles in northwest Atlantic shelf water

NASA Astrophysics Data System (ADS)

Longhurst, A. R.; Koike, I.; Li, W. K. W.; Rodriguez, J.; Dickie, P.; Kepay, P.; Partensky, F.; Bautista, B.; Ruiz, J.; Wells, M.; Bird, D. F.

1992-01-01

The existence of numerous (1.0 × 10 7 ml -1) sub-micron particles has been confirmed in northwest Atlantic shelf water. These particles were counted independently by two different resistive-pulse instruments, and their existence confirmed by our ability to reduce their numbers by ultracentrifugation, serial dilution and surface coagulation in a bubbling column. There are important implications for the dynamics of DOM in seawater if, as seems probable, these particles represent a fraction of "dissolved" organic material in seawater.

Improving the Thermal, Radial and Temporal Accuracy of the Analytical Ultracentrifuge through External References

PubMed Central

Ghirlando, Rodolfo; Balbo, Andrea; Piszczek, Grzegorz; Brown, Patrick H.; Lewis, Marc S.; Brautigam, Chad A.; Schuck, Peter; Zhao, Huaying

2013-01-01

Sedimentation velocity (SV) is a method based on first-principles that provides a precise hydrodynamic characterization of macromolecules in solution. Due to recent improvements in data analysis, the accuracy of experimental SV data emerges as a limiting factor in its interpretation. Our goal was to unravel the sources of experimental error and develop improved calibration procedures. We implemented the use of a Thermochron iButton® temperature logger to directly measure the temperature of a spinning rotor, and detected deviations that can translate into an error of as much as 10% in the sedimentation coefficient. We further designed a precision mask with equidistant markers to correct for instrumental errors in the radial calibration, which were observed to span a range of 8.6%. The need for an independent time calibration emerged with use of the current data acquisition software (Zhao et al., doi 10.1016/j.ab.2013.02.011) and we now show that smaller but significant time errors of up to 2% also occur with earlier versions. After application of these calibration corrections, the sedimentation coefficients obtained from eleven instruments displayed a significantly reduced standard deviation of ∼ 0.7 %. This study demonstrates the need for external calibration procedures and regular control experiments with a sedimentation coefficient standard. PMID:23711724

Improving the thermal, radial, and temporal accuracy of the analytical ultracentrifuge through external references.

PubMed

Ghirlando, Rodolfo; Balbo, Andrea; Piszczek, Grzegorz; Brown, Patrick H; Lewis, Marc S; Brautigam, Chad A; Schuck, Peter; Zhao, Huaying

2013-09-01

Sedimentation velocity (SV) is a method based on first principles that provides a precise hydrodynamic characterization of macromolecules in solution. Due to recent improvements in data analysis, the accuracy of experimental SV data emerges as a limiting factor in its interpretation. Our goal was to unravel the sources of experimental error and develop improved calibration procedures. We implemented the use of a Thermochron iButton temperature logger to directly measure the temperature of a spinning rotor and detected deviations that can translate into an error of as much as 10% in the sedimentation coefficient. We further designed a precision mask with equidistant markers to correct for instrumental errors in the radial calibration that were observed to span a range of 8.6%. The need for an independent time calibration emerged with use of the current data acquisition software (Zhao et al., Anal. Biochem., 437 (2013) 104-108), and we now show that smaller but significant time errors of up to 2% also occur with earlier versions. After application of these calibration corrections, the sedimentation coefficients obtained from 11 instruments displayed a significantly reduced standard deviation of approximately 0.7%. This study demonstrates the need for external calibration procedures and regular control experiments with a sedimentation coefficient standard. Published by Elsevier Inc.

Trimerization Dictates Solution Opalescence of a Monoclonal Antibody.

PubMed

Yang, Teng-Chieh; Langford, Alex Jacob; Kumar, Sandeep; Ruesch, John Carl; Wang, Wei

2016-08-01

Opalescence, sometimes observed in antibody solutions, is thought to be mediated by light scattering of soluble oligomers or insoluble particulates. However, mechanistic features, such as stoichiometry and self-association affinity of oligomeric species related to opalescence, are poorly understood. Here, opalescence behavior of a monoclonal antibody (mAb-1) solution was studied over a wide range of solution conditions including different protein concentrations, pH, and in the presence or absence of salt. Hydrodynamic and thermodynamic properties of mAb-1 solutions were studied by analytical ultracentrifugation and dynamic light scattering. Opalescence in mAb-1 solutions is pH and concentration dependent. The degree of opalescence correlates with reversible monomer-trimer equilibrium detected by analytical ultracentrifugation. Increased trimer formation corresponds to increased opalescence in mAb-1 solutions at higher pH and protein concentrations. Addition of NaCl shifts this equilibrium toward monomer and reduces solution opalescence. This study demonstrates that opalescence in mAb-1 solutions does not arise from the light scattering of monomer or random molecular self-associations but is strongly correlated with a specific self-association stoichiometry and affinity. Importantly, at pH 5.5 (far below isoelectric point of mAb-1), the solution is not opalescent and with nonideal behavior. This study also dissects several parameters to describe the hydrodynamic and thermodynamic nonideality. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Dissociation of glucocerebrosidase dimer in solution by its co-factor, saposin C

DOE PAGES

Gruschus, James M.; Jiang, Zhiping; Yap, Thai Leong; ...

2015-01-16

Mutations in the gene for the lysosomal enzyme glucocerebrosidase (GCase) cause Gaucher disease and are the most common risk factor for Parkinson disease (PD). Analytical ultracentrifugation of 8 μM GCase shows equilibrium between monomer and dimer forms. However, in the presence of its co-factor saposin C (Sap C), only monomer GCase is seen. Isothermal calorimetry confirms that Sap C associates with GCase in solution in a 1:1 complex (K d = 2.1 ± 1.1 μM). Saturation cross-transfer NMR determined that the region of Sap C contacting GCase includes residues 63–66 and 74–76, which is distinct from the region known tomore » enhance GCase activity. Because α-synuclein (α-syn), a protein closely associated with PD etiology, competes with Sap C for GCase binding, its interaction with GCase was also measured by ultracentrifugation and saturation cross-transfer. Unlike Sap C, binding of α-syn to GCase does not affect multimerization. However, adding α-syn reduces saturation cross-transfer from Sap C to GCase, confirming displacement. To explore where Sap C might disrupt multimeric GCase, GCase x-ray structures were analyzed using the program PISA, which predicted stable dimer and tetramer forms. In conclusion, for the most frequently predicted multimer interface, the GCase active sites are partially buried, suggesting that Sap C might disrupt the multimer by binding near the active site.« less

Mechanistic Implications of the Unique Structural Features and Dimerization of the Cytoplasmic Domain of the Pseudomonas Sigma Regulator, PupR

DOE PAGES

Jensen, Jaime L.; Balbo, Andrea; Neau, David B.; ...

2015-09-29

Gram-negative bacteria tightly regulate intracellular levels of iron, an essential nutrient. To ensure this tight regulation, some outer membrane TonB-dependent transporters (TBDTs) that are responsible for iron import stimulate their own transcription in response to extracellular binding by an iron-laden siderophore. This process is mediated by an inner membrane sigma regulator protein (an anti-sigma factor) that transduces an unknown periplasmic signal from the TBDT to release an intracellular sigma factor from the inner membrane, which ultimately upregulates TBDT transcription. Here we use the Pseudomonas putida ferric-pseudobactin BN7/BN8 sigma regulator, PupR, as a model system to understand the molecular mechanism ofmore » this conserved class of sigma regulators. We have determined the X-ray crystal structure of the cytoplasmic anti-sigma domain (ASD) of PupR to 2.0 Å. Size exclusion chromatography, small angle X-ray scattering, and sedimentation velocity analytical ultracentrifugation, all indicate that in contrast to other ASDs, the PupR-ASD exists as a dimer in solution. Mutagenesis of residues at the dimer interface identified from the crystal structure disrupts dimerization and protein stability, as determined by sedimentation velocity analytical ultracentrifugation and thermal denaturation circular dichroism spectroscopy. Lastly, these combined results suggest that this type of inner membrane sigma regulator may utilize an unusual mechanism to sequester their cognate sigma factors and prevent transcription activation.« less

Both Reversible Self-Association and Structural Changes Underpin Molecular Viscoelasticity of mAb Solutions.

PubMed

Sarangapani, Prasad S; Weaver, Justin; Parupudi, Arun; Besong, Tabot M D; Adams, Gary G; Harding, Stephen E; Manikwar, Prakash; Castellanos, Maria M; Bishop, Steven M; Pathak, Jai A

2016-12-01

The role of antibody structure (conformation) in solution rheology is probed. It is demonstrated here that pH-dependent changes in the tertiary structure of 2 mAb solutions lead to viscoelasticity and not merely a shear viscosity (η) increase. Steady shear flow curves on mAb solutions are reported over broad pH (3.0 ≤ pH ≤ 8.7) and concentration (2 mg/mL ≤ c ≤ 120 mg/mL) ranges to comprehensively characterize their rheology. Results are interpreted using size exclusion chromatography, differential scanning calorimetry, analytical ultracentrifugation, near-UV circular dichroism, and dynamic light scattering. Changes in tertiary structure with concentration lead to elastic yield stress and increased solution viscosity in solution of "mAb1." These findings are supported by dynamic light scattering and differential scanning calorimetry, which show increased hydrodynamic radius of mAb1 at low pH and a reduced melting temperature T m , respectively. Conversely, another molecule at 120 mg/mL solution concentration is a strong viscoelastic gel due to perturbed tertiary structure (seen in circular dichroism) at pH 3.0, but the same molecule responds as a viscous liquid due to reversible self-association at pH 7.4 (verified by analytical ultracentrifugation). Both protein-protein interactions and structural perturbations govern pH-dependent viscoelasticity of mAb solutions. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Mechanistic Implications of the Unique Structural Features and Dimerization of the Cytoplasmic Domain of the Pseudomonas Sigma Regulator, PupR

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

Jensen, Jaime L.; Balbo, Andrea; Neau, David B.

Gram-negative bacteria tightly regulate intracellular levels of iron, an essential nutrient. To ensure this tight regulation, some outer membrane TonB-dependent transporters (TBDTs) that are responsible for iron import stimulate their own transcription in response to extracellular binding by an iron-laden siderophore. This process is mediated by an inner membrane sigma regulator protein (an anti-sigma factor) that transduces an unknown periplasmic signal from the TBDT to release an intracellular sigma factor from the inner membrane, which ultimately upregulates TBDT transcription. Here we use the Pseudomonas putida ferric-pseudobactin BN7/BN8 sigma regulator, PupR, as a model system to understand the molecular mechanism ofmore » this conserved class of sigma regulators. We have determined the X-ray crystal structure of the cytoplasmic anti-sigma domain (ASD) of PupR to 2.0 Å. Size exclusion chromatography, small angle X-ray scattering, and sedimentation velocity analytical ultracentrifugation, all indicate that in contrast to other ASDs, the PupR-ASD exists as a dimer in solution. Mutagenesis of residues at the dimer interface identified from the crystal structure disrupts dimerization and protein stability, as determined by sedimentation velocity analytical ultracentrifugation and thermal denaturation circular dichroism spectroscopy. Lastly, these combined results suggest that this type of inner membrane sigma regulator may utilize an unusual mechanism to sequester their cognate sigma factors and prevent transcription activation.« less

Cooperativity and specificity of association of a designed transmembrane peptide.

PubMed Central

Gratkowski, Holly; Dai, Qing-Hong; Wand, A Joshua; DeGrado, William F; Lear, James D

2002-01-01

Thermodynamics studies aimed at quantitatively characterizing free energy effects of amino acid substitutions are not restricted to two state systems, but do require knowing the number of states involved in the equilibrium under consideration. Using analytical ultracentrifugation and NMR methods, we show here that a membrane-soluble peptide, MS1, designed by modifying the sequence of the water-soluble coiled-coil GCN4-P1, exhibits a reversible monomer-dimer-trimer association in detergent micelles with a greater degree of cooperativity in C14-betaine than in dodecyl phosphocholine detergents. PMID:12202385

Label-Free Quantitation of Ribosomal Proteins from Bacillus subtilis for Antibiotic Research.

PubMed

Schäkermann, Sina; Prochnow, Pascal; Bandow, Julia E

2017-01-01

Current research is focusing on ribosome heterogeneity as a response to changing environmental conditions and stresses, such as antibiotic stress. Altered stoichiometry and composition of ribosomal proteins as well as association of additional protein factors are mechanisms for shaping the protein expression profile or hibernating ribosomes. Here, we present a method for the isolation of ribosomes to analyze antibiotic-induced changes in the composition of ribosomes in Bacillus subtilis or other bacteria. Ribosomes and associated proteins are isolated by ultracentrifugation and proteins are identified and quantified using label-free mass spectrometry.

Analysis of Extracellular Vesicles in the Tumor Microenvironment.

PubMed

Al-Nedawi, Khalid; Read, Jolene

2016-01-01

Extracellular vesicles (ECV) are membrane compartments shed from all types of cells in various physiological and pathological states. In recent years, ECV have gained an increasing interest from the scientific community for their role as an intercellular communicator that plays important roles in modifying the tumor microenvironment. Multiple techniques have been established to collect ECV from conditioned media of cell culture or physiological fluids. The gold standard methodology is differential centrifugation. Although alternative techniques exist to collect ECV, these techniques have not proven suitable as a substitution for the ultracentrifugation procedure.

[Analysis and Control of in Vivo Kinetics of Exosomes for the Development of Exosome-based DDS].

PubMed

Takahashi, Yuki; Nishikawa, Makiya; Takakura, Yoshinobu

2016-01-01

  Exosomes are secretory membrane vesicles containing lipids, proteins, and nucleic acids. They act as intercellular transporters by delivering their components to exosome recipient cells. Based on their endogenous delivery system properties, exosomes are expected to become drug delivery systems (DDS) for various molecules such as nucleic acid-based drugs. Important factors such as drug loading to exosomes, production, and pharmacokinetics of exosomes need to be considered for the development of exosome-based DDS. Of these, the pharmacokinetics of exosomes have rarely been studied, probably because of the lack of quantitative evaluation methods of in vivo exosomal pharmacokinetics. We selected lactadherin as an exosome tropic protein and developed it as a fusion protein with Gaussia luciferase to label exosomes for in vivo imaging. In addition, a fusion protein of lactadherin and streptavidin was developed, and the tissue distribution of exosomes was quantitatively evaluated by radiolabeling the exosomes using (125)I-labeled biotin. Using labeled exosomes, we found that intravenously injected exosomes were rapidly cleared from the systemic circulation by macrophages. In addition, the exosomes were mainly distributed to the liver, lung, and spleen. We also examined the effect of exosome isolation methods on their physicochemical and pharmacokinetic properties. We found that exosomes collected by the ultracentrifugation-based density-gradient method were more dispersed than exosomes collected by other methods, including the ultracentrifugation-based pelleting method. The gradient method is more time-consuming than others; therefore the development of a more efficient method for exosome isolation will advance the development of exosome-based DDS.

The coxsackievirus-adenovirus receptor reveals complex homophilic and heterophilic interactions on neural cells.

PubMed

Patzke, Christopher; Max, Klaas E A; Behlke, Joachim; Schreiber, Jadwiga; Schmidt, Hannes; Dorner, Armin A; Kröger, Stephan; Henning, Mechthild; Otto, Albrecht; Heinemann, Udo; Rathjen, Fritz G

2010-02-24

The coxsackievirus-adenovirus receptor (CAR) is a member of the Ig superfamily strongly expressed in the developing nervous system. Our histological investigations during development reveal an initial uniform distribution of CAR on all neural cells with a concentration on membranes that face the margins of the nervous system (e.g., the basal laminae and the ventricular side). At more advanced stages, CAR becomes downregulated and restricted to specific regions including areas rich in axonal and dendritic surfaces. To study the function of CAR on neural cells, we used the fiber knob of the adenovirus, extracellular CAR domains, blocking antibodies to CAR, as well as CAR-deficient neural cells. Blocking antibodies were found to inhibit neurite extension in retina organ and retinal explant cultures, whereas the application of the recombinant fiber knob of the adenovirus subtype Ad2 or extracellular CAR domains promoted neurite extension and adhesion to extracellular matrices. We observed a promiscuous interaction of CAR with extracellular matrix glycoproteins, which was deduced from analytical ultracentrifugation experiments, affinity chromatography, and adhesion assays. The membrane proximal Ig domain of CAR, termed D2, was found to bind to a fibronectin fragment, including the heparin-binding domain 2, which promotes neurite extension of wild type, but not of CAR-deficient neural cells. In contrast to heterophilic interactions, homophilic association of CAR involves both Ig domains, as was revealed by ultracentrifugation, chemical cross-linking, and adhesion studies. The results of these functional and binding studies are correlated to a U-shaped homodimer of the complete extracellular domains of CAR detected by x-ray crystallography.

Structure of the mouse glucocorticoid receptor: rapid analysis by size-exclusion high-performance liquid chromatography.

PubMed

LaPointe, M C; Chang, C H; Vedeckis, W V

1986-04-22

Gel-exclusion high-performance liquid chromatography (HPLC) has been used to separate the untransformed from the transformed glucocorticoid receptor (GC-R) extracted from mouse AtT-20 cells. With 200 mM potassium phosphate as the eluent, an efficient separation of the forms of the GC-R is attained in 15-20 min. The untransformed cytosolic GC-R elutes from the column with a Stokes radius (Rs) of 8.2-8.6 nm, as do the molybdate-stabilized GC-R, the purified untransformed GC-R, and the cross-linked cytosolic GC-R. GC-R transformed in vitro by either ammonium sulfate precipitation, KCl treatment, or G-25 chromatography elutes with an Rs of 5.7-6 nm. Also, GC-R extracted from the nucleus with either 0.3 M KCl or 2 mM sodium tungstate, or purified by two cycles of DNA-cellulose chromatography, has an Rs of 5.5-6.3 nm. The data are identical either in the presence or in the absence of 20 mM Na2MoO4, suggesting that molybdate is not causing aggregation to produce a larger Rs value than that of the native receptor. Vertical tube rotor sucrose gradient ultracentrifugation of cytosol produces three forms of the GC-R: 9.1 S, 5.2 S, and 3.8 S. Sequential analysis of the GC-R forms by HPLC and vertical tube rotor ultracentrifugation and vice versa allows for the hydrodynamic determination of molecular weight within a very short time period (2-3 h total).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of leucine zipper complexes by electrospray ionization mass spectrometry.

PubMed Central

Wendt, H.; Dürr, E.; Thomas, R. M.; Przybylski, M.; Bosshard, H. R.

1995-01-01

The development of "soft" ionization methods has enabled the mass spectrometric analysis of higher-order structural features of proteins. We have applied electrospray ionization mass spectrometry (ESI-MS) to the analysis of the number and composition of polypeptide chains in homomeric and heteromeric leucine zippers. In comparison with other methods that have been used to analyze leucine zippers, such as analytical ultracentrifugation, gel chromatography, or electrophoretic band shift assays, ESI-MS is very fast and highly sensitive and provides a straightforward way to distinguish between homomeric and heteromeric coiled-coil structures. ESI-MS analyses were carried out on the parallel dimeric leucine zipper domain GCN4-p1 of the yeast transcription factor GCN4 and on three synthetic peptides with the sequences Ac-EYEALEKKLAAX1EAKX2QALEKKLEALEHG-amide: peptide LZ (X1, X2 = Leu), peptide LZ(12A) (X1 = Ala, X2 = Leu), and peptide LZ(16N) (X1 = Leu, X2 = Asn). Equilibrium ultracentrifugation analysis showed that LZ forms a trimeric coiled coil and this could be confirmed unequivocally by ESI-MS as could the dimeric nature of GCN4-p1. The formation of heteromeric two- and three-stranded leucine zippers composed of chains from LZ and LZ(12A), or from GCN4-p1 and LZ, was demonstrated by ESI-MS and confirmed by fluorescence quenching experiments on fluorescein-labeled peptides. The results illustrate the adaptability and flexibility of the leucine zipper motif, properties that could be useful to the design of specific protein assemblies by way of coiled-coil domains. PMID:8520482

Superacid-Surfactant Exchange: Enabling Nondestructive Dispersion of Full-Length Carbon Nanotubes in Water.

PubMed

Wang, Peng; Kim, Mijin; Peng, Zhiwei; Sun, Chuan-Fu; Mok, Jasper; Lieberman, Anna; Wang, YuHuang

2017-09-26

Attaining aqueous solutions of individual, long single-walled carbon nanotubes is a critical first step for harnessing the extraordinary properties of these materials. However, the widely used ultrasonication-ultracentrifugation approach and its variants inadvertently cut the nanotubes into short pieces. The process is also time-consuming and difficult to scale. Here we present an unexpectedly simple solution to this decade-old challenge by directly neutralizing a nanotube-chlorosulfonic acid solution in the presence of sodium deoxycholate. This straightforward superacid-surfactant exchange eliminates the need for both ultrasonication and ultracentrifugation altogether, allowing aqueous solutions of individual nanotubes to be prepared within minutes and preserving the full length of the nanotubes. We found that the average length of the processed nanotubes is more than 350% longer than sonicated controls, with a significant fraction approaching ∼9 μm, a length that is limited by only the raw material. The nondestructive nature is manifested by an extremely low density of defects, bright and homogeneous photoluminescence in the near-infrared, and ultrahigh electrical conductivity in transparent thin films (130 Ω/sq at 83% transmittance), which well exceeds that of indium tin oxide. Furthermore, we demonstrate that our method is fully compatible with established techniques for sorting nanotubes by their electronic structures and can also be readily applied to graphene. This surprisingly simple method thus enables nondestructive aqueous solution processing of high-quality carbon nanomaterials at large-scale and low-cost with the potential for a wide range of fundamental studies and applications, including, for example, transparent conductors, near-infrared imaging, and high-performance electronics.

Isolation and Characterization of a Toxic Moiety of Low Molecular Weight from Clostridium botulinum Type A

PubMed Central

Gerwing, Julia; Dolman, Claude E.; Bains, Hardial S.

1965-01-01

Gerwing, Julia (The University of British Columbia, Vancouver, B.C., Canada), Claude E. Dolman, and Hardial S. Bains. Isolation and characterization of a toxic moiety of low molecular weight from Clostridium botulinum type A. J. Bacteriol. 89:1383–1386. 1965.—A toxic moiety of low molecular weight has been isolated from a type A strain of Clostridium botulinum, by a method involving ammonium sulfate precipitation and elution through diethylaminoethyl cellulose at pH 5.6. By means of electrophoresis and ultracentrifugation, the toxic substance was shown to be homogeneous; a molecular weight of 12,200 was calculated. Images PMID:14293025

A gradient-free method for the purification of infective dengue virus for protein-level investigations.

PubMed

Jensen, Stephanie M; Nguyen, Celina T; Jewett, John C

2016-09-01

Dengue virus (DENV) is a mosquito-transmitted flavivirus that infects approximately 100 million people annually. Multi-day protocols for purification of DENV reduce the infective titer due to viral sensitivity to both temperature and pH. Herein we describe a 5-h protocol for the purification of all DENV serotypes, utilizing traditional gradient-free ultracentrifugation followed by selective virion precipitation. This protocol allows for the separation of DENV from contaminating proteins - including intact C6/36 densovirus, for the production of infective virus at high concentration for protein-level analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Sedimentation Coefficient, Frictional Coefficient, and Molecular Weight: A Preparative Ultracentrifuge Experiment for the Advanced Undergraduate Laboratory.

ERIC Educational Resources Information Center

Halsall, H. B.; Wermeling, J. R.

1982-01-01

Describes an experiment using a high-speed preparative centrifuge and calculator to demonstrate effects of the frictional coefficient of a macromolecule on its rate of transport in a force field and to estimate molecular weight of the macromolecule using an empirical relationship. Background information, procedures, and discussion of results are…

Isolation of biologically-active exosomes from human plasma.

PubMed

Muller, Laurent; Hong, Chang-Sook; Stolz, Donna B; Watkins, Simon C; Whiteside, Theresa L

2014-09-01

Effects of exosomes present in human plasma on immune cells have not been examined in detail. Immunological studies with plasma-derived exosomes require their isolation by procedures involving ultracentrifugation. These procedures were largely developed using supernatants of cultured cells. To test biologic activities of plasma-derived exosomes, methods are necessary that ensure adequate recovery of exosome fractions free of contaminating larger vesicles, cell fragments and protein/nucleic acid aggregates. Here, an optimized method for exosome isolation from human plasma/serum specimens of normal controls (NC) or cancer patients and its advantages and pitfalls are described. To remove undesirable plasma-contaminating components, ultrafiltration of differentially-centrifuged plasma/serum followed by size-exclusion chromatography prior to ultracentrifugation facilitated the removal of contaminants. Plasma or serum was equally acceptable as a source of exosomes based on the recovered protein levels (in μg protein/mL plasma) and TEM image quality. Centrifugation on sucrose density gradients led to large exosome losses. Fresh plasma was the best source of morphologically-intact exosomes, while the use of frozen/thawed plasma decreased exosome purity but not their biologic activity. Treatments of frozen plasma with DNAse, RNAse or hyaluronidase did not improve exosome purity and are not recommended. Cancer patients' plasma consistently yielded more isolated exosomes than did NCs' plasma. Cancer patients' exosomes also mediated higher immune suppression as evidenced by decreased CD69 expression on responder CD4+ T effector cells. Thus, the described procedure yields biologically-active, morphologically-intact exosomes that have reasonably good purity without large protein losses and can be used for immunological, biomarker and other studies. Copyright © 2014 Elsevier B.V. All rights reserved.

The effects of near-UV radiation on elasmobranch lens cytoskeletal actin.

PubMed

Zigman, S; Rafferty, N S; Scholz, D L; Lowe, K

1992-08-01

The role of near-UV radiation as a cytoskeletal actin-damaging agent was investigated. Two procedures were used to analyse fresh smooth dogfish (Mustelus canis) eye lenses that were incubated for up to 22 hr in vitro, with elasmobranch Ringer's medium, and with or without exposure to a near-UV lamp (emission principally at 365 nm; irradiance of 2.5 mW cm-2). These were observed histologically using phalloidin-rhodamine specific staining and by transmission electron microscopy. In addition, solutions of purified polymerized rabbit muscle actin were exposed to the same UV conditions and depolymerization was assayed by ultracentrifugation and high-pressure liquid chromatography. While the two actins studied do differ very slightly in some amino acid sequences, they would react physically nearly identically. The results showed that dogfish lenses developed superficial opacities due to near-UV exposure. Whole mounts of lens epithelium exhibited breakdown of actin filaments in the basal region of the cells within 18 hr of UV exposure. TEM confirmed the breakdown of actin filaments due to UV exposure. SDS-PAGE and immunoblotting positively identified actin in these cells. Direct exposure of purified polymerized muscle actin in polymerizing buffer led to an increase in actin monomer of approximately 25% in the UV-exposed solutions within 3-18 hr, whether assayed by ultracentrifugation or HPLC. The above indicates that elasmobranch lens epithelial cells contain UV-labile actin filaments, and that near-UV radiation, as is present in the sunlit environment, can break down the actin structure in these cells. Furthermore, breakdown of purified polymerized muscle actin does occur due to near-UV light exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

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

Prof. P. Somasundaran

The aim of the project is to develop and evaluate efficient novel surfactant mixtures for enhanced oil recovery. Preliminary ultra-filtration tests suggest that two kinds of micelles may exist in binary surfactant mixtures at different concentrations. Due to the important role played in interfacial processes by micelles as determined by their structures, focus of the current work is on the delineation of the relationship between such aggregate structures and chemical compositions of the surfactants. A novel analytical centrifuge application is explored to generate information on structures of different surfactants aggregates. In this report, optical systems, typical output of the analyticalmore » ultracentrifuge results and four basic experiments are discussed. Initial sedimentation velocity investigations were conducted using nonyl phenol ethoxylated decyl ether (NP-10) to choose the best analytical protocol, calculate the partial specific volume and obtain information on sedimentation coefficient, aggregation mass of micelles. The partial specific volume was calculated to be 0.920. Four softwares: Optima{trademark} XL-A/XL-I data analysis software, DCDT+, Svedberg and SEDFIT, were compared for the analysis of sedimentation velocity experimental data. The sedimentation coefficient and aggregation number of NP-10 micelles obtained using the first three softwares at 25 C are 209, 127, and 111, respectively. The last one is closest to the result from Light Scattering. The reason for the differences in numbers obtained using the three softwares is discussed. Based on these tests, Svedberg and SEDFIT analysis are chosen for further studies. This approach using the analytical ultracentrifugation offers an unprecedented opportunity now to obtain important information on mixed micelles and their role in interfacial processes.« less

High-resolution, submicron particle size distribution analysis using gravitational-sweep sedimentation.

PubMed Central

Mächtle, W

1999-01-01

Sedimentation velocity is a powerful tool for the analysis of complex solutions of macromolecules. However, sample turbidity imposes an upper limit to the size of molecular complexes currently amenable to such analysis. Furthermore, the breadth of the particle size distribution, combined with possible variations in the density of different particles, makes it difficult to analyze extremely complex mixtures. These same problems are faced in the polymer industry, where dispersions of latices, pigments, lacquers, and emulsions must be characterized. There is a rich history of methods developed for the polymer industry finding use in the biochemical sciences. Two such methods are presented. These use analytical ultracentrifugation to determine the density and size distributions for submicron-sized particles. Both methods rely on Stokes' equations to estimate particle size and density, whereas turbidity, corrected using Mie's theory, provides the concentration measurement. The first method uses the sedimentation time in dispersion media of different densities to evaluate the particle density and size distribution. This method works provided the sample is chemically homogeneous. The second method splices together data gathered at different sample concentrations, thus permitting the high-resolution determination of the size distribution of particle diameters ranging from 10 to 3000 nm. By increasing the rotor speed exponentially from 0 to 40,000 rpm over a 1-h period, size distributions may be measured for extremely broadly distributed dispersions. Presented here is a short history of particle size distribution analysis using the ultracentrifuge, along with a description of the newest experimental methods. Several applications of the methods are provided that demonstrate the breadth of its utility, including extensions to samples containing nonspherical and chromophoric particles. PMID:9916040

Tissue Factor Coagulant Activity is Regulated by the Plasma Membrane Microenvironment.

PubMed

Yu, Yuanjie; Böing, Anita N; Hau, Chi M; Hajji, Najat; Ruf, Wolfram; Sturk, Auguste; Nieuwland, Rienk

2018-06-01

 Tissue factor (TF) can be present in a non-coagulant and coagulant form. Whether the coagulant activity is affected by the plasma membrane microenvironment is unexplored.  This article studies the presence and coagulant activity of human TF in plasma membrane micro-domains.  Plasma membranes were isolated from human MIA PaCa2 cells, MDA-MB-231 cells and human vascular smooth muscle cells by Percoll gradient ultracentrifugation after cell disruption. Plasma membranes were fractionated by OptiPrep gradient ultracentrifugation, and the presence of TF, flotillin, caveolin, clathrin, protein disulphide isomerase (PDI), TF pathway inhibitor (TFPI) and phosphatidylserine (PS) were determined.  Plasma membranes contain two detergent-resistant membrane (DRM) compartments differing in density and biochemical composition. High-density DRMs (DRM-H) have a density ( ρ ) of 1.15 to 1.20 g/mL and contain clathrin, whereas low-density DRMs (DRM-L) have a density between 1.09 and 1.13 g/mL and do not contain clathrin. Both DRMs contain TF, flotillin and caveolin. PDI is detectable in DRM-H, TFPI is not detectable in either DMR-H or DRM-L and PS is detectable in DRM-L. The DRM-H-associated TF (> 95% of the TF antigen) lacks detectable coagulant activity, whereas the DRM-L-associated TF triggers coagulation. This coagulant activity is inhibited by lactadherin and thus PS-dependent, but seemed insensitive to 16F16, an inhibitor of PDI.  Non-coagulant and coagulant TF are present within different types of DRMs in the plasma membrane, and the composition of these DRMs may affect the TF coagulant activity. Schattauer GmbH Stuttgart.

Extracellular plasma RNA from colon cancer patients is confined in a vesicle-like structure and is mRNA-enriched

PubMed Central

García, José Miguel; García, Vanesa; Peña, Cristina; Domínguez, Gemma; Silva, Javier; Diaz, Raquel; Espinosa, Pablo; Citores, Maria Jesús; Collado, Manuel; Bonilla, Félix

2008-01-01

Little is yet known about the origin and protective mechanism of free nucleic acids in plasma. We investigated the possibility of these free nucleic acids being particle associated. Plasma samples from colon cancer patients and cell culture media were subjected to various antibody incubations, ultracentrifugation, and RNA extraction protocols for total RNA, epithelial RNA, and mRNA. Flow cytometry using a Ber-EP4 antibody and confocal laser microscopy after staining with propidium iodide were also performed. mRNA levels of the LISCH7 and SDHA genes were determined in cells and in culture media. Ber-EP4 antibody and polystyrene beads coated with oligo dT sequences were employed. We observed that, after incubation, total RNA and mRNA were always detected after membrane digestion, and that epithelial RNA was detected before this procedure. In ultracentrifugation, mRNA was caught in the supernatant only if a former lysis mediated or in the pellet if there was no previous digestion. Flow cytometry determinations showed that antibody-coated microbeads keep acellular structures bearing epithelial antigens apart. Confocal laser microscopy made 1- to 2-μm-diameter particles perceptible in the vicinity of magnetic polystyrene beads. Relevant differences were observed between mRNA of cells and culture media, as there was a considerable difference in LISCH7 mRNA levels between HT29 and IMR90 cell co-cultures and their culture media. Our results support the view that extracellular RNA found in plasma from cancer patients circulates in association with or is protected in a multiparticle complex, and that an active release mechanism by tumor cells may be a possible origin. PMID:18456845

P2X1 receptors localized in lipid rafts mediate ATP motor responses in the human vas deferens longitudinal muscles.

PubMed

Donoso, María Verónica; Norambuena, Andrés; Navarrete, Camilo; Poblete, Inés; Velasco, Alfredo; Huidobro-Toro, Juan Pablo

2014-02-01

To assess the role of the P2X1 receptors (P2X1R) in the longitudinal and circular layers of the human vas deferens, ex vivo-isolated strips or rings were prepared from tissue biopsies to record isometric contractions. To ascertain its membrane distribution, tissue extracts were analyzed by immunoblotting following sucrose gradient ultracentrifugation. ATP, alpha,beta-methylene ATP, or electrical field stimulation elicited robust contractions of the longitudinal layer but not of the circular layer which demonstrated inconsistent responses. Alpha,beta-methylene ATP generated stronger and more robust contractions than ATP. In parallel, prostatic segments of the rat vas deferens were examined. The motor responses in both species were not sustained but decayed within the first minute, showing desensitization to additional applications. Cross-desensitization was established between alpha,beta-methylene ATP or ATP-evoked contractions and electrical field stimulation-induced contractions. Full recovery of the desensitized motor responses required more than 30 min and showed a similar pattern in human and rat tissues. Immunoblot analysis of the human vas deferens extracts revealed a P2X1R oligomer of approximately 200 kDa under nonreducing conditions, whereas dithiothreitol-treated extracts showed a single band of approximately 70 kDa. The P2X1R was identified in ultracentrifugation fractions containing 15%-29% sucrose; the receptor localized in the same fractions as flotillin-1, indicating that it regionalized into smooth muscle lipid rafts. In conclusion, ATP plays a key role in human vas deferens contractile responses of the longitudinal smooth muscle layer, an effect mediated through P2X1Rs.

Feline Calicivirus, Murine Norovirus, Porcine Sapovirus, and Tulane Virus Survival on Postharvest Lettuce.

PubMed

Esseili, Malak A; Saif, Linda J; Farkas, Tibor; Wang, Qiuhong

2015-08-01

Human norovirus (HuNoV) is the leading cause of foodborne illnesses, with an increasing number of outbreaks associated with leafy greens. Because HuNoV cannot be routinely cultured, culturable feline calicivirus (FCV), murine norovirus (MNV), porcine sapovirus (SaV), and Tulane virus (TV) have been used as surrogates. These viruses are generated in different cell lines as infected cell lysates, which may differentially affect their stability. Our objective was to uniformly compare the survival of these viruses on postharvest lettuce while evaluating the effects of cell lysates on their survival. Viruses were semipurified from cell lysates by ultrafiltration or ultracentrifugation followed by resuspension in sterile water. Virus survival was examined before and after semipurification: in suspension at room temperature (RT) until day 28 and on lettuce leaves stored at RT for 3 days or at 4°C for 7 and 14 days. In suspension, both methods significantly enhanced the survival of all viruses. On lettuce, the survival of MNV in cell lysates was similar to that in water, under all storage conditions. In contrast, the survival of FCV, SaV, and TV was differentially enhanced, under different storage conditions, by removing cell lysates. Following semipurification, viruses showed similar persistence to each other on lettuce stored under all conditions, with the exception of ultracentrifugation-purified FCV, which showed a higher inactivation rate than MNV at 4°C for 14 days. In conclusion, the presence of cell lysates in viral suspensions underestimated the survivability of these surrogate viruses, while viral semipurification revealed similar survivabilities on postharvest lettuce leaves. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

[Characteristics of exosomes andmicroparticles discovered in human tears].

PubMed

Grigor'eva, A E; Tamkovich, S N; Eremina, A V; Tupikin, A E; Kabilov, M R; Chernykh, V V; Vlassov, V V; Laktionov, P P; Ryabchikova, E I

2016-01-01

Exosomes represent a sort of extracellular vesicles, which transfer molecular signals in organism and possess markers of producing cells. Our study was aimed at search of exosomes in the tears of healthy humans, confirmation of their nature and examination of exosome morphological and molecular-biological characteristics. The tears (110-340 ml) were collected from 24 healthy donors (aged 46-60 years); individual probes were centrifuged at 20000 g for 15 min to pellet cell debris. The supernatants were examined in electron microscope using negative staining; and they were also used for isolation and purification of the exosomes by filtration (100 nm pore-size) and double ultracentrifugation (90 min at 100000 g, 4°C). The "pellets" were subjected to electron microscopy, immunolabeling. The RNA and DNA were isolated from the samples, and their sizes were evaluated by capillary electrophoresis, the concentration and localization of nucleic acids were determined. Sequencing of DNA was performed using MiSeq ("Illumina", USA), data were analyzed using CLC GW 7.5 ("Qiagen", USA). Sequences were mapped on human genome (hg19). Electron microscopy revealed in supernatants of the tears cell debris, spherical microparticles (20-40 nm), membrane vesicles and macromolecular aggregates. The "pellets" obtained after ultracentrifugation, contained microparticles (17%), spherical and cup-shaped EVs (40-100 nm, 83%), which were positive for CD63, CD9 and CD24 receptors (specific markers of exosomes). Our study showed presence of high amount of exosomes in human tears, and relation of the exosomes with RNA (size less than 200 nt) and DNA (size was 3-9 kb). Sequencing of the DNA showed that about 92% of the reads mapped to human genome.

Friend Leukemogenic Virus-neutralizing Antibody from Mouse Ascitic Fluid

PubMed Central

March, R. W.; Chirigos, M. A.; Hook, W. A.; Burka, B. L.

1967-01-01

Ascitic fluid antibody produced in C57/B1 mice immunized with Friend leukemogenic virus exhibited potent neutralizing activity. In vitro neutralization tests revealed that a mean neutralization index of 3.0 was achieved, and it was shown by sucrose gradient ultracentrifugation that this antibody resembled the 7S type. A mean yield of 6.7 ml of fluid per mouse per weekly paracentesis was obtained over an 8-week period. The ascitic fluid antibody to Friend virus was also active in vivo. Mice given antibody 3, 5, 7, and 9 days after infection with Friend virus did not develop the splenomegaly characteristic of Friend disease. PMID:16349755

THE SEDIMENTATION PROPERTIES OF THE SKIN-SENSITIZING ANTIBODIES OF RAGWEED-SENSITIVE PATIENTS

PubMed Central

Andersen, Burton R.; Vannier, Wilton E.

1964-01-01

The sedimentation coefficients of the skin-sensitizing antibodies to ragweed were evaluated by the moving partition cell method and the sucrose density gradient method. The most reliable results were obtained by sucrose density gradient ultracentrifugation which showed that the major portion of skin-sensitizing antibodies to ragweed sediment with an average value of 7.7S (7.4 to 7.9S). This is about one S unit faster than γ-globulins (6.8S). The data from the moving partition cell method are in agreement with these results. Our studies failed to demonstrate heterogeneity of the skin-sensitizing antibodies with regard to sedimentation rate. PMID:14194391

Role of Escherichia coli dnaG function in coliphage M13 DNA synthesis

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

Dasgupta, S.; Mitra, S.

Examination of the role of Escherichia coli dnaG function in different stages of M13 phage DNA synthesis by ultracentrifugal analysis of intracellular phage DNA in a thermosensitive dnaG mutant shows that: (a) the formation of parental double-strand replicative-form DNA (rfDNA) from the infecting virus is independent of dnaG function; (b) the synthesis of progeny rfDNA requires dnaG product; (c) after a pool of rfDNA is made up, dnaG function is not required for the progeny single-strand DNA (ssDNA) synthesis. The ssDNAs produced under nonpermissive condition are mostly circular and biologically functional.

Biochemical and structural studies of fish lymphocystis disease virions isolated from skin tumours of Pleuronectes.

PubMed

Samalecos, C

1986-06-01

Fish lymphocystis disease viruses (FLDV) were isolated directly from lymphocystis disease lesions of various flatfish species and further purified. Subunits could be identified only after the purified virus was disrupted. In combination with different types of treatment, Nonidet-P40, dithiothreitol, proteases digestion and after ultrasonication and ultracentrifugation, the inner region of FLDV was studied. The purified virus was used for isolation of the virus nucleoid and for further study of the viral genome. Contour length measurements of 20 DNA molecules gave an average length of 40.44 +/- 3.2 micron. Lines of precipitation between isolated nucleoid material and FLDV-antibodies were shown by immunoelectrophoresis.

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

P. Somasundaran

The aim of the project is to develop a knowledge base to help the design of enhanced processes for mobilizing and extracting untrapped oil. We emphasize evaluation of novel surfactant mixtures and obtaining optimum combinations of the surfactants for efficient chemical flooding EOR processes. In this regard, an understanding of the aggregate shape, size and structure is crucial since these properties govern the crude oil removal efficiency. During the three-year period, the adsorption and aggregation behavior of sugar-based surfactants and their mixtures with other types of surfactants have been studied. Sugar-based surfactants are made from renewable resources, nontoxic and biodegradable.more » They are miscible with water and oil. These environmentally benign surfactants feature high surface activity, good salinity, calcium and temperature tolerance, and unique adsorption behavior. They possess the characteristics required for oil flooding surfactants and have the potential for replacing currently used surfactants in oil recovery. A novel analytical ultracentrifugation technique has been successfully employed for the first time, to characterize the aggregate species present in mixed micellar solution due to its powerful ability to separate particles based on their size and shape and monitor them simultaneously. Analytical ultracentrifugation offers an unprecedented opportunity to obtain important information on mixed micelles, structure-performance relationship for different surfactant aggregates in solution and their role in interfacial processes. Initial sedimentation velocity investigations were conducted using nonyl phenol ethoxylated decyl ether (NP-10) to choose the best analytical protocol, calculate the partial specific volume and obtain information on sedimentation coefficient, aggregation mass of micelles. Four softwares: OptimaTM XL-A/XL-I data analysis software, DCDT+, Svedberg and SEDFIT, were compared for the analysis of sedimentation velocity

Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

PubMed Central

Surya, Wahyu; Torres, Jaume

2015-01-01

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and under physiological conditions. This makes AUC a method of choice to quantitatively assess stoichiometry and thermodynamics of homo- and hetero-association that are transient and reversible in biochemical processes. In the modality of sedimentation equilibrium (SE), a balance between diffusion and sedimentation provides a profile as a function of radial distance that depends on a specific association model. Herein, a detailed SE protocol is described to determine the size and monomer-monomer association energy of a small membrane protein oligomer using an analytical ultracentrifuge. AUC-ES is label-free, only based on physical principles, and can be used on both water soluble and membrane proteins. An example is shown of the latter, the small hydrophobic (SH) protein in the human respiratory syncytial virus (hRSV), a 65-amino acid polypeptide with a single α-helical transmembrane (TM) domain that forms pentameric ion channels. NMR-based structural data shows that SH protein has two protonatable His residues in its transmembrane domain that are oriented facing the lumen of the channel. SE experiments have been designed to determine how pH affects association constant and the oligomeric size of SH protein. While the pentameric form was preserved in all cases, its association constant was reduced at low pH. These data are in agreement with a similar pH dependency observed for SH channel activity, consistent with a lumenal orientation of the two His residues in SH protein. The latter may experience electrostatic repulsion and reduced oligomer stability at low pH. In summary, this method is applicable whenever quantitative information on subtle protein-protein association changes in physiological conditions have to be measured.   PMID:25867485

Sedimentation equilibrium of a small oligomer-forming membrane protein: effect of histidine protonation on pentameric stability.

PubMed

Surya, Wahyu; Torres, Jaume

2015-04-02

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and under physiological conditions. This makes AUC a method of choice to quantitatively assess stoichiometry and thermodynamics of homo- and hetero-association that are transient and reversible in biochemical processes. In the modality of sedimentation equilibrium (SE), a balance between diffusion and sedimentation provides a profile as a function of radial distance that depends on a specific association model. Herein, a detailed SE protocol is described to determine the size and monomer-monomer association energy of a small membrane protein oligomer using an analytical ultracentrifuge. AUC-ES is label-free, only based on physical principles, and can be used on both water soluble and membrane proteins. An example is shown of the latter, the small hydrophobic (SH) protein in the human respiratory syncytial virus (hRSV), a 65-amino acid polypeptide with a single α-helical transmembrane (TM) domain that forms pentameric ion channels. NMR-based structural data shows that SH protein has two protonatable His residues in its transmembrane domain that are oriented facing the lumen of the channel. SE experiments have been designed to determine how pH affects association constant and the oligomeric size of SH protein. While the pentameric form was preserved in all cases, its association constant was reduced at low pH. These data are in agreement with a similar pH dependency observed for SH channel activity, consistent with a lumenal orientation of the two His residues in SH protein. The latter may experience electrostatic repulsion and reduced oligomer stability at low pH. In summary, this method is applicable whenever quantitative information on subtle protein-protein association changes in physiological conditions have to be measured.

The sedimentation properties of ferritins. New insights and analysis of methods of nanoparticle preparation

PubMed Central

May, Carrie A.; Grady, John K.; Laue, Thomas M.; Poli, Maura; Arosio, Paolo; Chasteen, N. Dennis

2010-01-01

Background Ferritin exhibits complex behavior in the ultracentrifuge due to variability in iron core size among molecules. A comprehensive study was undertaken to develop procedures for obtaining more uniform cores and assessing their homogeneity. Methods Analytical ultracentrifugation was used to measure the mineral core size distributions obtained by adding iron under high- and low-flux conditions to horse spleen (apoHoSF) and human H-chain (apoHuHF) apoferritins. Results More uniform core sizes are obtained with the homopolymer human H-chain ferritin than with the heteropolymer horse spleen HoSF protein in which subpopulations of HoSF molecules with varying iron content are observed. A binomial probability distribution of H- and L-subunits among protein shells qualitatively accounts for the observed subpopulations. The addition of Fe2+ to apoHuHF produces iron core particle size diameters from 3.8 ± 0.3 to 6.2 ± 0.3 nm. Diameters from 3.4 ± 0.6 to 6.5 ± 0.6 nm are obtained with natural HoSF after sucrose gradient fractionation. The change in the sedimentation coefficient as iron accumulates in ferritin suggests that the protein shell contracts ~10% to a more compact structure, a finding consistent with published electron micrographs. The physicochemical parameters for apoHoSF (15%/85% H/L subunits) are M = 484,120 g/mol, ν̄ = 0.735mL/g, s20,w = 17.0 S and D20,W = 3.21 × 10−7 cm2/s; and for apoHuHF M = 506,266 g/mol, ν̄ = 0.724 mL/g, s20,w = 18.3 S and D20,w = 3.18 × 10−7 cm2/s. Significance The methods presented here should prove useful in the synthesis of size controlled nanoparticles of other minerals. PMID:20307627

Nanosized UCMSC-derived extracellular vesicles but not conditioned medium exclusively inhibit the inflammatory response of stimulated T cells: implications for nanomedicine.

PubMed

Monguió-Tortajada, Marta; Roura, Santiago; Gálvez-Montón, Carolina; Pujal, Josep Maria; Aran, Gemma; Sanjurjo, Lucía; Franquesa, Marcel la; Sarrias, Maria-Rosa; Bayes-Genis, Antoni; Borràs, Francesc E

2017-01-01

Undesired immune responses have drastically hampered outcomes after allogeneic organ transplantation and cell therapy, and also lead to inflammatory diseases and autoimmunity. Umbilical cord mesenchymal stem cells (UCMSCs) have powerful regenerative and immunomodulatory potential, and their secreted extracellular vesicles (EVs) are envisaged as a promising natural source of nanoparticles to increase outcomes in organ transplantation and control inflammatory diseases. However, poor EV preparations containing highly-abundant soluble proteins may mask genuine vesicular-associated functions and provide misleading data. Here, we used Size-Exclusion Chromatography (SEC) to successfully isolate EVs from UCMSCs-conditioned medium. These vesicles were defined as positive for CD9, CD63, CD73 and CD90, and their size and morphology characterized by NTA and cryo-EM. Their immunomodulatory potential was determined in polyclonal T cell proliferation assays, analysis of cytokine profiles and in the skewing of monocyte polarization. In sharp contrast to the non-EV containing fractions, to the complete conditioned medium and to ultracentrifuged pellet, SEC-purified EVs from UCMSCs inhibited T cell proliferation, resembling the effect of parental UCMSCs. Moreover, while SEC-EVs did not induce cytokine response, the non-EV fractions, conditioned medium and ultracentrifuged pellet promoted the secretion of pro-inflammatory cytokines by polyclonally stimulated T cells and supported Th17 polarization. In contrast, EVs did not induce monocyte polarization, but the non-EV fraction induced CD163 and CD206 expression and TNF-α production in monocytes. These findings increase the growing evidence confirming that EVs are an active component of MSC's paracrine immunosuppressive function and affirm their potential for therapeutics in nanomedicine. In addition, our results highlight the importance of well-purified and defined preparations of MSC-derived EVs to achieve the immunosuppressive

Analysis of antibody aggregate content at extremely high concentrations using sedimentation velocity with a novel interference optics.

PubMed

Schilling, Kristian; Krause, Frank

2015-01-01

Monoclonal antibodies represent the most important group of protein-based biopharmaceuticals. During formulation, manufacturing, or storage, antibodies may suffer post-translational modifications altering their physical and chemical properties. Such induced conformational changes may lead to the formation of aggregates, which can not only reduce their efficiency but also be immunogenic. Therefore, it is essential to monitor the amount of size variants to ensure consistency and quality of pharmaceutical antibodies. In many cases, antibodies are formulated at very high concentrations > 50 g/L, mostly along with high amounts of sugar-based excipients. As a consequence, all routine aggregation analysis methods, such as size-exclusion chromatography, cannot monitor the size distribution at those original conditions, but only after dilution and usually under completely different solvent conditions. In contrast, sedimentation velocity (SV) allows to analyze samples directly in the product formulation, both with limited sample-matrix interactions and minimal dilution. One prerequisite for the analysis of highly concentrated samples is the detection of steep concentration gradients with sufficient resolution: Commercially available ultracentrifuges are not able to resolve such steep interference profiles. With the development of our Advanced Interference Detection Array (AIDA), it has become possible to register interferograms of solutions as highly concentrated as 150 g/L. The other major difficulty encountered at high protein concentrations is the pronounced non-ideal sedimentation behavior resulting from repulsive intermolecular interactions, for which a comprehensive theoretical modelling has not yet been achieved. Here, we report the first SV analysis of highly concentrated antibodies up to 147 g/L employing the unique AIDA ultracentrifuge. By developing a consistent experimental design and data fit approach, we were able to provide a reliable estimation of the minimum

Reference values assessment in a Mediterranean population for small dense low-density lipoprotein concentration isolated by an optimized precipitation method.

PubMed

Fernández-Cidón, Bárbara; Padró-Miquel, Ariadna; Alía-Ramos, Pedro; Castro-Castro, María José; Fanlo-Maresma, Marta; Dot-Bach, Dolors; Valero-Politi, José; Pintó-Sala, Xavier; Candás-Estébanez, Beatriz

2017-01-01

High serum concentrations of small dense low-density lipoprotein cholesterol (sd-LDL-c) particles are associated with risk of cardiovascular disease (CVD). Their clinical application has been hindered as a consequence of the laborious current method used for their quantification. Optimize a simple and fast precipitation method to isolate sd-LDL particles and establish a reference interval in a Mediterranean population. Forty-five serum samples were collected, and sd-LDL particles were isolated using a modified heparin-Mg 2+ precipitation method. sd-LDL-c concentration was calculated by subtracting high-density lipoprotein cholesterol (HDL-c) from the total cholesterol measured in the supernatant. This method was compared with the reference method (ultracentrifugation). Reference values were estimated according to the Clinical and Laboratory Standards Institute and The International Federation of Clinical Chemistry and Laboratory Medicine recommendations. sd-LDL-c concentration was measured in serums from 79 subjects with no lipid metabolism abnormalities. The Passing-Bablok regression equation is y = 1.52 (0.72 to 1.73) + 0.07 x (-0.1 to 0.13), demonstrating no significant statistical differences between the modified precipitation method and the ultracentrifugation reference method. Similarly, no differences were detected when considering only sd-LDL-c from dyslipidemic patients, since the modifications added to the precipitation method facilitated the proper sedimentation of triglycerides and other lipoproteins. The reference interval for sd-LDL-c concentration estimated in a Mediterranean population was 0.04-0.47 mmol/L. An optimization of the heparin-Mg 2+ precipitation method for sd-LDL particle isolation was performed, and reference intervals were established in a Spanish Mediterranean population. Measured values were equivalent to those obtained with the reference method, assuring its clinical application when tested in both normolipidemic and dyslipidemic

Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography.

PubMed

Blans, Kristine; Hansen, Maria S; Sørensen, Laila V; Hvam, Michael L; Howard, Kenneth A; Möller, Arne; Wiking, Lars; Larsen, Lotte B; Rasmussen, Jan T

2017-01-01

Studies have suggested that nanoscale extracellular vesicles (EV) in human and bovine milk carry immune modulatory properties which could provide beneficial health effects to infants. In order to assess the possible health effects of milk EV, it is essential to use isolates of high purity from other more abundant milk structures with well-documented bioactive properties. Furthermore, gentle isolation procedures are important for reducing the risk of generating vesicle artefacts, particularly when EV subpopulations are investigated. In this study, we present two isolation approaches accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV-marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides and presents a phospholipid profile differing from milk fat globules surrounded by epithelial cell plasma membrane. Moreover, the milk EV fractions are enriched in RNA with distinct and diverging profiles from milk fat globules. Collectively, our data supports that successful milk EV isolation can be accomplished in few steps without the use of ultracentrifugation, as the presented isolation approaches based on SEC effectively isolates EV in both human and bovine milk.

Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography

PubMed Central

Blans, Kristine; Hansen, Maria S.; Sørensen, Laila V.; Hvam, Michael L.; Howard, Kenneth A.; Möller, Arne; Wiking, Lars; Larsen, Lotte B.; Rasmussen, Jan T.

2017-01-01

ABSTRACT Studies have suggested that nanoscale extracellular vesicles (EV) in human and bovine milk carry immune modulatory properties which could provide beneficial health effects to infants. In order to assess the possible health effects of milk EV, it is essential to use isolates of high purity from other more abundant milk structures with well-documented bioactive properties. Furthermore, gentle isolation procedures are important for reducing the risk of generating vesicle artefacts, particularly when EV subpopulations are investigated. In this study, we present two isolation approaches accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV-marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides and presents a phospholipid profile differing from milk fat globules surrounded by epithelial cell plasma membrane. Moreover, the milk EV fractions are enriched in RNA with distinct and diverging profiles from milk fat globules. Collectively, our data supports that successful milk EV isolation can be accomplished in few steps without the use of ultracentrifugation, as the presented isolation approaches based on SEC effectively isolates EV in both human and bovine milk. PMID:28386391

Functionalization of quantum rods with oligonucleotides for programmable assembly with DNA origami

NASA Astrophysics Data System (ADS)

Doane, Tennyson L.; Alam, Rabeka; Maye, Mathew M.

2015-02-01

The DNA-mediated self-assembly of CdSe/CdS quantum rods (QRs) onto DNA origami is described. Two QR types with unique optical emission and high polarization were synthesized, and then functionalized with oligonucleotides (ssDNA) using a novel protection-deprotection approach, which harnessed ssDNA's tailorable rigidity and denaturation temperature to increase DNA coverage by reducing non-specific coordination and wrapping. The QR assembly was programmable, and occurred at two different assembly zones that had capture strands in parallel alignment. QRs with different optical properties were assembled, opening up future studies on orientation dependent QR FRET. The QR-origami conjugates could be purified via gel electrophoresis and sucrose gradient ultracentrifugation. Assembly yields, QR stoichiometry and orientation, as well as energy transfer implications were studied in light of QR distances, origami flexibility, and conditions.The DNA-mediated self-assembly of CdSe/CdS quantum rods (QRs) onto DNA origami is described. Two QR types with unique optical emission and high polarization were synthesized, and then functionalized with oligonucleotides (ssDNA) using a novel protection-deprotection approach, which harnessed ssDNA's tailorable rigidity and denaturation temperature to increase DNA coverage by reducing non-specific coordination and wrapping. The QR assembly was programmable, and occurred at two different assembly zones that had capture strands in parallel alignment. QRs with different optical properties were assembled, opening up future studies on orientation dependent QR FRET. The QR-origami conjugates could be purified via gel electrophoresis and sucrose gradient ultracentrifugation. Assembly yields, QR stoichiometry and orientation, as well as energy transfer implications were studied in light of QR distances, origami flexibility, and conditions. Electronic supplementary information (ESI) available: Experimental conditions, DNA origami blueprint and

Molecular Basis of Differential B-Pentamer Stability of Shiga Toxins 1 and 2

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

Conrady, Deborah G.; Flagler, Michael J.; Friedmann, David R.

2012-06-27

Escherichia coli strain O157:H7 is a major cause of food poisoning that can result in severe diarrhea and, in some cases, renal failure. The pathogenesis of E. coli O157:H7 is in large part due to the production of Shiga toxin (Stx), an AB{sub 5} toxin that consists of a ribosomal RNA-cleaving A-subunit surrounded by a pentamer of receptor-binding B subunits. There are two major isoforms, Stx1 and Stx2, which differ dramatically in potency despite having 57% sequence identity. Animal studies and epidemiological studies show Stx2 is associated with more severe disease. Although the molecular basis of this difference is unknown,more » data suggest it is associated with the B-subunit. Mass spectrometry studies have suggested differential B-pentamer stability between Stx1 and Stx2. We have examined the relative stability of the B-pentamers in solution. Analytical ultracentrifugation using purified B-subunits demonstrates that Stx2B, the more deadly isoform, shows decreased pentamer stability compared to Stx1B (EC{sub 50} = 2.3 {micro}M vs. EC{sub 50} = 0.043 {micro}M for Stx1B). X-ray crystal structures of Stx1B and Stx2B identified a glutamine in Stx2 (versus leucine in Stx1) within the otherwise strongly hydrophobic interface between B-subunits. Interchanging these residues switches the stability phenotype of the B-pentamers of Stx1 and Stx2, as demonstrated by analytical ultracentrifugation and circular dichroism. These studies demonstrate a profound difference in stability of the B-pentamers in Stx1 and Stx2, illustrate the mechanistic basis for this differential stability, and provide novel reagents to test the basis for differential pathogenicity of these toxins.« less

von Willebrand's disease antigen II. A new plasma and platelet antigen deficient in severe von Willebrand's disease.

PubMed Central

Montgomery, R R; Zimmerman, T S

1978-01-01

Factor VIII-related antigen (VIIIag) is deficient in plasma and platelets of patients with severe von Willebrand's disease. This study reports a second von Willebrand's disease antigen (vWagII), distinct from VIIIag, that is also deficient in the platelets and plasma of patients with severe von Willebrand's disease. VIIIag and vWagII are separable by molecular exclusion chromatography, sucrose density gradient ultracentrifugation, and crossed immunoelectrophoresis. They show reactions of immunologic nonidentity with each other, and thus, do not share a precursor-product relationship. vWagII is released from normal platelets during blood clotting, accounting for a fourfold higher concentration of vWagII in serum over plasma. Images PMID:307007

CENTRIFUGES

DOEpatents

Beams, J.W.; Snoddy, L.B.

1960-08-01

Damping bearings for use on the shafts of an ultracentrifuge were designed which are capable of passing through critical angular speeds. The shaft extending from one end of the rotor is journaled in fixed-plain bearings mounted on annular resilient shock-absorbing elements to dampen small vibrations. The shaft at the other end of the rotor is journaled in two damper-bearing assemblies which are so spaced on the shaft that a vibration node can at no time exist at both bearing assemblies. These bearings are similar to the other bearings except that the bearing housings are slidably mounted on the supporting structure for movement transverse to the rotational axis of the rotor.

Ultra-high resolution crystal structure of recombinant caprine β-lactoglobulin.

PubMed

Crowther, Jennifer M; Lassé, Moritz; Suzuki, Hironori; Kessans, Sarah A; Loo, Trevor S; Norris, Gillian E; Hodgkinson, Alison J; Jameson, Geoffrey B; Dobson, Renwick C J

2014-11-03

β-Lactoglobulin (βlg) is the most abundant whey protein in the milks of ruminant animals. While bovine βlg has been subjected to a vast array of studies, little is known about the caprine ortholog. We present an ultra-high resolution crystal structure of caprine βlg complemented by analytical ultracentrifugation and small-angle X-ray scattering data. In both solution and crystalline states caprine βlg is dimeric (K(D)<5 μM); however, our data suggest a flexible quaternary arrangement of subunits within the dimer. These structural findings will provide insight into relationships among structural, processing, nutritional and immunological characteristics that distinguish cow's and goat's milk. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Solvent extraction of oil-sand components for determination of trace elements by neutron activation analysis

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

Jacobs, F.S.; Filby, R.H.

Instrumental neutron activation analysis was used to measure the concentrations of 30 elements in Athabasca oil sands and oil-sand components. The oil sands were separated into solid residue, bitumen, and fines by Soxhlet extraction with toluene-bitumen extract. The mineral content of the extracted bitumen was dependent on the treatment of the oil sand prior to extraction. The geochemically important and organically associated trace element contents of the bitumen (and asphaltenes) were determined by subtracting the mineral contributions from the total measured concentrations. The method allows analysis of the bitumen without the necessity of ultracentrifugation or membrane filtration, which might removemore » geochemically important components of the bitumen. The method permits classification of trace elements into organic and inorganic combinations.« less

Cell-Free Translation of Integral Membrane Proteins into Unilamelar Liposomes

PubMed Central

Goren, Michael A.; Nozawa, Akira; Makino, Shin-ichi; Wrobel, Russell L.; Fox, Brian G.

2018-01-01

Wheat germ cell-free translation is shown to be an effective method to produce integral membrane proteins in the presence of unilamelar liposomes. In this chapter, we describe the expression vectors, preparation of mRNA, two types of cell-free translation reactions performed in the presence of liposomes, a simple and highly efficient purification of intact proteoliposomes using density gradient ultracentrifugation, and some of the types of characterization studies that are facilitated by this facile preparative approach. The in vitro transfer of newly translated, membrane proteins into liposomes compatible with direct measurements of their catalytic function is contrasted with existing approaches to extract membrane proteins from biological membranes using detergents and subsequently transfer them back to liposomes for functional studies. PMID:19892197

Complex of vitamins and antioxidants protects low-density lipoproteins in blood plasma from free radical oxidation and activates antioxidants enzymes in erythrocytes from patients with coronary heart disease.

PubMed

Konovalova, G G; Lankin, V Z; Tikhaze, A K; Nezhdanova, I B; Lisina, M O; Kukharchuk, V V

2003-08-01

We studied the effect of a complex containing antioxidant vitamins C and E, provitamin A, and antioxidant element selenium on the contents of primary (lipid peroxides) and secondary products (malonic dialdehyde) of free radical lipid oxidation in low-density lipoproteins isolated from the plasma of patients with coronary heart disease and hypercholesterolemia by means of preparative ultracentrifugation. Activity of key antioxidant enzymes in the blood was measured during treatment with the antioxidant preparation. Combination treatment with antioxidant vitamins and antioxidant element selenium sharply decreased the contents of primary and secondary free radical oxidation products in circulating low-density lipoproteins and increased activity of antioxidant enzymes in erythrocytes. Activities of superoxide dismutase and selenium-containing glutathione peroxidase increased 1 and 2 months after the start of therapy, respectively.

Application of pulsed-gradient Fourier transform nuclear magnetic resonance to the study of self-diffusion of phospholipid vesicles.

PubMed

McDonald, G G; Vanderkooi, J M

1975-05-20

A pulsed-gradient Fourier transform nuclear magnetic resonance (NMR) technique was appplied to the study of diffusion of phospholipid vesicles. The diffusion coefficient of dimyristoyllecithin vesicles (DML) in a D2O-phospahte buffer at 37 degrees is D = 1.9 TIMES 10(-6) cm2/sec. In a solution made viscous by DNA addition, the diffusion coefficient of DML vesicles was 3.5 times 10(-7) cm2/sec. These values compare favorably with the diffusion rate for liposomes as determined by ultracentrifugation and by Stokes law calculation. The data suggest that DML diffusion is controlled primarily by whole liposome migration as opposed to movement of individual molecules within the liposome, liposome rotation, or fast exchange between lecithin molecules in solution and in vesicles.

Evaluation of synthetic/reconstituted high-density lipoproteins as delivery vehicles for paclitaxel.

PubMed

McConathy, Walter J; Nair, Maya P; Paranjape, Sulabha; Mooberry, Linda; Lacko, Andras G

2008-02-01

Reconstituted (synthetic) high-density lipoprotein particles carrying paclitaxel (rHDL/PTX) were prepared with substantially higher PTX content than reported earlier. The rHDL/PTX complexes seemed to be primarily spherical nanoparticles when examined via electron microscopy, with a constant composition, molecular weight and exceptional stability even after ultracentrifugation and storage for up to 6 months. The rHDL/PTX nanoparticles had superior cytotoxicity against several cancer cell lines (MCF7, DU145, OV1063 and OVCAR-3), the half maximal inhibitory concentration (IC50) having been found to be 5-20 times lower than that of the free drug. Studies with mice showed that the rHDL/PTX nanoparticles were substantially better tolerated than the corresponding dosages of either Taxol or Abraxane.

An efficient method and device for transfer of semisolid materials into solid-state NMR spectroscopy rotors.

PubMed

Hisao, Grant S; Harland, Michael A; Brown, Robert A; Berthold, Deborah A; Wilson, Thomas E; Rienstra, Chad M

2016-04-01

The study of mass-limited biological samples by magic angle spinning (MAS) solid-state NMR spectroscopy critically relies upon the high-yield transfer of material from a biological preparation into the MAS rotor. This issue is particularly important for maintaining biological activity and hydration of semi-solid samples such as membrane proteins in lipid bilayers, pharmaceutical formulations, microcrystalline proteins and protein fibrils. Here we present protocols and designs for rotor-packing devices specifically suited for packing hydrated samples into Pencil-style 1.6 mm, 3.2 mm standard, and 3.2 mm limited speed MAS rotors. The devices are modular and therefore readily adaptable to other rotor and/or ultracentrifugation tube geometries. Copyright © 2016 Elsevier Inc. All rights reserved.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of the phosphotriesterase from Mycobacterium tuberculosis.

PubMed

Zhang, Liang; Chen, Ruyi; Dong, Zhe; Li, Xin

2013-01-01

Organophosphates (OPs) are extremely toxic compounds that are used as insecticides or even as chemical warfare agents. Phosphotriesterases (PHPs) are responsible for the detoxification of OPs by catalysing their degradation. Almost 100 PHP structures have been solved to date, yet the crystal structure of the phosphotriesterase from Mycobacterium tuberculosis (mPHP) remains unavailable. This study reports the first crystallization of mPHP. The crystal belonged to space group C222(1), with unit-cell parameters a = 68.03, b = 149.60, c = 74.23 Å, α = β = γ = 90°. An analytical ultracentrifugation experiment suggested that mPHP exists as a dimer in solution, even though one molecule is calculated to be present in the asymmetric unit according to the structural data.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of the phosphotriesterase from Mycobacterium tuberculosis

PubMed Central

Zhang, Liang; Chen, Ruyi; Dong, Zhe; Li, Xin

2013-01-01

Organophosphates (OPs) are extremely toxic compounds that are used as insecticides or even as chemical warfare agents. Phosphotriesterases (PHPs) are responsible for the detoxification of OPs by catalysing their degradation. Almost 100 PHP structures have been solved to date, yet the crystal structure of the phosphotriesterase from Mycobacterium tuberculosis (mPHP) remains unavailable. This study reports the first crystallization of mPHP. The crystal belonged to space group C2221, with unit-cell parameters a = 68.03, b = 149.60, c = 74.23 Å, α = β = γ = 90°. An analytical ultracentrifugation experiment suggested that mPHP exists as a dimer in solution, even though one molecule is calculated to be present in the asymmetric unit according to the structural data. PMID:23295488

Bone Marrow Mesenchymal Stem Cell-Derived CD63+ Exosomes Transport Wnt3a Exteriorly and Enhance Dermal Fibroblast Proliferation, Migration, and Angiogenesis In Vitro.

PubMed

McBride, Jeffrey D; Rodriguez-Menocal, Luis; Guzman, Wellington; Candanedo, Ambar; Garcia-Contreras, Marta; Badiavas, Evangelos V

2017-10-01

Wnts are secreted glycoproteins that regulate stem cell self-renewal, differentiation, and cell-to-cell communication during embryonic development and in adult tissues. Bone marrow mesenchymal stem cells (BM-MSCs) have been shown to stimulate dermis repair and regeneration; however, it is unclear how BM-MSCs may modulate downstream Wnt signaling. While recent reports implicate that Wnt ligands and Wnt messenger RNAs (such as Wnt4) exist within the interior compartment of exosomes, it has been debated whether or not Wnts exist on the exterior surface of exosomes to travel in the extracellular space. To help answer this question, we utilized flow cytometry of magnetic beads coated with anti-CD63 antibodies and found, for the first time, that Wnt3a protein is detectable exteriorly on CD63 + exosomes derived from BM-MSCs over-secreting Wnt3a into serum-free conditioned media (Wnt3a CM). Our data suggest that CD63 + exosomes significantly help transport exterior Wnt3a signal to recipient cells to promote fibroblast and endothelial functions. During purification of exosomes, we unexpectedly found that use of ultracentrifugation alone significantly decreased the ability to detect exteriorly bound Wnt3a on CD63 + exosomes, however, polyethylene glycol (PEG)-mediated exosome-enrichment before exosome-purification (with ultracentrifugation into a sucrose cushion) resulted in exosomes more likely to retain exterior Wnt3a detectability and downstream Wnt/beta-catenin activity. Our findings indicate the important role that purification methods may have on stem cell-derived Wnt-exosome activity in downstream assays. The ability for BM-MSC Wnt3a CM and exosomes to stimulate dermal fibroblast proliferation and migration, and endothelial angiogenesis in vitro, was significantly decreased after CD63 + -exosome depletion or knockdown of Wnt coreceptor LRP6 in recipient cells, suggesting both are required for optimal Wnt-exosome activity in our system. Thus, BM-MSC-derived CD63

Changes in localization and density of CD63-positive exosome-like substances in the hen oviduct with artificial insemination and their effect on sperm viability.

PubMed

Huang, A; Isobe, N; Yoshimura, Y

2017-10-01

Avian sperm are stored in the sperm storage tubules (SSTs) of the hen oviduct for a prolonged period. However, the precise mechanisms by which sperm are kept alive in the SSTs are still not fully understood. The aim of this study was to determine whether exosomes are secreted by SST cells and play a role in the survival of sperm. Utero-vaginal junction (UVJ) tissue from approximately 50 wk old White Leghorn hens was collected before (control group) and after intravaginal insemination with seminal plasma (SP group) or semen (AI group). The samples were used to prepare frozen sections and total protein extraction. The localization of the CD63, an exosome marker, was determined by immunohistochemistry and its protein level in the UVJ mucosal tissues was examined by Western blot. Exosomes were isolated from the culture media of UVJ and vaginal mucosa cells by ultracentrifugation and characterized by SDS-PAGE and Western blot. The viability and motility of sperm incubated with exosomes were also examined. CD63 was localized in the apical region of UVJ mucosal epithelium cells and SST cells of control, SP, and AI groups. The CD63 protein decreased in SST cells surrounding resident sperm and tended to appear in the SST lumen in the AI group. The protein level of CD63 in UVJ mucosal tissues was significantly higher in the AI group than control. The CD63 protein (approximately 75 kDa) was detected in ultracentrifugation pellets from the culture medium of UVJ and vagina cells. The viability of sperm incubated with 1 μg/μl vaginal exosomes was significantly decreased but was not affected by UVJ exosomes. These results suggest that exosomes were synthesized by SST cells and may be secreted into SST lumen when sperm were stored in SSTs. The role of SST exosomes in sperm storage needs to be examined further. Copyright © 2017 Elsevier Inc. All rights reserved.

Pilot scale production of highly efficacious and stable enterovirus 71 vaccine candidates.

PubMed

Chou, Ai-Hsiang; Liu, Chia-Chyi; Chang, Cheng-Peng; Guo, Meng-Shin; Hsieh, Shih-Yang; Yang, Wen-Hsueh; Chao, Hsin-Ju; Wu, Chien-Long; Huang, Ju-Lan; Lee, Min-Shi; Hu, Alan Yung-Chi; Lin, Sue-Chen; Huang, Yu-Yun; Hu, Mei-Hua; Chow, Yen-Hung; Chiang, Jen-Ron; Chang, Jui-Yuan; Chong, Pele

2012-01-01

Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia and now is being recognized as an important neurotropic virus. Effective medications and prophylactic vaccine against EV71 infection are urgently needed. Based on the success of inactivated poliovirus vaccine, a prototype chemically inactivated EV71 vaccine candidate has been developed and currently in human phase 1 clinical trial. In this report, we present the development of a serum-free cell-based EV71 vaccine. The optimization at each step of the manufacturing process was investigated, characterized and quantified. In the up-stream process development, different commercially available cell culture media either containing serum or serum-free was screened for cell growth and virus yield using the roller-bottle technology. VP-SFM serum-free medium was selected based on the Vero cell growth profile and EV71 virus production. After the up-stream processes (virus harvest, diafiltration and concentration), a combination of gel-filtration liquid chromatography and/or sucrose-gradient ultracentrifugation down-stream purification processes were investigated at a pilot scale of 40 liters each. Although the combination of chromatography and sucrose-gradient ultracentrifugation produced extremely pure EV71 infectious virus particles, the overall yield of vaccine was 7-10% as determined by a VP2-based quantitative ELISA. Using chromatography as the downstream purification, the virus yield was 30-43%. To retain the integrity of virus neutralization epitopes and the stability of the vaccine product, the best virus inactivation was found to be 0.025% formalin-treatment at 37 °C for 3 to 6 days. Furthermore, the formalin-inactivated virion vaccine candidate was found to be stable for >18 months at 4 °C and a microgram of viral proteins formulated with alum adjuvant could induce strong virus-neutralizing antibody responses in mice, rats, rabbits, and non-human primates. These

SEPARATION AND CHARACTERIZATION OF HUMAN SERUM CHYLOMICRONS

PubMed Central

Scanu, Angelo; Page, Irvine H.

1959-01-01

Chylomicrons were separated by low and high speed ultracentrifugation from lipemic sera of human subjects in the absorptive phase. The final chylomicron preparation was free from other serum components and contained a small constant amount of protein, approximately 2 per cent of the chylomicron fraction. Electrophoresis, immunochemical analysis, and absorption experiments identified the protein component as derived from a mixture of beta and alpha1 serum lipoproteins. Large aliquots of an emulsion of serum freed of chylomicrons and coconut oil were incubated at 37°C. for 2 hours and ultracentrifuged as in the preparation of chylomicrons. The fat particles now showed the presence of minute amounts of beta and alpha1 serum lipoproteins in almost the same proportion as found in chylomicrons. "Finger prints" of delipidized samples of chylomicrons and particles from serum-coconut oil emulsion gave similar, although not identical patterns. The data on "clearing factor" activity corroborated the finding that serum alpha1 lipoproteins are contained in chylomicrons and particles from serum-coconut oil emulsion. These two lipide particles, partially delipidized, were both able to activate a "clearing factor" system in vitro, a property exhibited only by intact or partially delipidized alpha1 serum lipoproteins. Clearing activity was satisfactorily determined by using an emulsion of coconut oil mixed in agar as a substrate to give an opaque gel, in which the diffusing enzyme showed its activity by areas of clearing. The results obtained by this technique were in agreement with those based on fall in optical density and non-esterified fatty acid production. Chemical analysis of serum chylomicrons showed a concentration of cholesterol and phospholipides higher than could be accounted for by the attached beta and alpha1 serum lipoproteins. On the basis of these results the assumption is made that in the blood stream small amounts of serum lipoproteins, by a process of

Preliminary Evaluation of Cesium Distribution for Wet Sieving Process Planned for Soil Decontamination in Japan - 13104

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

Enokida, Y.; Tanada, Y.; Hirabayashi, D.

2013-07-01

For the purpose of decontaminating radioactive cesium from a huge amount of soil, which has been estimated to be 1.2x10{sup 8} m{sup 3} by excavating to a 5-cm depth from the surface of Fukushima Prefecture where a severe nuclear accident occurred at TEPCO's power generating site and has emitted a significant amount of radioactive materials, mainly radioactive cesium, a wet sieving process was selected as one of effective methods available in Japan. Some private companies have demonstrated this process for soil treatment in the Fukushima area by testing at their plants. The results were very promising, and a full-fledged applicationmore » is expected to follow. In the present study, we spiked several aqueous samples containing soil collected from an industrial wet sieving plant located near our university for the recycling of construction wastes with non-radioactive cesium hydroxide. The present study provides scientific data concerning the effectiveness in volume reduction of the contaminated soil by a wet sieving process as well as the cesium distribution between the liquid phase and clay minerals for each sub-process of the full-scale one, but a simulating plant equipped with a process of coagulating sedimentation and operational safety fundamentals for the plant. Especially for the latter aspect, the study showed that clay minerals of submicron size strongly bind a high content of cesium, which was only slightly removed by coagulation with natural sedimentation (1 G) nor centrifugal sedimentation (3,700 G) and some of the cesium may be transferred to the effluent or recycled water. By applying ultracentrifugation (257,000 G), most of submicron clay minerals containing cesium was removed, and the cesium amount which might be transferred to the effluent or recycled water, could be reduced to less than 2.3 % of the original design by the addition of a cesium barrier consisting of ultracentrifugation or a hollow fiber membrane. (authors)« less

Characterization of recombinant dihydrodipicolinate synthase from the bread wheat Triticum aestivum.

PubMed

Gupta, Ruchi; Hogan, Campbell J; Perugini, Matthew A; Soares da Costa, Tatiana P

2018-05-09

Recombinant wheat DHDPS was produced for the first time in milligram quantities and shown to be an enzymatically active tetramer in solution using analytical ultracentrifugation and small angle X-ray scattering. Wheat is an important cereal crop with an extensive role in global food supply. Given our rapidly growing population, strategies to increase the nutritional value and production of bread wheat are of major significance in agricultural science to satisfy our dietary requirements. Lysine is one of the most limiting essential amino acids in wheat, thus, a thorough understanding of lysine biosynthesis is of upmost importance to improve its nutritional value. Dihydrodipicolinate synthase (DHDPS; EC 4.3.3.7) catalyzes the first committed step in the lysine biosynthesis pathway of plants. Here, we report for the first time the expression and purification of recombinant DHDPS from the bread wheat Triticum aestivum (Ta-DHDPS). The optimized protocol yielded 36 mg of > 98% pure recombinant Ta-DHDPS per liter of culture. Enzyme kinetic studies demonstrate that the recombinant Ta-DHDPS has a K M (pyruvate) of 0.45 mM, K M (l-aspartate-4-semialdehyde) of 0.07 mM, k cat of 56 s -1 , and is inhibited by lysine (IC 50 LYS of 0.033 mM), which agree well with previous studies using labor-intensive purification from wheat suspension cultures. We subsequently employed circular dichroism spectroscopy, analytical ultracentrifugation and small angle X-ray scattering to show that the recombinant enzyme is folded with 60% α/β structure and exists as a 7.5 S tetrameric species with a R g of 33 Å and D max of 118 Å. This study is the first to report the biophysical properties of the recombinant Ta-DHDPS in aqueous solution and offers an excellent platform for future studies aimed at improving nutritional value and primary production of bread wheat.

Liver lipase and high-density lipoprotein. Lipoprotein changes after incubation of human serum with rat liver lipase.

PubMed

Groot, P H; Scheek, L M; Jansen, H

1983-05-16

Human sera were incubated with rat liver lipase after inactivation of lecithin:cholesterol acyltransferase, and the changes in serum lipoprotein composition were measured. In the presence of liver lipase serum triacylglycerol and phosphatidylcholine were hydrolyzed. The main changes in the concentrations of these lipids were found in the high-density lipoprotein fraction. Subfractionation of high-density lipoprotein by rate-zonal ultracentrifugation showed a prominent decrease in all constituents of high-density lipoprotein2, a smaller decrease in the 'light' high-density lipoprotein3 and an increase in the 'heavy' high-density lipoprotein3. These data support a concept in which liver lipase is involved in high-density lipoprotein2 phospholipid and triacylglycerol catabolism and suggest that as a result of this action high-density lipoprotein2 is converted into high-density lipoprotein3.

[Comparative protein analytic studies of various intravenous 7S-immunoglobulin preparations].

PubMed

Fateh-Moghadam, A; Wick, M; Simon, H

1984-02-01

Seven different commercial intravenous 7S-immunoglobulin preparations have been examined by electrophoresis, immunoelectrophoresis, quantitative determination of immunoglobulins and other proteins, gel chromatography and analytical ultracentrifugation. No significant difference concerning IgG and monomeric immunoglobulin concentrations was observed. The content of IgM, dimers and polymers showed slight, that of IgA considerable differences. All immunoglobulin preparations comply with the European Pharmacopoea requirements. The rate of adverse reactions should be equally low due to similar dimer and polymer contents. IgA-free preparations are considered to be more suitable in primary hypogammaglobulinaemia whereas IgA-containing preparations could be of benefit in acquired hypogammaglobulinaemia. While the presence of an intact immunoglobulin molecule is thought to be essential for its full therapeutic efficacy, the influence of the preparation method is still in debate.

Preparation of synaptic plasma membrane and postsynaptic density proteins using a discontinuous sucrose gradient.

PubMed

Bermejo, Marie Kristel; Milenkovic, Marija; Salahpour, Ali; Ramsey, Amy J

2014-09-03

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960's, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

Application of zinc chloride precipitation method for rapid isolation and concentration of infectious Pectobacterium spp. and Dickeya spp. lytic bacteriophages from surface water and plant and soil extracts.

PubMed

Czajkowski, Robert; Ozymko, Zofia; Lojkowska, Ewa

2016-01-01

This is the first report describing precipitation of bacteriophage particles with zinc chloride as a method of choice to isolate infectious lytic bacteriophages against Pectobacterium spp. and Dickeya spp. from environmental samples. The isolated bacteriophages are ready to use to study various (ecological) aspects of bacteria-bacteriophage interactions. The method comprises the well-known precipitation of phages from aqueous extracts of the test material by addition of ZnCl2, resuscitation of bacteriophage particles in Ringer's buffer to remove the ZnCl2 excess and a soft agar overlay assay with the host bacterium to isolate infectious individual phage plaques. The method requires neither an enrichment step nor other steps (e. g., PEG precipitation, ultrafiltration, or ultracentrifugation) commonly used in other procedures and results in isolation of active viable bacteriophage particles.

Asymmetric resonance Raman excitation profiles and violation of the Condon approximation in single-wall carbon nanotubes

NASA Astrophysics Data System (ADS)

Doorn, Stephen; Duque, Juan; Telg, Hagen; Chen, Hang; Swan, Anna; Haroz, Erik; Kono, Junichiro; Tu, Xiaomin; Zheng, Ming

2012-02-01

DNA wrapping-based ion exchange chromatography and density gradient ultracentrifugation provide nanotube samples highly enriched in single chiralities. We present resonance Raman excitation profiles for the G-band of several single chirality semiconducting and metallic species. The expected incoming and outgoing resonance peaks are observed in the profiles, but contrary to long-held assumptions, the outgoing resonance is always significantly weaker than the ingoing resonance peak. This strong asymmetry in the profiles arises from a violation of the Condon approximation [1]. Results will be discussed in the context of theoretical models that suggest significant coordinate dependence in the transition dipole (non-Condon effects). The generality of the behavior across semiconducting and metallic types, nanotube family, phonon mode, and Eii will be demonstrated. [4pt] [1] J. Duque et. al., ACS Nano, 5, 5233 (2011).

Production and Purification of a Staphylococcus epidermidis Bacteriocin

PubMed Central

Jetten, A. M.; Vogels, G. D.; de Windt, F.

1972-01-01

Liquid cultures of Staphylococcus epidermidis 1580 contained rather small amounts of a bacteriocin, staphylococcin 1580, which was found both in the supernatant fluid and in the cell pellet. It could be extracted from the cells with 5% NaCl solution. The staphylococcin production could not be induced by ultraviolet irradiation or treatment with mitomycin C. Bacteria grown on semisolid medium produced a much larger amount of the compound with a high specific activity. The staphylococcin was purified by ammonium sulfate precipitation, ultracentrifugation, and chromatography on Sephadex columns. The purified material was homogeneous on polyacrylamide gel electrophoresis. The molecular weight was between 150,000 and 400,000. The bacteriocin was composed of protein, carbohydrate, and lipid and consisted of subunits exhibiting a molecular weight of about 20,000. Images PMID:5079063

Structural changes of TasA in biofilm formation of Bacillus subtilis

PubMed Central

Diehl, Anne; Roske, Yvette; Ball, Linda; Chowdhury, Anup; Hiller, Matthias; Molière, Noel; Kramer, Regina; Stöppler, Daniel; Worth, Catherine L.; Schlegel, Brigitte; Leidert, Martina; Cremer, Nils; Erdmann, Natalja; Lopez, Daniel; Stephanowitz, Heike; Krause, Eberhard; Schmieder, Peter; Akbey, Ümit; Oschkinat, Hartmut

2018-01-01

Microorganisms form surface-attached communities, termed biofilms, which can serve as protection against host immune reactions or antibiotics. Bacillus subtilis biofilms contain TasA as major proteinaceous component in addition to exopolysaccharides. In stark contrast to the initially unfolded biofilm proteins of other bacteria, TasA is a soluble, stably folded monomer, whose structure we have determined by X-ray crystallography. Subsequently, we characterized in vitro different oligomeric forms of TasA by NMR, EM, X-ray diffraction, and analytical ultracentrifugation (AUC) experiments. However, by magic-angle spinning (MAS) NMR on live biofilms, a swift structural change toward only one of these forms, consisting of homogeneous and protease-resistant, β-sheet–rich fibrils, was observed in vivo. Thereby, we characterize a structural change from a globular state to a fibrillar form in a functional prokaryotic system on the molecular level. PMID:29531041

A simple tagging system for protein encapsulation.

PubMed

Seebeck, Florian P; Woycechowsky, Kenneth J; Zhuang, Wei; Rabe, Jürgen P; Hilvert, Donald

2006-04-12

Molecular containers that encapsulate specific cargo can be useful for many natural and non-natural processes. We report a simple system, based on charge complementarity, for the encapsulation of appropriately tagged proteins within an engineered, proteinaceous capsid. Four negative charges per monomer were added to the lumazine synthase from Aquifex aeolicus (AaLS). The capsids formed by the engineered AaLS associate with green fluorescent protein bearing a positively charged deca-arginine tag upon coproduction in Escherichia coli. Analytical ultracentrifugation and scanning force microscopy studies indicated that the engineered AaLS retains the ability to form capsids, but that their average size was substantially increased. The success of this strategy demonstrates that both the container and guest components of protein-based encapsulation systems can be convergently designed in a straightforward manner, which may help to extend their versatility.

Preferential assembly of heteromeric kainate and AMPA receptor amino terminal domains

PubMed Central

Lomash, Suvendu; Chittori, Sagar; Glasser, Carla

2017-01-01

Ion conductivity and the gating characteristics of tetrameric glutamate receptor ion channels are determined by their subunit composition. Competitive homo- and hetero-dimerization of their amino-terminal domains (ATDs) is a key step controlling assembly. Here we measured systematically the thermodynamic stabilities of homodimers and heterodimers of kainate and AMPA receptors using fluorescence-detected sedimentation velocity analytical ultracentrifugation. Measured affinities span many orders of magnitude, and complexes show large differences in kinetic stabilities. The association of kainate receptor ATD dimers is generally weaker than the association of AMPA receptor ATD dimers, but both show a general pattern of increased heterodimer stability as compared to the homodimers of their constituents, matching well physiologically observed receptor combinations. The free energy maps of AMPA and kainate receptor ATD dimers provide a framework for the interpretation of observed receptor subtype combinations and possible assembly pathways. PMID:29058671

Preferential assembly of heteromeric kainate and AMPA receptor amino terminal domains.

PubMed

Zhao, Huaying; Lomash, Suvendu; Chittori, Sagar; Glasser, Carla; Mayer, Mark L; Schuck, Peter

2017-10-23

Ion conductivity and the gating characteristics of tetrameric glutamate receptor ion channels are determined by their subunit composition. Competitive homo- and hetero-dimerization of their amino-terminal domains (ATDs) is a key step controlling assembly. Here we measured systematically the thermodynamic stabilities of homodimers and heterodimers of kainate and AMPA receptors using fluorescence-detected sedimentation velocity analytical ultracentrifugation. Measured affinities span many orders of magnitude, and complexes show large differences in kinetic stabilities. The association of kainate receptor ATD dimers is generally weaker than the association of AMPA receptor ATD dimers, but both show a general pattern of increased heterodimer stability as compared to the homodimers of their constituents, matching well physiologically observed receptor combinations. The free energy maps of AMPA and kainate receptor ATD dimers provide a framework for the interpretation of observed receptor subtype combinations and possible assembly pathways.

The RSV F and G glycoproteins interact to form a complex on the surface of infected cells

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

Low, Kit-Wei; Tan, Timothy; Ng, Ken

2008-02-08

In this study, the interaction between the respiratory syncytial virus (RSV) fusion (F) protein, attachment (G) protein, and small hydrophobic (SH) proteins was examined. Immunoprecipitation analysis suggested that the F and G proteins exist as a protein complex on the surface of RSV-infected cells, and this conclusion was supported by ultracentrifugation analysis that demonstrated co-migration of surface-expressed F and G proteins. Although our analysis provided evidence for an interaction between the G and SH proteins, no evidence was obtained for a single protein complex involving all three of the virus proteins. These data suggest the existence of multiple virus glycoproteinmore » complexes within the RSV envelope. Although the stimulus that drives RSV-mediated membrane fusion is unknown, the association between the G and F proteins suggest an indirect role for the G protein in this process.« less

Alteration in lipid composition of plasma membranes of sensitive and resistant Guerin carcinoma cells due to the action of free and liposomal form of cisplatin.

PubMed

Naleskina, L A; Todor, I N; Nosko, M M; Lukianova, N Y; Pivnyuk, V M; Chekhun, V F

2013-09-01

To study in vivo changes of lipid composition of plasma membranes of sensitive and resistant to cisplatin Guerin carcinoma cells under influence of free and liposomal cisplatin forms. The isolation of plasma membranes from parental (sensitive) and resistant to cisplatin Guerin carcinoma cells was by differential ultracentrifugation in sucrose density gradient. Lipids were detected by method of thin-layer chromatography. It was determined that more effective action of cisplatin liposomal form on resistant cells is associated with essential abnormalities of conformation of plasma membrane due to change of lipid components and architectonics of rafts. It results in the increase of membrane fluidity. Reconstructions in lipid composition of plasma membranes of cisplatin-resistant Guerin carcinoma cells provide more intensive delivery of drug into the cells, increase of its concentration and more effective interaction with cellular structural elements.

Comparison of Methods for Characterizing Nonideal Solute Self-Association by Sedimentation Equilibrium

PubMed Central

Scott, David J.; Winzor, Donald J.

2009-01-01

Abstract We have examined in detail analytical solutions of expressions for sedimentation equilibrium in the analytical ultracentrifuge to describe self-association under nonideal conditions. We find that those containing the radial dependence of total solute concentration that incorporate the Adams-Fujita assumption for composition-dependence of activity coefficients reveal potential shortcomings for characterizing such systems. Similar deficiencies are shown in the use of the NONLIN software incorporating the same assumption about the interrelationship between activity coefficients for monomer and polymer species. These difficulties can be overcome by iterative analyses incorporating expressions for the composition-dependence of activity coefficients predicted by excluded volume considerations. A recommendation is therefore made for the replacement of current software packages by programs that incorporate rigorous statistical-mechanical allowance for thermodynamic nonideality in sedimentation equilibrium distributions reflecting solute self-association. PMID:19651047

Effect of high hydrostatic pressure and whey proteins on the disruption of casein micelle isolates.

PubMed

Harte, Federico M; Gurram, Subba Rao; Luedecke, Lloyd O; Swanson, Barry G; Barbosa-Cánovas, Gustavo V

2007-11-01

High hydrostatic pressure disruption of casein micelle isolates was studied by analytical ultracentrifugation and transmission electron microscopy. Casein micelles were isolated from skim milk and subjected to combinations of thermal treatment (85 degrees C, 20 min) and high hydrostatic pressure (up to 676 MPa) with and without whey protein added. High hydrostatic pressure promoted extensive disruption of the casein micelles in the 250 to 310 MPa pressure range. At pressures greater than 310 MPa no further disruption was observed. The addition of whey protein to casein micelle isolates protected the micelles from high hydrostatic pressure induced disruption only when the mix was thermally processed before pressure treatment. The more whey protein was added (up to 5 g/l) the more the protection against high hydrostatic pressure induced micelle disruption was observed in thermally treated samples subjected to 310 MPa.

Protein and nitrogen composition of equine (Equus caballus) milk during early lactation.

PubMed

Zicker, S C; Lonnerdal, B

1994-01-01

Separation of whey protein from casein in equine milk was achieved by adjustment of pH to 4.3 without addition of calcium, and by ultracentrifugation at 189,000 g for 1 hr. True protein, whey protein, and casein decreased significantly during the first 28 days of lactation with the magnitude of decrease being greatest for whey protein. The proportion of nitrogen in whey protein:casein decreased from 85:15 to 54:46 during the 28 day time period. The concentration of non-protein nitrogen remained relatively constant at 500 mg nitrogen/l but increased in proportion from 2 to 13% of the total nitrogen during the first 28 days of lactation. These results illustrate the unique nitrogen composition of equine milk, which is intermediate between human and ruminant milk, and how it changes during early lactation.

Isolation, crystallization in the macrogravitation field, preliminary X-ray investigation of uridine phosphorylase from Escherichia coli K-12.

PubMed

Mikhailov, A M; Smirnova, E A; Tsuprun, V L; Tagunova, I V; Vainshtein, B K; Linkova, E V; Komissarov, A A; Siprashvili, Z Z; Mironov, A S

1992-03-01

Uridine phosphorylase (UPH) from Escherichia coli K-12 has been purified to near homogeneity from a strain harbouring the udp gene, encoding UPH, on a multicopy plasmid. UPH was purified to electrophoretic homogeneity with the specific activity 230 units/mg with a recovery of 80%, yielding 120 mg of enzyme from 3g cells. Crystals of enzyme suitable for X-ray diffraction analysis were obtained in a preparative ultracentrifuge. The packing of the molecules in the crystals may be described by the space group P2(1)2(1)2(1) with the unit cell constants a = 90.4; b = 128.8; c = 136.8 A. There is one molecule per asymmetric unit, Vm = 2.4. These crystals diffract to at least 2.5-2.7 A resolution. The hexameric structure of UPH was directly demonstrated by electron microscopy study and image processing.

Effects of gravity on ontogeny in animals.

NASA Technical Reports Server (NTRS)

Pitts, G. C.

1973-01-01

Inversion or clinostat rotation of frogs' eggs demonstrated a critical period of enhanced gravity-sensitivity prior to the first cleavage. These results were corroborated by centrifugation studies which localized the period of maximal sensitivity at approximately 20 minutes post-fertilization. Eggs of various invertebrates suspended in aqueous solution proved capable of normal development following brief ultracentrifugation. Among fly larvae, grasshopper nymphs, turtles, mice, rats, and chickens, growth rates were inversely related to G-force, and maximal chronic acceleration tolerated was inversely related to body size. Body composition data demonstrated the importance of separately evaluating fat and the fat-free portion of the body in studies of the effect of acceleration on growth in homeotherms. The attempt to evaluate the effect of weightlessness on the development of frogs' eggs in Biosatellite 2 was inconclusive for technical reasons.

[Studies on purification and properties of antagonistic protein from bacteria SS02 of Paenibacillus daejeonensis].

PubMed

Zhu, Kai; Zhang, Xiao-Yu; Ren, Zhi; Feng, Ding-Sheng; Wang, Yi-Ding

2007-07-01

The antifungal, anti-bacterical, anti-brine shrimp activities of SD22 isolated from Paenibacillus daejeonensis Bacteria SS02 were studied. The separation steps included ultracentrifugation, ultrafiltration and (NH4)2SO4 fractional precipitation, further purification was performed by SephadexG-75 and DEAE-32 chromatography. Its molecular weight determined by SDS-PAGE was 56.0 kD and its isoelectfic point was 6.4. SD22 was thermostable to some extent and stable to ultraviolet, but sensitive to some of the enzyme. SD22 could kill most pathogens from propagation, such as Rhizoctonia cerealis, Sclerotinia sclerotiorum Physalospora piricala, Trichodema viride, Gliocladium viride, Curvularia leaf spot, Fusarium sp, Fusarium head blight, Beauveria Bassiana, Escherichia coli, Staphylococcus aureus, Bacillus subtilis , Candidal vaginitis, Fusarium oxysporum Schl. emend. Sayder & Hansem et al. The results will be helpful to find out a novel antifungal protein.

Calcium phosphate nanoparticles as versatile carrier for small and large molecules across cell membranes

NASA Astrophysics Data System (ADS)

Sokolova, Viktoriya; Rotan, Olga; Klesing, Jan; Nalbant, Perihan; Buer, Jan; Knuschke, Torben; Westendorf, Astrid M.; Epple, Matthias

2012-06-01

The successful transport of molecules across the cell membrane is a key point in biology and medicine. In most cases, molecules alone cannot penetrate the cell membrane, therefore an efficient carrier is needed. Calcium phosphate nanoparticles (diameter: 100-250 nm, depending on the functionalization) were loaded with fluorescent oligonucleotides, peptide, proteins, antibodies, polymers or porphyrins and characterized by dynamic light scattering, nanoparticle tracking analysis and scanning electron microscopy. Any excess of molecules was removed by ultracentrifugation, and the dissolved molecules at the same concentration were used as control. The uptake of such fluorescence-labeled nanoparticles into HeLa cells was monitored by fluorescence microscopy and confocal laser scanning microscopy. Calcium phosphate nanoparticles were able to transport all molecules across the cell membrane, whereas the dissolved molecules alone were taken up only to a very small extent or even not at all.

Intestinal secretion of immunoglobulins and antibodies to Escherichia coli in the pig

PubMed Central

Porter, P.; Noakes, D. E.; Allen, W. D.

1970-01-01

Immunoglobulins and antibodies against Escherichia coli 0141 have been studied in porcine intestinal secretions obtained from Thiry Vella loops prepared in the mid jejunum of 4 animals. The molecular size of the secreted immunoglobulins were investigated by gel filtration and sucrose density gradient ultra-centrifugation. Intestinal IgM was found to have 7S characteristics and intestinal IgA mainly 11S characteristics similar to secretory IgA isolated from porcine milk. Immune inhibition studies with rabbit anti-IgA-globulin serum produced complete elimination of E. coli 0141 antibodies detected by direct haemagglutination. In one animal incomplete antibody assayed by antiglobulin haemagglutination was identified in fractions associated with IgM and IgG. Immunofluorescent studies were made to correlate immunoglobulins in the small intestinal tissue with weaning. ImagesFIG. 1FIG. 2FIG. 7 PMID:4193669

Conformational changes of the N-terminal part of Mason-Pfizer monkey virus p12 protein during multimerization

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

Knejzlik, Zdenek; Ulbrich, Pavel; Strohalm, Martin

2009-10-10

The Mason-Pfizer monkey virus is a prototype Betaretrovirus with the defining characteristic that it assembles spherical immature particles from Gag-related polyprotein precursors within the cytoplasm of the infected cell. It was shown previously that the N-terminal part of the Gag p12 domain (wt-Np12) is required for efficient assembly. However, the precise role for p12 in mediating Gag-Gag interaction is still poorly understood. In this study we employed detailed circular dichroism spectroscopy, electron microscopy and ultracentrifugation analyses of recombinant wt-Np12 prepared by in vitro transcription and translation. The wt-Np12 domain fragment forms fibrillar structures in a concentration-dependent manner. Assembly into fibersmore » is linked to a conformational transition from unfolded or another non-periodical state to alpha-helix during multimerization.« less

UC/MALDI-MS analysis of HDL; evidence for density-dependent post-translational modifications

NASA Astrophysics Data System (ADS)

Johnson, Jeffery D.; Henriquez, Ronald R.; Tichy, Shane E.; Russell, David H.; McNeal, Catherine J.; Macfarlane, Ronald D.

2007-12-01

The purpose of this study is to determine whether the nature of the post-translational modifications of the major apolipoproteins of HDL is different for density-distinct subclasses. These subclasses were separated by ultracentrifugation using a novel density-forming solute to yield a high-resolution separation. The serum of two subjects, a control with a normolipidemic profile and a subject with diagnosed cardiovascular disease, was studied. Aliquots of three HDL subclasses were analyzed by MALDI and considerable differences were seen when comparing density-distinct subclasses and also when comparing the two subjects. A detailed analysis of the post-translational modification pattern of apoA-1 shows evidence of considerable protease activity, particularly in the more dense fractions. We conclude that part of the heterogeneity of the population of HDL particles is due to density-dependent protease activity.

Construction of a Chassis for a Tripartite Protein-Based Molecular Motor.

PubMed

Small, Lara S R; Bruning, Marc; Thomson, Andrew R; Boyle, Aimee L; Davies, Roberta B; Curmi, Paul M G; Forde, Nancy R; Linke, Heiner; Woolfson, Derek N; Bromley, Elizabeth H C

2017-06-16

Improving our understanding of biological motors, both to fully comprehend their activities in vital processes, and to exploit their impressive abilities for use in bionanotechnology, is highly desirable. One means of understanding these systems is through the production of synthetic molecular motors. We demonstrate the use of orthogonal coiled-coil dimers (including both parallel and antiparallel coiled coils) as a hub for linking other components of a previously described synthetic molecular motor, the Tumbleweed. We use circular dichroism, analytical ultracentrifugation, dynamic light scattering, and disulfide rearrangement studies to demonstrate the ability of this six-peptide set to form the structure designed for the Tumbleweed motor. The successful formation of a suitable hub structure is both a test of the transferability of design rules for protein folding as well as an important step in the production of a synthetic protein-based molecular motor.

Binding of the cyclic AMP receptor protein of Escherichia coli to RNA polymerase.

PubMed

Pinkney, M; Hoggett, J G

1988-03-15

Fluorescence polarization studies were used to study the interaction of a fluorescein-labelled conjugate of the Escherichia coli cyclic AMP receptor protein (F-CRP) and RNA polymerase. Under conditions of physiological ionic strength, F-CRP binds to RNA polymerase holoenzyme in a cyclic AMP-dependent manner; the dissociation constant was about 3 microM in the presence of cyclic AMP and about 100 microM in its absence. Binding to core RNA polymerase under the same conditions was weak (Kdiss. approx. 80-100 microM) and independent of cyclic AMP. Competition experiments established that native CRP and F-CRP compete for the same binding site on RNA polymerase holoenzyme and that the native protein binds about 3 times more strongly than does F-CRP. Analytical ultracentrifuge studies showed that CRP binds predominantly to the monomeric rather than the dimeric form of RNA polymerase.

The elusive structural role of ubiquitinated histones.

PubMed

Moore, Susan C; Jason, Laure; Ausió, Juan

2002-01-01

It is increasingly apparent that histone posttranslational modifications are important in chromatin structure and dynamics. However, histone ubiquitination has received little attention. Histones H1, H3, H2A, and H2B can be ubiquitinated in vivo, but the most prevalent are uH2A and uH2B. The size of this modification suggests some sort of structural impact. Physiological observations suggest that ubiquitinated histones may have multiple functions and structural effects. Ubiquitinated histones have been correlated with transcriptionally active DNA, implying that it may prevent chromatin folding or help maintain an open conformation. Also, in some organisms during spermiogenesis, a process involving extensive chromatin remodeling, uH2A levels increase just prior to histone replacement by protamines. Determination of chromatin's structural changes resulting from histone ubiquitination is therefore important. Recent work using reconstituted nucleosomes and chromatin fibers containing uH2A indicate that in the absence of linker histones, ubiquitination has little structural impact. DNase I digests and analytical ultracentrifugation of reconstituted ubiquitinated nucleosomes show no structural differences. Solubility assays using reconstituted chromatin fibers in the presence of divalent ions demonstrate that uH2A fibers are slightly more prone to aggregation than controls, and analytical ultracentrifugation results with different MgCl2 and NaCl concentrations determined that chromatin folding is not affected by this modification. Additional work to assess possible synergistic affects with histone acetylation also precludes any structural implications. Protamine displacement experiments concluded that the presence of uH2A does not significantly affect the ability of the protamines to displace histones. In addition, uH2A does not interfere with histone H1 binding to the nucleosome. While work with uH2B remains insufficient to come to any definitive conclusions about its

Reduction of spiked porcine circovirus during the manufacture of a Vero cell-derived vaccine.

PubMed

Lackner, Cornelia; Leydold, Sandra M; Modrof, Jens; Farcet, Maria R; Grillberger, Leopold; Schäfer, Birgit; Anderle, Heinz; Kreil, Thomas R

2014-04-11

Porcine circovirus-1 (PCV1) was recently identified as a contaminant in live Rotavirus vaccines, which was likely caused by contaminated porcine trypsin. The event triggered the development of new regulatory guidance on the use of porcine trypsin which shall ensure that cell lines and porcine trypsin in use are free from PCV1. In addition, manufacturing processes of biologicals other than live vaccines include virus clearance steps that may prevent and mitigate any potential virus contamination of product. In this work, artificial spiking of down-scaled models for the manufacturing process of an inactivated pandemic influenza virus vaccine were used to investigate inactivation of PCV1 and the physico-chemically related porcine parvovirus (PPV) by formalin and ultraviolet-C (UV-C) treatment as well as removal by the purification step sucrose gradient ultracentrifugation. A PCV1 infectivity assay, using a real-time PCR infectivity readout was established. The formalin treatment (0.05% for 48h) showed substantial inactivation for both PCV1 and PPV with reduction factors of 3.0log10 and 6.8log10, respectively, whereas UV-C treatment resulted in complete PPV (≥5.9log10) inactivation already at a dose of 13mJ/cm but merely 1.7log10 at 24mJ/cm(2) for PCV1. The UV-C inactivation results with PPV were confirmed using minute virus of mice (MVM), indicating that parvoviruses are far more sensitive to UV-C than PCV1. The sucrose density gradient ultracentrifugation also contributed to PCV1 clearance with a reduction factor of 2log10. The low pH treatment during the production of procine trypsin was investigated and showed effective inactivation for both PCV1 (4.5log10) and PPV (6.4log10). In conclusion, PCV1 in general appears to be more resistant to virus inactivation than PPV. Still, the inactivated pandemic influenza vaccine manufacturing process provides for robust virus reduction, in addition to the already implemented testing for PCV1 to avoid any contaminations

Structural changes of TasA in biofilm formation of Bacillus subtilis.

PubMed

Diehl, Anne; Roske, Yvette; Ball, Linda; Chowdhury, Anup; Hiller, Matthias; Molière, Noel; Kramer, Regina; Stöppler, Daniel; Worth, Catherine L; Schlegel, Brigitte; Leidert, Martina; Cremer, Nils; Erdmann, Natalja; Lopez, Daniel; Stephanowitz, Heike; Krause, Eberhard; van Rossum, Barth-Jan; Schmieder, Peter; Heinemann, Udo; Turgay, Kürşad; Akbey, Ümit; Oschkinat, Hartmut

2018-03-27

Microorganisms form surface-attached communities, termed biofilms, which can serve as protection against host immune reactions or antibiotics. Bacillus subtilis biofilms contain TasA as major proteinaceous component in addition to exopolysaccharides. In stark contrast to the initially unfolded biofilm proteins of other bacteria, TasA is a soluble, stably folded monomer, whose structure we have determined by X-ray crystallography. Subsequently, we characterized in vitro different oligomeric forms of TasA by NMR, EM, X-ray diffraction, and analytical ultracentrifugation (AUC) experiments. However, by magic-angle spinning (MAS) NMR on live biofilms, a swift structural change toward only one of these forms, consisting of homogeneous and protease-resistant, β-sheet-rich fibrils, was observed in vivo. Thereby, we characterize a structural change from a globular state to a fibrillar form in a functional prokaryotic system on the molecular level. Copyright © 2018 the Author(s). Published by PNAS.

Modular structure of the full-length DNA gyrase B subunit revealed by small-angle X-ray scattering.

PubMed

Costenaro, Lionel; Grossmann, J Günter; Ebel, Christine; Maxwell, Anthony

2007-03-01

DNA gyrase, the only topoisomerase able to introduce negative supercoils into DNA, is essential for bacterial transcription and replication; absent from humans, it is a successful target for antibacterials. From biophysical experiments in solution, we report a structural model at approximately 12-15 A resolution of the full-length B subunit (GyrB). Analytical ultracentrifugation shows that GyrB is mainly a nonglobular monomer. Ab initio modeling of small-angle X-ray scattering data for GyrB consistently yields a "tadpole"-like envelope. It allows us to propose an organization of GyrB into three domains-ATPase, Toprim, and Tail-based on their crystallographic and modeled structures. Our study reveals the modular organization of GyrB and points out its potential flexibility, needed during the gyrase catalytic cycle. It provides important insights into the supercoiling mechanism by gyrase and suggests new lines of research.

Self-homodimerization of an actinoporin by disulfide bridging reveals implications for their structure and pore formation.

PubMed

Valle, Aisel; Pérez-Socas, Luis Benito; Canet, Liem; Hervis, Yadira de la Patria; de Armas-Guitart, German; Martins-de-Sa, Diogo; Lima, Jônatas Cunha Barbosa; Souza, Adolfo Carlos Barros; Barbosa, João Alexandre Ribeiro Gonçalves; de Freitas, Sonia Maria; Pazos, Isabel Fabiola

2018-04-26

The Trp111 to Cys mutant of sticholysin I, an actinoporin from Stichodactyla helianthus sea anemone, forms a homodimer via a disulfide bridge. The purified dimer is 193 times less hemolytic than the monomer. Ultracentrifugation, dynamic light scattering and size-exclusion chromatography demonstrate that monomers and dimers are the only independent oligomeric states encountered. Indeed, circular dichroism and fluorescence spectroscopies showed that Trp/Tyr residues participate in homodimerization and that the dimer is less thermostable than the monomer. A homodimer three-dimensional model was constructed and indicates that Trp147/Tyr137 are at the homodimer interface. Spectroscopy results validated the 3D-model and assigned 85° to the disulfide bridge dihedral angle responsible for dimerization. The homodimer model suggests that alterations in the membrane/carbohydrate-binding sites in one of the monomers, as result of dimerization, could explain the decrease in the homodimer ability to form pores.

Incorporation of adenylate cyclase into membranes of giant liposomes using membrane fusion with recombinant baculovirus-budded virus particles.

PubMed

Mori, Takaaki; Kamiya, Koki; Tomita, Masahiro; Yoshimura, Tetsuro; Tsumoto, Kanta

2014-06-01

Recombinant transmembrane adenylate cyclase (AC) was incorporated into membranes of giant liposomes using membrane fusion between liposomes and baculovirus-budded virus (BV). AC genes were constructed into transfer vectors in a form fused with fluorescent protein or polyhistidine at the C-terminus. The recombinant BVs were collected by ultracentrifugation and AC expression was verified using western blotting. The BVs and giant liposomes generated using gentle hydration were fused under acidic conditions; the incorporation of AC into giant liposomes was demonstrated by confocal laser scanning microscopy through the emission of fluorescence from their membranes. The AC-expressing BVs were also fused with liposomes containing the substrate (ATP) with/without a specific inhibitor (SQ 22536). An enzyme immunoassay on extracts of the sample demonstrated that cAMP was produced inside the liposomes. This procedure facilitates direct introduction of large transmembrane proteins into artificial membranes without solubilization.

Preparation of a highly concentrated, completely monomeric, active sarcoplasmic reticulum Ca2+-ATPase.

PubMed

Lüdi, H; Hasselbach, W

1985-11-21

Sarcoplasmic reticulum vesicles from fast skeletal muscle were partially delipidated with sodium cholate at high ionic strength and sedimented in a discontinuous sucrose gradient. Phospholipid content was reduced from 0.777 mumol/mg protein to 0.242 mumol/mg protein. As judged from gel electrophoresis and high pressure liquid gel chromatography, accessory proteins were removed during centrifugation and the Ca2+-ATPase was obtained in an almost pure form. Addition of myristoylglycerophosphocholine (1 mg/mg protein) reactivates ATPase and dinitrophenylphosphatase activity to the same degree obtained with native vesicles. Using the analytical ultracentrifuge it could be demonstrated that the reactivated Ca2+-ATPase was present exclusively in a monomeric state. These results were obtained at high and low ionic strength and up to a protein concentration of 10 mg/ml. Therefore this preparation should be very useful to investigate differences between oligomeric and monomeric Ca2+-ATPase.

Interaction of Myosin Phosphatase Target Subunit (MYPT1) with Myosin Phosphatase-RhoA Interacting Protein (MRIP): A Role of Glutamic Acids in the Interaction.

PubMed

Lee, Eunhee; Stafford, Walter F

2015-01-01

Scaffold proteins bind to and functionally link protein members of signaling pathways. Interaction of the scaffold proteins, myosin phosphatase target subunit (MYPT1) and myosin phosphatase-RhoA interacting protein (MRIP), causes co-localization of myosin phosphatase and RhoA to actomyosin. To examine biophysical properties of interaction of MYPT1 with MRIP, we employed analytical ultracentrifugation and surface plasmon resonance. In regard to MRIP, its residues 724-837 are sufficient for the MYPT1/MRIP interaction. Moreover, MRIP binds to MYPT1 as either a monomer or a dimer. With respect to MYPT1, its leucine repeat region, LR (residues 991-1030) is sufficient to account for the MYPT1/MRIP interaction. Furthermore, point mutations that replace glutamic acids 998-1000 within LR reduced the binding affinity toward MRIP. This suggests that the glutamic acids of MYPT1 play an important role in the interaction.

Guine pig liver L-asparaginase. Separation, purification, and intracellular localisation of two distinct enzymatic activities.

PubMed

Rogez, J C; Plaquet, R; Biserte, G

1975-12-18

Two distinct L-asparaginase (EC 3.5.1.1) activities were detected in guinea pig liver: Asparaginase 1 and Asparaginase 2. Asparaginase 1 has been purified 272 fold from the crude homogenate; its molecular weight was evaluated by gel filtration to be about 150 000. The purified preparation was shown to be homogeneous by cellulose acetate strip and polyacrylamide disc-gel electrophoresis. Asparaginase 2 has been purified 63.5 fold from the crude homogenate. Its molecular weight was evaluated by gel filtration to be about 21 500. Cellulose acetate strip electrophoresis demonstrated two bands, one of which corresponded to Asparaginase 1 and the other to Asparaginase 2. Cellular fractionation in the ultracentrifuge, showed Asparaginase 1 to be present only in the cytosol fraction. Asparaginase 2 which was unstable at 105 000 X g seemed mostly localized in the mitochondria and secondarily in the cytoplasmic fraction.

Adsorption Studies of Gadolinium ion on Graphitic Carbon Nitride

NASA Astrophysics Data System (ADS)

Kuila, S. K.; Kundu, T. K.

2018-03-01

Bulk graphitic carbon nitride (g-C3N4) is synthesized by thermal decomposition of urea and used as an adsorbent for gadolinium ion (Gd3+) from aqueous solution. Adsorption capacity of g-C3N4 is found to be influenced by initial Gd3+ concentration, solution pH and contact time. Adsorbed Gd3+is separated from g-C3N4 by ultracentrifuge. Initial and Gd ion accumulated g-C3N4 adsorbent are characterized by X-ray diffraction technique (XRD) for phase identification, UV-visible and Fourier transform infrared (FTIR) spectroscopy for adsorption characteristics and optical property, scanning electron microscopy (SEM) for morphological behaviour along with energy dispersive X-ray spectroscopy (EDS) for elemental study. HNO3(0.1M), NaOH (0.1M) and de-ionized water are used for desorption and around 97% quantitative recovery of Gd ion is observed.

Purification of Bacteriophages Using Anion-Exchange Chromatography.

PubMed

Vandenheuvel, Dieter; Rombouts, Sofie; Adriaenssens, Evelien M

2018-01-01

In bacteriophage research and therapy, most applications ask for highly purified phage suspensions. The standard technique for this is ultracentrifugation using cesium chloride gradients. This technique is cumbersome, elaborate and expensive. Moreover, it is unsuitable for the purification of large quantities of phage suspensions.The protocol described here, uses anion-exchange chromatography to bind phages to a stationary phase. This is done using an FLPC system, combined with Convective Interaction Media (CIM ® ) monoliths. Afterward, the column is washed to remove impurities from the CIM ® disk. By using a buffer solution with a high ionic strength, the phages are subsequently eluted from the column and collected. In this way phages can be efficiently purified and concentrated.This protocol can be used to determine the optimal buffers, stationary phase chemistry and elution conditions, as well as the maximal capacity and recovery of the columns.

Value of Osteoblast-Derived Exosomes in Bone Diseases.

PubMed

Ge, Min; Wu, Yingzhi; Ke, Ronghu; Cai, Tianyi; Yang, Junyi; Mu, Xiongzheng

2017-06-01

The authors' purpose is to reveal the value of osteoblast-derived exosomes in bone diseases. Microvesicles from supernatants of mouse Mc3t3 were isolated by ultracentrifugation and then the authors presented the protein profile by proteomics analysis. The authors detected a total number of 1536 proteins by mass spectrometry and found 172 proteins overlap with bone database. The Ingenuity Pathway Analysis shows network of "Skeletal and Muscular System Development and Function, Developmental Disorder, Hereditary Disorder" and pathway about osteogenesis. EFNB1 and transforming growth factor beta receptor 3 in the network, LRP6, bone morphogenetic protein receptor type-1, and SMURF1 in the pathway seemed to be valuable in the exosome research of related bone disease. The authors' study unveiled the content of osteoblast-derived exosome and discussed valuable protein in it which might provide novel prospective in bone diseases research.

Purification and properties of nitroalkane-oxidizing enzyme from Hansenula mrakii.

PubMed Central

Kido, T; Yamamoto, T; Soda, K

1976-01-01

A nitroalkane-oxidizing enzyme was purified about 1,300-fold from a cell extract of Hansenula mrakii grown in a medium containing nitroethane as the sole nitrogen source by ammonium sulfate fractionation, diethylaminoethyl-cellulose column chromatography, hydroxyapatite column chromatography, and Bio-Gel P-150 column chromatography. The enzyme was shown to be homogeneous upon acrylamide gel electrophoresis and ultracentrifugation. The enzyme exhibits absorption maxima at 274, 370, 415, and 440 nm and a shoulder at 470 nm. Balance studies showed that 2 mol of 2-nitropropane is converted into an equimolar amount of acetone and nitrite with the consumption of 1 mol of oxygen. Hydrogen peroxide is not formed in the enzyme reaction. In addition to 2-nitropropane, 1-nitropropane and nitroethane are oxidatively dentrified by the enzyme, but nitromethane is inert to the enzyme. The nitroalkanes are not oxidized under anaerobic conditions. Images PMID:947888

Dioxane contributes to the altered conformation and oligomerization state of a designed engrailed homeodomain variant

PubMed Central

Hom, Geoffrey K.; Lassila, J. Kyle; Thomas, Leonard M.; Mayo, Stephen L.

2005-01-01

Our goal was to compute a stable, full-sequence design of the Drosophila melanogaster engrailed homeodomain. Thermal and chemical denaturation data indicated the design was significantly more stable than was the wild-type protein. The data were also nearly identical to those for a similar, later full-sequence design, which was shown by NMR to adopt the homeodomain fold: a three-helix, globular monomer. However, a 1.65 Å crystal structure of the design described here turned out to be of a completely different fold: a four-helix, rodlike tetramer. The crystallization conditions included ~25% dioxane, and subsequent experiments by circular dichroism and sedimentation velocity analytical ultracentrifugation indicated that dioxane increases the helicity and oligomerization state of the designed protein. We attribute at least part of the discrepancy between the target fold and the crystal structure to the presence of a high concentration of dioxane. PMID:15741348

The small heat shock protein Hsp27 affects assembly dynamics and structure of keratin intermediate filament networks.

PubMed

Kayser, Jona; Haslbeck, Martin; Dempfle, Lisa; Krause, Maike; Grashoff, Carsten; Buchner, Johannes; Herrmann, Harald; Bausch, Andreas R

2013-10-15

The mechanical properties of living cells are essential for many processes. They are defined by the cytoskeleton, a composite network of protein fibers. Thus, the precise control of its architecture is of paramount importance. Our knowledge about the molecular and physical mechanisms defining the network structure remains scarce, especially for the intermediate filament cytoskeleton. Here, we investigate the effect of small heat shock proteins on the keratin 8/18 intermediate filament cytoskeleton using a well-controlled model system of reconstituted keratin networks. We demonstrate that Hsp27 severely alters the structure of such networks by changing their assembly dynamics. Furthermore, the C-terminal tail domain of keratin 8 is shown to be essential for this effect. Combining results from fluorescence and electron microscopy with data from analytical ultracentrifugation reveals the crucial role of kinetic trapping in keratin network formation. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Binding of the cyclic AMP receptor protein of Escherichia coli to RNA polymerase.

PubMed Central

Pinkney, M; Hoggett, J G

1988-01-01

Fluorescence polarization studies were used to study the interaction of a fluorescein-labelled conjugate of the Escherichia coli cyclic AMP receptor protein (F-CRP) and RNA polymerase. Under conditions of physiological ionic strength, F-CRP binds to RNA polymerase holoenzyme in a cyclic AMP-dependent manner; the dissociation constant was about 3 microM in the presence of cyclic AMP and about 100 microM in its absence. Binding to core RNA polymerase under the same conditions was weak (Kdiss. approx. 80-100 microM) and independent of cyclic AMP. Competition experiments established that native CRP and F-CRP compete for the same binding site on RNA polymerase holoenzyme and that the native protein binds about 3 times more strongly than does F-CRP. Analytical ultracentrifuge studies showed that CRP binds predominantly to the monomeric rather than the dimeric form of RNA polymerase. PMID:2839152

A new general method for the assessment of the molecular-weight distribution of polydisperse preparations. Its application to an intestinal epithelial glycoprotein and two dextran samples, and comparison with a monodisperse glycoprotein

PubMed Central

Gibbons, Richard A.; Dixon, Stephen N.; Pocock, David H.

1973-01-01

A specimen of intestinal glycoprotein isolated from the pig and two samples of dextran, all of which are polydisperse (that is, the preparations may be regarded as consisting of a continuous distribution of molecular weights), have been examined in the ultracentrifuge under meniscus-depletion conditions at equilibrium. They are compared with each other and with a glycoprotein from Cysticercus tenuicollis cyst fluid which is almost monodisperse. The quantity c−⅓ (c=concentration) is plotted against ξ (the reduced radius); this plot is linear when the molecular-weight distribution approximates to the `most probable', i.e. when Mn:Mw:Mz: M(z+1)....... is as 1:2:3:4: etc. The use of this plot, and related procedures, to evaluate qualitatively and semi-quantitatively molecular-weight distribution functions where they can be realistically approximated to Schulz distributions is discussed. The theoretical basis is given in an Appendix. PMID:4778265

Solution Structures of 2 : 1 And 1 : 1 DNA Polymerase - DNA Complexes Probed By Ultracentrifugation And Small-Angle X-Ray Scattering

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

Tang, K.H.; /Ohio State U.; Niebuhr, M.

2009-04-30

We report small-angle X-ray scattering (SAXS) and sedimentation velocity (SV) studies on the enzyme-DNA complexes of rat DNA polymerase {beta} (Pol {beta}) and African swine fever virus DNA polymerase X (ASFV Pol X) with one-nucleotide gapped DNA. The results indicated formation of a 2 : 1 Pol {beta}-DNA complex, whereas only 1 : 1 Pol X-DNA complex was observed. Three-dimensional structural models for the 2 : 1 Pol {beta}-DNA and 1 : 1 Pol X-DNA complexes were generated from the SAXS experimental data to correlate with the functions of the DNA polymerases. The former indicates interactions of the 8 kDamore » 5{prime}-dRP lyase domain of the second Pol {beta} molecule with the active site of the 1 : 1 Pol {beta}-DNA complex, while the latter demonstrates how ASFV Pol X binds DNA in the absence of DNA-binding motif(s). As ASFV Pol X has no 5{prime}-dRP lyase domain, it is reasonable not to form a 2 : 1 complex. Based on the enhanced activities of the 2 : 1 complex and the observation that the 8 kDa domain is not in an optimal configuration for the 5{prime}-dRP lyase reaction in the crystal structures of the closed ternary enzyme-DNA-dNTP complexes, we propose that the asymmetric 2 : 1 Pol {beta}-DNA complex enhances the function of Pol {beta}.« less

Self-interaction of NPM1 modulates multiple mechanisms of liquid-liquid phase separation.

PubMed

Mitrea, Diana M; Cika, Jaclyn A; Stanley, Christopher B; Nourse, Amanda; Onuchic, Paulo L; Banerjee, Priya R; Phillips, Aaron H; Park, Cheon-Gil; Deniz, Ashok A; Kriwacki, Richard W

2018-02-26

Nucleophosmin (NPM1) is an abundant, oligomeric protein in the granular component of the nucleolus with roles in ribosome biogenesis. Pentameric NPM1 undergoes liquid-liquid phase separation (LLPS) via heterotypic interactions with nucleolar components, including ribosomal RNA (rRNA) and proteins which display multivalent arginine-rich linear motifs (R-motifs), and is integral to the liquid-like nucleolar matrix. Here we show that NPM1 can also undergo LLPS via homotypic interactions between its polyampholytic intrinsically disordered regions, a mechanism that opposes LLPS via heterotypic interactions. Using a combination of biophysical techniques, including confocal microscopy, SAXS, analytical ultracentrifugation, and single-molecule fluorescence, we describe how conformational changes within NPM1 control valency and switching between the different LLPS mechanisms. We propose that this newly discovered interplay between multiple LLPS mechanisms may influence the direction of vectorial pre-ribosomal particle assembly within, and exit from the nucleolus as part of the ribosome biogenesis process.

An overview on genome organization of marine organisms.

PubMed

Costantini, Maria

2015-12-01

In this review we will concentrate on some general genome features of marine organisms and their evolution, ranging from vertebrate to invertebrates until unicellular organisms. Before genome sequencing, the ultracentrifugation in CsCl led to high resolution of mammalian DNA (without seeing at the sequence). The analytical profile of human DNA showed that the vertebrate genome is a mosaic of isochores, typically megabase-size DNA segments that belong in a small number of families characterized by different GC levels. The recent availability of a number of fully sequenced genomes allowed mapping very precisely the isochores, based on DNA sequences. Since isochores are tightly linked to biological properties such as gene density, replication timing and recombination, the new level of detail provided by the isochore map helped the understanding of genome structure, function and evolution. This led the current level of knowledge and to further insights. Copyright © 2015. Published by Elsevier B.V.

A centrifugation-based physicochemical characterization method for the interaction between proteins and nanoparticles

NASA Astrophysics Data System (ADS)

Bekdemir, Ahmet; Stellacci, Francesco

2016-10-01

Nanomedicine requires in-depth knowledge of nanoparticle-protein interactions. These interactions are studied with methods limited to large or fluorescently labelled nanoparticles as they rely on scattering or fluorescence-correlation signals. Here, we have developed a method based on analytical ultracentrifugation (AUC) as an absorbance-based, label-free tool to determine dissociation constants (KD), stoichiometry (Nmax), and Hill coefficient (n), for the association of bovine serum albumin (BSA) with gold nanoparticles. Absorption at 520 nm in AUC renders the measurements insensitive to unbound and aggregated proteins. Measurements remain accurate and do not become more challenging for small (sub-10 nm) nanoparticles. In AUC, frictional ratio analysis allows for the qualitative assessment of the shape of the analyte. Data suggests that small-nanoparticles/protein complexes significantly deviate from a spherical shape even at maximum coverage. We believe that this method could become one of the established approaches for the characterization of the interaction of (small) nanoparticles with proteins.

3D plasmonic nanobowl platform for the study of exosomes in solution

NASA Astrophysics Data System (ADS)

Lee, Changwon; Carney, Randy P.; Hazari, Sidhartha; Smith, Zachary J.; Knudson, Alisha; Robertson, Christopher S.; Lam, Kit S.; Wachsmann-Hogiu, Sebastian

2015-05-01

Thin silver film coated nanobowl Surface Enhanced Raman Spectroscopy (SERS) substrates are used to capture exosomes in solution for SERS measurements that can provide biochemical analysis of intact and ruptured exosomes. Exosomes derived via Total Exosome Isolation Reagent (TEIR) as well as ultracentrifugation (UC) from the SKOV3 cell line were analyzed. Spectra of exosomes derived via TEIR are dominated by a signal characteristic for the TEIR kit that needs to be subtracted for all measurements. Differences in SERS spectra recorded at different times during the drying of the exosome solution are statistically analyzed with Principal Component Analysis (PCA). At the beginning of the drying process, SERS spectra of exosomes exhibit peaks characteristic for both lipids and proteins. Later on during the drying process, new SERS peaks develop, suggesting that the initially intact exosome ruptures over time. This time-dependent evolution of SERS peaks enables analysis of exosomal membrane contents and the contents inside the exosomes.

Efficient method for preparation of highly purified lipopolysaccharides by hydrophobic interaction chromatography.

PubMed

Muck, A; Ramm, M; Hamburger, M

1999-09-10

A method for the efficient preparation of highly purified lipopolysaccharides (LPSs) by hydrophobic interaction chromatography (HIC) has been developed. The procedure can be used for the purification of cell wall bound LPSs after hot phenol-water extraction and for the isolation of extracellular LPSs from the supernatant, respectively. The method described has been tested with artificial mixtures containing LPSs, polysaccharide, protein and RNA and subsequently employed for the preparative purification of two LPSs of different origin, namely the extracellular LPS secreted by Escherichia coli E49 into the culture medium, and the cell wall bound LPS from Pseudomonas aeruginosa VA11465/1. Compared to currently used methods for LPS purification such as enzymatic digestion and ultracentrifugation, the chromatographic separation reported here combines superior purity with minimal loss of LPS, high reproducibility and simple handling. The removal of contaminants such as protein, RNA and polysaccharides and the recovery of LPSs were monitored by appropriate assays.

Primary Macroglobulinemia

PubMed Central

Edwards, A. M.; Costopoulous, L. B.; Bell, H. E.

1964-01-01

Clinical features presented by a patient with primary macroglobulinemia over a four-year period included cachexia, weight loss, bleeding tendency, anemia, lymphadenopathy, hepatosplenomegaly and recurrent pulmonary bacterial infections. Immunoelectrophoresis demonstrated the presence of a β2 macroglobulin which, on ultracentrifugation, was found to have a sedimentation constant of 14.9 S20, w; this macroglobulin constituted over 40% of the total serum protein. Postmortem findings included the typical “naked” lymphocyte infiltration of the reticuloendothelial system, with septic embolization from a terminal Gram-negative bacteremia, associated with a mesenteric vascular occlusion. A feature of particular interest was the antemortem appearance of gas in the portal venous system, shown on two abdominal scout radiographs taken one and two hours before death. The diagnostic significance of this rare radiologic sign is discussed. ImagesFig. 1Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8Fig. 9Fig. 10Fig. 11Fig. 12Fig. 13Fig. 14Fig. 15 PMID:14226112

Self-interaction of NPM1 modulates multiple mechanisms of liquid–liquid phase separation

DOE PAGES

Mitrea, Diana M.; Cika, Jaclyn A.; Stanley, Christopher B.; ...

2018-02-26

Nucleophosmin (NPM1) is an abundant, oligomeric protein in the granular component of the nucleolus with roles in ribosome biogenesis. Pentameric NPM1 undergoes liquid–liquid phase separation (LLPS) via heterotypic interactions with nucleolar components, including ribosomal RNA (rRNA) and proteins which display multivalent arginine-rich linear motifs (R-motifs), and is integral to the liquid-like nucleolar matrix. Here we show that NPM1 can also undergo LLPS via homotypic interactions between its polyampholytic intrinsically disordered regions, a mechanism that opposes LLPS via heterotypic interactions. Using a combination of biophysical techniques, including confocal microscopy, SAXS, analytical ultracentrifugation, and single-molecule fluorescence, we describe how conformational changes withinmore » NPM1 control valency and switching between the different LLPS mechanisms. We propose that this newly discovered interplay between multiple LLPS mechanisms may influence the direction of vectorial pre-ribosomal particle assembly within, and exit from the nucleolus as part of the ribosome biogenesis process.« less

Waterborne Colloidal Polymer/Silica Hybrid Dispersions and Their Assembly into Mesoporous Poly(melamine-formaldehyde) Xerogels.

PubMed

Schwarz, Dana; Weber, Jens

2015-08-04

The acid-catalyzed polycondensation of oligo(melamine-formaldehyde) in aqueous phase and in the presence of silica nanoparticles leads to a stable dispersion of coexisting silica and polymer nanoparticles. The dispersion can be processed into mesoporous xerogels (SBET ≈ 200 m(2) g(-1)), whose porosity can be enhanced by etching of silica up to specific surface areas of >400 m(2) g(-1). The formation mechanism and the characteristics of the hybrid dispersion are crucial to the materials derived from it and analyzed in detail using a variety of experimental techniques (electron and force microscopy, light and X-ray scattering, ultracentrifugation, and spectroscopy). The transformation of the dispersion into xerogels by electrostatic destabilization is described. Furthermore, the obtained materials are characterized with regard to their porosity and morphology using microscopy and porosimetry. The impact of selected synthesis parameters on the obtained properties is discussed, and it was found (most interestingly) that stable porosity was only observed if silica nanoparticles were present within the dispersion.

Effect of calcium ions on structure and stability of the C1q-like domain of otolin-1 from human and zebrafish.

PubMed

Hołubowicz, Rafał; Wojtas, Magdalena; Taube, Michał; Kozak, Maciej; Ożyhar, Andrzej; Dobryszycki, Piotr

2017-12-01

Otolin-1 is a collagen-like protein expressed in the inner ear of vertebrates. It provides an organic scaffold for otoliths in fish and otoconia in land vertebrates. In this study, the expression and purification procedure of C1q-like domain of otolin-1 from human and zebrafish was developed. The structure and stability of the proteins were investigated. The results of sedimentation velocity analytical ultracentrifugation and small-angle X-ray scattering indicated that the C1q-like domain of otolin-1 forms stable trimers in solution in the presence of calcium ions. It was also observed that calcium ions influenced the secondary structure of the proteins. C1q-like domains were stabilized by the calcium ions. The human variant was especially affected by the calcium ions. The results indicate the importance of the C1q-like domain for the assembly of the organic matrix of otoliths and otoconia. © 2017 Federation of European Biochemical Societies.

Tubulin Dimer Reversible Dissociation

PubMed Central

Schuck, Peter; Sackett, Dan L.

2016-01-01

Tubulins are evolutionarily conserved proteins that reversibly polymerize and direct intracellular traffic. Of the tubulin family only αβ-tubulin forms stable dimers. We investigated the monomer-dimer equilibrium of rat brain αβ-tubulin using analytical ultracentrifugation and fluorescence anisotropy, observing tubulin in virtually fully monomeric and dimeric states. Monomeric tubulin was stable for a few hours and exchanged into preformed dimers, demonstrating reversibility of dimer dissociation. Global analysis combining sedimentation velocity and fluorescence anisotropy yielded Kd = 84 (54–123) nm. Dimer dissociation kinetics were measured by analyzing the shape of the sedimentation boundary and by the relaxation of fluorescence anisotropy following rapid dilution of labeled tubulin, yielding koff in the range 10−3–10−2 s−1. Thus, tubulin dimers reversibly dissociate with moderately fast kinetics. Monomer-monomer association is much less sensitive than dimer-dimer association to solution changes (GTP/GDP, urea, and trimethylamine oxide). PMID:26934918

Cellular processing of gold nanoparticles: CE-ICP-MS evidence for the speciation changes in human cytosol.

PubMed

Legat, Joanna; Matczuk, Magdalena; Timerbaev, Andrei R; Jarosz, Maciej

2018-01-01

The cellular uptake of gold nanoparticles (AuNPs) may (or may not) affect their speciation, but information on the chemical forms in which the particles exist in the cell remains obscure. An analytical method based on the use of capillary electrophoresis hyphenated with inductively coupled plasma mass spectrometry (ICP-MS) has been proposed to shed light on the intracellular processing of AuNPs. It was observed that when being introduced into normal cytosol, the conjugates of 10-50 nm AuNPs with albumin evolved in human serum stayed intact. On the contrary, under simulated cancer cytosol conditions, the nanoconjugates underwent decomposition, the rate of which and the resulting metal speciation patterns were strongly influenced by particle size. The new peaks that appeared in ICP-MS electropherograms could be ascribed to nanosized species, as upon ultracentrifugation, they quantitatively precipitated whereas the supernatant showed only trace Au signals. Our present study is the first step to unravel a mystery of the cellular chemistry for metal-based nanomedicines.

Ensemble brightening and enhanced quantum yield in size-purified silicon nanocrystals

DOE PAGES

Miller, Joseph B.; Van Sickle, Austin R.; Anthony, Rebecca J.; ...

2012-07-18

Here, we report on the quantum yield, photoluminescence (PL) lifetime and ensemble photoluminescent stability of highly monodisperse plasma-synthesized silicon nanocrystals (SiNCs) prepared though density-gradient ultracentrifugation in mixed organic solvents. Improved size uniformity leads to a reduction in PL line width and the emergence of entropic order in dry nanocrystal films. We find excellent agreement with the anticipated trends of quantum confinement in nanocrystalline silicon, with a solution quantum yield that is independent of nanocrystal size for the larger fractions but decreases dramatically with size for the smaller fractions. We also find a significant PL enhancement in films assembled from themore » fractions, and we use a combination of measurement, simulation and modeling to link this ‘brightening’ to a temporally enhanced quantum yield arising from SiNC interactions in ordered ensembles of monodisperse nanocrystals. Using an appropriate excitation scheme, we exploit this enhancement to achieve photostable emission.« less

Brain-derived exosomes from dementia with Lewy bodies propagate α-synuclein pathology.

PubMed

Ngolab, Jennifer; Trinh, Ivy; Rockenstein, Edward; Mante, Michael; Florio, Jazmin; Trejo, Margarita; Masliah, Deborah; Adame, Anthony; Masliah, Eliezer; Rissman, Robert A

2017-06-09

Proteins implicated in neurodegenerative conditions such as Alzheimer's disease (AD) and Dementia with Lewy Bodies (DLB) have been identified in bodily fluids encased in extracellular vesicles called exosomes. Whether exosomes found in DLB patients can transmit pathology is not clear. In this study, exosomes were successfully harvested through ultracentrifugation from brain tissue from DLB and AD patients as well as non-diseased brain tissue. Exosomes extracted from brains diagnosed with either AD or DLB contained aggregate-prone proteins. Furthermore, injection of brain-derived exosomes from DLB patients into the brains of wild type mice induced α-synuclein (α-syn) aggregation. As assessed through immunofluorescent double labeling, α-syn aggregation was observed in MAP2 + , Rab5 + neurons. Using a neuronal cell line, we also identified intracellular α-syn aggregation mediated by exosomes is dependent on recipient cell endocytosis. Together, these data suggest that exosomes from DLB patients are sufficient for seeding and propagating α-syn aggregation in vivo.

Stepwise Assembly and Characterization of DNA Linked Two-Color Quantum Dot Clusters.

PubMed

Coopersmith, Kaitlin; Han, Hyunjoo; Maye, Mathew M

2015-07-14

The DNA-mediated self-assembly of multicolor quantum dot (QD) clusters via a stepwise approach is described. The CdSe/ZnS QDs were synthesized and functionalized with an amphiphilic copolymer, followed by ssDNA conjugation. At each functionalization step, the QDs were purified via gradient ultracentrifugation, which was found to remove excess polymer and QD aggregates, allowing for improved conjugation yields and assembly reactivity. The QDs were then assembled and disassembled in a stepwise manner at a ssDNA functionalized magnetic colloid, which provided a convenient way to remove unreacted QDs and ssDNA impurities. After assembly/disassembly, the clusters' optical characteristics were studied by fluorescence spectroscopy and the assembly morphology and stoichiometry was imaged via electron microscopy. The results indicate that a significant amount of QD-to-QD energy transfer occurred in the clusters, which was studied as a function of increasing acceptor-to-donor ratios, resulting in increased QD acceptor emission intensities compared to controls.

Soluble Amyloid-beta Aggregates from Human Alzheimer’s Disease Brains

PubMed Central

Esparza, Thomas J.; Wildburger, Norelle C.; Jiang, Hao; Gangolli, Mihika; Cairns, Nigel J.; Bateman, Randall J.; Brody, David L.

2016-01-01

Soluble amyloid-beta (Aβ) aggregates likely contribute substantially to the dementia that characterizes Alzheimer’s disease. However, despite intensive study of in vitro preparations and animal models, little is known about the characteristics of soluble Aβ aggregates in the human Alzheimer’s disease brain. Here we present a new method for extracting soluble Aβ aggregates from human brains, separating them from insoluble aggregates and Aβ monomers using differential ultracentrifugation, and purifying them >6000 fold by dual antibody immunoprecipitation. The method resulted in <40% loss of starting material, no detectible ex vivo aggregation of monomeric Aβ, and no apparent ex vivo alterations in soluble aggregate sizes. By immunoelectron microscopy, soluble Aβ aggregates typically appear as clusters of 10–20 nanometer diameter ovoid structures with 2-3 amino-terminal Aβ antibody binding sites, distinct from previously characterized structures. This approach may facilitate investigation into the characteristics of native soluble Aβ aggregates, and deepen our understanding of Alzheimer’s dementia. PMID:27917876

Lack of effect of acute repaglinide administration on postprandial lipaemia in patients with type 2 diabetes mellitus.

PubMed

Tentolouris, N; Kolia, M; Tselepis, A D; Perea, D; Kitsou, E; Kyriaki, D; Tambaki, A P; Karabina, S P; Sala, C; Fragoulopoulos, E; Katsilambros, N

2003-09-01

The effect of acute repaglinide administration (2 mg) on postprandial glycaemia and lipaemia has been examined in 20 subjects with type 2 diabetes mellitus. Each subject received in the morning, after a 12 to 14 h fast, a standard mixed meal (total energy 783 kcal), preceded by one tablet of 2 mg repaglinide or placebo. Chylomicrons and chylomicron-deficient plasma were prepared by ultracentrifugation. Triglyceride levels in CM fraction (CM-triglycerides) in total plasma as well as in CM-deficient plasma (non-CM-triglycerides) were determined. A significant reduction in postprandial glycaemia was observed after repaglinide administration compared to placebo ( p < 0.001). Plasma concentrations of total triglycerides, CM-triglycerides, non-CM-triglycerides, free fatty acids and the other plasma lipids measured, were not significantly different between the two phases of the study. It is concluded that, in contrast to sulphonylureas, acute repaglinide administration does not improve postprandial lipaemia in patients with type 2 diabetes.

Structural Characterization of the Boca/Mesd Maturation Factors for LDL-Receptor-Type beta Propeller Domains

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

M Collins; W Hendrickson

2011-12-31

Folding and trafficking of low-density lipoprotein receptor (LDLR) family members, which play essential roles in development and homeostasis, are mediated by specific chaperones. The Boca/Mesd chaperone family specifically promotes folding and trafficking of the YWTD {beta} propeller-EGF domain pair found in the ectodomain of all LDLR members. Limited proteolysis, NMR spectroscopy, analytical ultracentrifugation, and X-ray crystallography were used to define a conserved core composed of a structured domain that is preceded by a disordered N-terminal region. High-resolution structures of the ordered domain were determined for homologous proteins from three metazoans. Seven independent protomers reveal a novel ferrodoxin-like superfamily fold withmore » two distinct {beta} sheet topologies. A conserved hydrophobic surface forms a dimer interface in each crystal, but these differ substantially at the atomic level, indicative of nonspecific hydrophobic interactions that may play a role in the chaperone activity of the Boca/Mesd family.« less

Synthesis and characterization of novel polymers from non-petroleum sources for use in enhanced oil recovery. Progress report, July 1,1981-June 30, 1982. [Starch-g-polyacrylamide; polysaccharides and acrylamides; Schardinger-. beta. -dextrin and acrylamide

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

Butler, G.B.; Hogen-Esch, T.E.

1982-01-01

Annual progress reports are presented for the following tasks: (1) synthesis and structural characterization of polysaccharide-based graft copolymers for use in tertiary oil recovery; (2) determination of physical properties of the polymers and their solutions, and screening of the polymers to determine their utility in oil recovery. Over the past year synthesis and characterization studies have continued in the following five areas: (1) starch-g-polyacrylamide (ST-g-PAM) copolymers; (2) graft copolymers of other polysaccharides (gum arabic, yellow dextrin, pectin, okra polysaccharide, and guar gum) and acrylamides; (3) a naturally occurring polysaccharide extracted from okra (Akro); (4) graft copolymers of Schardinger-..beta..-dextrin and acrylamidemore » (SD-g-PAM); (5) chemical degradation of ST-g-PAM and SD-g-PAM copolymers. For physical properties studies, the following areas were investigated: (1) characterization of copolymers by ultracentrifugation, size exclusion chromatography and nucleophore membrane filtration; (2) rheological studies on copolymers; and (3) statistical analysis of variables in graft copolymerization. (ATT)« less

Localization of cellulose synthase in Acetobacter xylinum

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

Bureau, T.E.

1987-01-01

The cytoplasmic and outer membranes of Acetobacter xylinum (ATCC 53582) were isolated by discontinuous sucrose density ultracentrifugation. Both lysozyme and trypsin were required for efficient crude membrane separation. Primary dehydrogenases and NADH oxidase were used as cytoplasmic membrane markers, and 2-keto-3-deoxy-octulosonic acid was used to identify the outer membranes. Cellulose synthetase activity was assayed as the conversion of radioactivity from UDP-(/sup 14/C)glucose into an alkali-insoluble ..beta..-1,4-D-(/sup 14/C)glucan. The cellulosic nature of the product was demonstrated by enzymatic hydrolysis followed by thin-layer chromatography, and by methylation analysis followed by thin-layer chromatography and gas chromatography-mass spectroscopy. X-ray diffraction analysis indicated that themore » in vitro product is cellulose II which is in contrast to the in vivo product, namely cellulose I. In addition, no microfibrillar morphology could be observed from negative stained and metal shadowed preparations of the in vitro product.« less

Citrus limon-derived nanovesicles inhibit cancer cell proliferation and suppress CML xenograft growth by inducing TRAIL-mediated cell death

PubMed Central

Raimondo, Stefania; Naselli, Flores; Fontana, Simona; Monteleone, Francesca; Lo Dico, Alessia; Saieva, Laura; Zito, Giovanni; Flugy, Anna; Manno, Mauro; Di Bella, Maria Antonietta; De Leo, Giacomo; Alessandro, Riccardo

2015-01-01

Nanosized vesicles are considered key players in cell to cell communication, thus influencing physiological and pathological processes, including cancer. Nanovesicles have also been found in edible-plants and have shown therapeutic activity in inflammatory bowel diseases; however information on their role in affecting cancer progression is missing. Our study identify for the first time a fraction of vesicles from lemon juice (Citrus limon L.), obtained as a result of different ultracentrifugation, with density ranging from 1,15 to 1,19 g/ml and specific proteomic profile. By using an in vitro approach, we show that isolated nanovesicles inhibit cancer cell proliferation in different tumor cell lines, by activating a TRAIL-mediated apoptotic cell death. Furthermore, we demonstrate that lemon nanovesicles suppress CML tumor growth in vivo by specifically reaching tumor site and by activating TRAIL-mediated apoptotic cell processes. Overall, this study suggests the possible use of plant-edible nanovesicles as a feasible approach in cancer treatment. PMID:26098775

How can macromolecular crowding inhibit biological reactions? The enhanced formation of DNA nanoparticles

PubMed Central

Hou, Sen; Trochimczyk, Piotr; Sun, Lili; Wisniewska, Agnieszka; Kalwarczyk, Tomasz; Zhang, Xuzhu; Wielgus-Kutrowska, Beata; Bzowska, Agnieszka; Holyst, Robert

2016-01-01

In contrast to the already known effect that macromolecular crowding usually promotes biological reactions, solutions of PEG 6k at high concentrations stop the cleavage of DNA by HindIII enzyme, due to the formation of DNA nanoparticles. We characterized the DNA nanoparticles and probed the prerequisites for their formation using multiple techniques such as fluorescence correlation spectroscopy, dynamic light scattering, fluorescence analytical ultracentrifugation etc. In >25% PEG 6k solution, macromolecular crowding promotes the formation of DNA nanoparticles with dimensions of several hundreds of nanometers. The formation of DNA nanoparticles is a fast and reversible process. Both plasmid DNA (2686 bp) and double-stranded/single-stranded DNA fragment (66bp/nt) can form nanoparticles. We attribute the enhanced nanoparticle formation to the depletion effect of macromolecular crowding. This study presents our idea to enhance the formation of DNA nanoparticles by macromolecular crowding, providing the first step towards a final solution to efficient gene therapy. PMID:26903405

How can macromolecular crowding inhibit biological reactions? The enhanced formation of DNA nanoparticles.

PubMed

Hou, Sen; Trochimczyk, Piotr; Sun, Lili; Wisniewska, Agnieszka; Kalwarczyk, Tomasz; Zhang, Xuzhu; Wielgus-Kutrowska, Beata; Bzowska, Agnieszka; Holyst, Robert

2016-02-23

In contrast to the already known effect that macromolecular crowding usually promotes biological reactions, solutions of PEG 6k at high concentrations stop the cleavage of DNA by HindIII enzyme, due to the formation of DNA nanoparticles. We characterized the DNA nanoparticles and probed the prerequisites for their formation using multiple techniques such as fluorescence correlation spectroscopy, dynamic light scattering, fluorescence analytical ultracentrifugation etc. In >25% PEG 6k solution, macromolecular crowding promotes the formation of DNA nanoparticles with dimensions of several hundreds of nanometers. The formation of DNA nanoparticles is a fast and reversible process. Both plasmid DNA (2686 bp) and double-stranded/single-stranded DNA fragment (66 bp/nt) can form nanoparticles. We attribute the enhanced nanoparticle formation to the depletion effect of macromolecular crowding. This study presents our idea to enhance the formation of DNA nanoparticles by macromolecular crowding, providing the first step towards a final solution to efficient gene therapy.

Structures of the Signal Recognition Particle Receptor from the Archaeon Pyrococcus furiosus: Implications for the Targeting Step at the Membrane

PubMed Central

Egea, Pascal F.; Tsuruta, Hiro; de Leon, Gladys P.; Napetschnig, Johanna; Walter, Peter; Stroud, Robert M.

2008-01-01

In all organisms, a ribonucleoprotein called the signal recognition particle (SRP) and its receptor (SR) target nascent proteins from the ribosome to the translocon for secretion or membrane insertion. We present the first X-ray structures of an archeal FtsY, the receptor from the hyper-thermophile Pyrococcus furiosus (Pfu), in its free and GDP•magnesium-bound forms. The highly charged N-terminal domain of Pfu-FtsY is distinguished by a long N-terminal helix. The basic charges on the surface of this helix are likely to regulate interactions at the membrane. A peripheral GDP bound near a regulatory motif could indicate a site of interaction between the receptor and ribosomal or SRP RNAs. Small angle X-ray scattering and analytical ultracentrifugation indicate that the crystal structure of Pfu-FtsY correlates well with the average conformation in solution. Based on previous structures of two sub-complexes, we propose a model of the core of archeal and eukaryotic SRP•SR targeting complexes. PMID:18978942

Design of a minimal protein oligomerization domain by a structural approach.

PubMed

Burkhard, P; Meier, M; Lustig, A

2000-12-01

Because of the simplicity and regularity of the alpha-helical coiled coil relative to other structural motifs, it can be conveniently used to clarify the molecular interactions responsible for protein folding and stability. Here we describe the de novo design and characterization of a two heptad-repeat peptide stabilized by a complex network of inter- and intrahelical salt bridges. Circular dichroism spectroscopy and analytical ultracentrifugation show that this peptide is highly alpha-helical and 100% dimeric tinder physiological buffer conditions. Interestingly, the peptide was shown to switch its oligomerization state from a dimer to a trimer upon increasing ionic strength. The correctness of the rational design principles used here is supported by details of the atomic structure of the peptide deduced from X-ray crystallography. The structure of the peptide shows that it is not a molten globule but assumes a unique, native-like conformation. This de novo peptide thus represents an attractive model system for the design of a molecular recognition system.

Design of a minimal protein oligomerization domain by a structural approach.

PubMed Central

Burkhard, P.; Meier, M.; Lustig, A.

2000-01-01

Because of the simplicity and regularity of the alpha-helical coiled coil relative to other structural motifs, it can be conveniently used to clarify the molecular interactions responsible for protein folding and stability. Here we describe the de novo design and characterization of a two heptad-repeat peptide stabilized by a complex network of inter- and intrahelical salt bridges. Circular dichroism spectroscopy and analytical ultracentrifugation show that this peptide is highly alpha-helical and 100% dimeric tinder physiological buffer conditions. Interestingly, the peptide was shown to switch its oligomerization state from a dimer to a trimer upon increasing ionic strength. The correctness of the rational design principles used here is supported by details of the atomic structure of the peptide deduced from X-ray crystallography. The structure of the peptide shows that it is not a molten globule but assumes a unique, native-like conformation. This de novo peptide thus represents an attractive model system for the design of a molecular recognition system. PMID:11206050

Transport of large breakdown products of dietary protein through the gut wall.

PubMed Central

Hemmings, W A; Williams, E W

1978-01-01

Ferritin or tritium labelled immunoglobulin G may, by electron microscopy, be demonstrated entering, within, and leaving the epithelial cells. Quantitative studies using various proteins labelled with radioiodine show that large amounts of protein bound radioactivity may be demonstrated in the tissues after feeding the labelled protein to adult rats by stomach tube. The molecular size of this material as determined by sugar gradient ultracentrifugation of tissue extracts ranges when IgG is fed from 50,000-20,000 Daltons. The material retains its ability to react as antigen with antisera specific to the original molecule: precipitation reactions may be obtained in gels and quantitative studies show that cnosiderable amounts of the protein-bound radioactivity are still specifically precipitable. Such studies have been carried out with alpha-gliadin as well as bovine IgG. At 100 days old rats may absorb as much as 40% of a dose of bovine IgG in the form of these large molecular breakdown products. PMID:680603

The pressure-induced, lactose-dependent changes in the composition and size of casein micelles.

PubMed

Wang, Pengjie; Jin, Shaoming; Guo, Huiyuan; Zhao, Liang; Ren, Fazheng

2015-04-15

The effects of lactose on the changes in the composition and size of casein micelles induced by high-pressure treatment and the related mechanism of action were investigated. Dispersions of ultracentrifuged casein micelle pellets with 0-10% (w/v) lactose were subjected to high pressure (400 MPa) at 20 °C for 40 min. The results indicated that the level of non-sedimentable caseins was positively related to the amount of lactose added prior to pressure treatment, and negatively correlated to the size. A mechanism for the pressure-induced, lactose-dependent changes in the casein micelles is proposed. Lactose inhibits the hydrophobic interactions between the micellar fragments during or after pressure release, through the hydrophilic layer formed by their hydrogen bonds around the micellar fragments. In addition, lactose does not favour the association between calcium and the casein aggregates after pressure release. Due to these two functions, lactose inhibited the formation of larger micelles after pressure treatment. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparative Proteomics of Human Monkeypox and Vaccinia Intracellular Mature and Extracellular Enveloped Virions

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

Manes, Nathan P.; Estep, Ryan D.; Mottaz, Heather M.

2008-03-07

Orthopoxviruses are the largest and most complex of the animal viruses. In response to the recent emergence of monkeypox in Africa and the threat of smallpox bioterrorism, virulent (monkeypox virus) and benign (vaccinia virus) orthopoxviruses were proteomically compared with the goal of identifying proteins required for pathogenesis. Orthopoxviruses were grown in HeLa cells to two different viral forms (intracellular mature virus and extracellular enveloped virus), purified by sucrose gradient ultracentrifugation, denatured using RapiGest™ surfactant, and digested with trypsin. Unfractionated samples and strong cation exchange HPLC fractions were analyzed by reversed-phase LC-MS/MS, and analyses of the MS/MS spectra using SEQUEST® andmore » X! Tandem resulted in the identification of hundreds of monkeypox, vaccinia, and copurified host proteins. The unfractionated samples were additionally analyzed by LC-MS on an LTQ-Orbitrap™, and the accurate mass and elution time tag approach was used to perform quantitative comparisons. Possible pathophysiological roles of differentially expressed orthopoxvirus genes are discussed.« less

Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA

PubMed Central

Mori, Tetsuya; Saveliev, Sergei V.; Xu, Yao; Stafford, Walter F.; Cox, Michael M.; Inman, Ross B.; Johnson, Carl H.

2002-01-01

KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecA/DnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecA/DnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns. PMID:12477935

Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA.

PubMed

Mori, Tetsuya; Saveliev, Sergei V; Xu, Yao; Stafford, Walter F; Cox, Michael M; Inman, Ross B; Johnson, Carl H

2002-12-24

KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecADnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecADnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns.

Effects of UVA irradiation, aryl azides, and reactive oxygen species on the orthogonal inactivation of the human immunodeficiency virus (HIV-1)

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

Belanger, Julie M.; Raviv, Yossef; Viard, Mathias

2011-08-15

Previously we reported that hydrophobic aryl azides partition into hydrophobic regions of the viral membrane of enveloped viruses and inactivate the virus upon UVA irradiation for 2 min. Prolonged irradiation (15 min) resulted in viral protein aggregation as visualized via Western blot analysis, due to reactive oxygen species (ROS) formation, with preservation of the surface antigenic epitopes. Herein, we demonstrate that these aggregates show detergent resistance and that this property may be useful towards the creation of a novel orthogonal virus inactivation strategy for use in preparing experimental vaccines. When ROS-modified HIV virus preparations were treated with 1% Triton X-100,more » there was an increase in the percent of viral proteins (gp41, p24) in the viral pellet after ultracentrifugation through sucrose. Transmission electron microscopy (TEM) of these detergent-resistant pellets shows some recognizable virus fragments, and immunoprecipitation studies of the gp41 aggregates suggest the aggregation is covalent in nature, involving short-range interactions.« less

A conserved tripeptide in CNG and HCN channels regulates ligand gating by controlling C-terminal oligomerization.

PubMed

Zhou, Lei; Olivier, Nelson B; Yao, Huan; Young, Edgar C; Siegelbaum, Steven A

2004-12-02

Cyclic nucleotides directly enhance the opening of the tetrameric CNG and HCN channels, although the mechanism remains unclear. We examined why HCN and certain CNG subunits form functional homomeric channels, whereas other CNG subunits only function in heteromeric channels. The "defect" in the CNGA4 subunit that prevents its homomeric expression was localized to its C-linker, which connects the transmembrane domain to the binding domain and contains a tripeptide that decreases the efficacy of ligand gating. Remarkably, replacement of the homologous HCN tripeptide with the CNGA4 sequence transformed cAMP into an inverse agonist that inhibits HCN channel opening. Using analytical ultracentrifugation, we identified the structural basis for this gating switch: whereas cAMP normally enhances the assembly of HCN C-terminal domains into a tetrameric gating ring, inclusion of the CNGA4 tripeptide reversed this action so that cAMP now causes gating ring disassembly. Thus, ligand gating depends on the dynamic oligomerization of C-terminal binding domains.

Proteomic analysis of exosomes from human neural stem cells by flow field-flow fractionation and nanoflow liquid chromatography-tandem mass spectrometry.

PubMed

Kang, Dukjin; Oh, Sunok; Ahn, Sung-Min; Lee, Bong-Hee; Moon, Myeong Hee

2008-08-01

Exosomes, small membrane vesicles secreted by a multitude of cell types, are involved in a wide range of physiological roles such as intercellular communication, membrane exchange between cells, and degradation as an alternative to lysosomes. Because of the small size of exosomes (30-100 nm) and the limitations of common separation procedures including ultracentrifugation and flow cytometry, size-based fractionation of exosomes has been challenging. In this study, we used flow field-flow fractionation (FlFFF) to fractionate exosomes according to differences in hydrodynamic diameter. The exosome fractions collected from FlFFF runs were examined by transmission electron microscopy (TEM) to morphologically confirm their identification as exosomes. Exosomal lysates of each fraction were digested and analyzed using nanoflow LC-ESI-MS-MS for protein identification. FIFFF, coupled with mass spectrometry, allows nanoscale size-based fractionation of exosomes and is more applicable to primary cells and stem cells since it requires much less starting material than conventional gel-based separation, in-gel digestion and the MS-MS method.

Self-interaction of NPM1 modulates multiple mechanisms of liquid–liquid phase separation

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

Mitrea, Diana M.; Cika, Jaclyn A.; Stanley, Christopher B.

Nucleophosmin (NPM1) is an abundant, oligomeric protein in the granular component of the nucleolus with roles in ribosome biogenesis. Pentameric NPM1 undergoes liquid–liquid phase separation (LLPS) via heterotypic interactions with nucleolar components, including ribosomal RNA (rRNA) and proteins which display multivalent arginine-rich linear motifs (R-motifs), and is integral to the liquid-like nucleolar matrix. Here we show that NPM1 can also undergo LLPS via homotypic interactions between its polyampholytic intrinsically disordered regions, a mechanism that opposes LLPS via heterotypic interactions. Using a combination of biophysical techniques, including confocal microscopy, SAXS, analytical ultracentrifugation, and single-molecule fluorescence, we describe how conformational changes withinmore » NPM1 control valency and switching between the different LLPS mechanisms. We propose that this newly discovered interplay between multiple LLPS mechanisms may influence the direction of vectorial pre-ribosomal particle assembly within, and exit from the nucleolus as part of the ribosome biogenesis process.« less

Classification of hyperlipidaemias and hyperlipoproteinaemias*

PubMed Central

1970-01-01

Many studies of atherosclerosis have indicated hyperlipidaemia as a predisposing factor to vascular disease. The relationship holds even for mild degrees of hyperlipidaemia, a fact that underlines the importance of this category of disorders. Both primary and secondary hyperlipidaemias represent such a variety of abnormalities that an internationally acceptable provisional classification is highly desirable in order to facilitate communication between scientists with different backgrounds. The present memorandum presents such a classification; it briefly describes the criteria for diagnosis of the main types of hyperlipidaemia as well as the methods of their determination. Because lipoproteins offer more information than analysis of plasma lipids (most of the plasma lipids being bound to various proteins), the classification is based on lipoprotein analyses by electrophoresis and ultracentrifugation. Simpler methods, however, such as the observation of plasma and measurements of cholesterol and triglycerides, are used to the fullest possible extent in determining the lipoprotein patterns. PMID:4930042

Purification, crystallization and X-ray crystallographic analysis of a putative exopolyphosphatase from Zymomonas mobilis

PubMed Central

Zhang, Aili; Guo, Erhong; Qian, Lanfang; Tang, Nga-Yeung; Watt, Rory M.; Bartlam, Mark

2016-01-01

Exopolyphosphatase (PPX) enzymes degrade inorganic polyphosphate (poly-P), which is essential for the survival of microbial cells in response to external stresses. In this study, a putative exopolyphosphatase from Zymomonas mobilis (ZmPPX) was crystallized. Crystals of the wild-type enzyme diffracted to 3.3 Å resolution and could not be optimized further. The truncation of 29 amino acids from the N-terminus resulted in crystals that diffracted to 1.8 Å resolution. The crystals belonged to space group C2, with unit-cell parameters a = 122.0, b = 47.1, c = 89.5 Å, α = γ = 90, β = 124.5°. An active-site mutant that crystallized in the same space group and with similar unit-cell parameters diffracted to 1.56 Å resolution. One molecule was identified per asymmetric unit. Analytical ultracentrifugation confirmed that ZmPPX forms a dimer in solution. It was confirmed that ZmPPX possesses exopolyphosphatase activity against a synthetic poly-P substrate. PMID:26919520

Purification, crystallization and X-ray crystallographic analysis of a putative exopolyphosphatase from Zymomonas mobilis.

PubMed

Zhang, Aili; Guo, Erhong; Qian, Lanfang; Tang, Nga-Yeung; Watt, Rory M; Bartlam, Mark

2016-03-01

Exopolyphosphatase (PPX) enzymes degrade inorganic polyphosphate (poly-P), which is essential for the survival of microbial cells in response to external stresses. In this study, a putative exopolyphosphatase from Zymomonas mobilis (ZmPPX) was crystallized. Crystals of the wild-type enzyme diffracted to 3.3 Å resolution and could not be optimized further. The truncation of 29 amino acids from the N-terminus resulted in crystals that diffracted to 1.8 Å resolution. The crystals belonged to space group C2, with unit-cell parameters a = 122.0, b = 47.1, c = 89.5 Å, α = γ = 90, β = 124.5°. An active-site mutant that crystallized in the same space group and with similar unit-cell parameters diffracted to 1.56 Å resolution. One molecule was identified per asymmetric unit. Analytical ultracentrifugation confirmed that ZmPPX forms a dimer in solution. It was confirmed that ZmPPX possesses exopolyphosphatase activity against a synthetic poly-P substrate.

Mapping of Adenovirus of serotype 3 fibre interaction to desmoglein 2 revealed a novel 'non-classical' mechanism of viral receptor engagement.

PubMed

Vassal-Stermann, Emilie; Mottet, Manon; Ducournau, Corinne; Iseni, Frédéric; Vragniau, Charles; Wang, Hongjie; Zubieta, Chloe; Lieber, André; Fender, Pascal

2018-05-30

High-affinity binding of the trimeric fibre protein to a cell surface primary receptor is a common feature shared by all adenovirus serotypes. Recently, a long elusive species B adenovirus receptor has been identified. Desmoglein 2 (DSG2) a component of desmosomal junction, has been reported to interact at high affinity with Human adenoviruses HAd3, HAd7, HAd11 and HAd14. Little is known with respect to the molecular interactions of adenovirus fibre with the DSG2 ectodomain. By using different DSG2 ectodomain constructs and biochemical and biophysical experiments, we report that the third extracellular cadherin domain (EC3) of DSG2 is critical for HAd3 fibre binding. Unexpectedly, stoichiometry studies using multi-angle laser light scattering (MALLS) and analytical ultra-centrifugation (AUC) revealed a non-classical 1:1 interaction (one DSG2 per trimeric fibre), thus differentiating 'DSG2-interacting' adenoviruses from other protein receptor interacting adenoviruses in their infection strategy.

Isolation of Polysomal RNA for Analyzing Stress-Responsive Genes Regulated at the Translational Level in Plants.

PubMed

Li, Yong-Fang; Mahalingam, Ramamurthy; Sunkar, Ramanjulu

2017-01-01

Alteration of gene expression is an essential mechanism, which allows plants to respond and adapt to adverse environmental conditions. Transcriptome and proteome analyses in plants exposed to abiotic stresses revealed that protein levels are not correlated with the changes in corresponding mRNAs, indicating regulation at translational level is another major regulator for gene expression. Analysis of translatome, which refers to all mRNAs associated with ribosomes, thus has the potential to bridge the gap between transcriptome and proteome. Polysomal RNA profiling and recently developed ribosome profiling (Ribo-seq) are two main methods for translatome analysis at global level. Here, we describe the classical procedure for polysomal RNA isolation by sucrose gradient ultracentrifugation followed by highthroughput RNA-seq to identify genes regulated at translational level. Polysomal RNA can be further used for a variety of downstream applications including Northern blot analysis, qRT-PCR, RNase protection assay, and microarray-based gene expression profiling.

High-rate nano-crystalline Li 4Ti 5O 12 attached on carbon nano-fibers for hybrid supercapacitors

NASA Astrophysics Data System (ADS)

Naoi, Katsuhiko; Ishimoto, Shuichi; Isobe, Yusaku; Aoyagi, Shintaro

A lithium titanate (Li 4Ti 5O 12)-based electrode which can operate at unusually high current density (300 C) was developed as negative electrode for hybrid capacitors. The high-rate Li 4Ti 5O 12 electrode has a unique nano-structure consisting of unusually small nano-crystalline Li 4Ti 5O 12 (ca. 5-20 nm) grafted onto carbon nano-fiber anchors (nc-Li 4Ti 5O 12/CNF). This nano-structured nc-Li 4Ti 5O 12/CNF composite are prepared by simple sol-gel method under ultra-centrifugal force (65,000 N) followed by instantaneous annealing at 900 °C for 3 min. A model hybrid capacitor cell consisting of a negative nc-Li 4Ti 5O 12/CNF composite electrode and a positive activated carbon electrode showed high energy density of 40 Wh L -1 and high power density of 7.5 kW L -1 comparable to conventional EDLCs.

[Efficient extraction of transmembrane proteins using ProteoExtract Transmembrane Protein Extraction Kit].

PubMed

Błachnio, Karina

2010-01-01

Detergents commonly used for solubilization of membrane proteins may be ionic or non-ionic. Exposing membrane proteins to detergents, however, can adversely affect their native structure, which can be a major hindrance for functional studies. This is especially true for proteins with multiple transmembrane domains. The ProteoExtract Transmembrane Protein Extraction Kit (TM-PEK), offered by Merck, provides a detergent-free novel reagents to enable the mild and efficient extraction of proteins containing seven transmembrane domains, such as GPCRs (G-Protein Coupled Receptors) e.g.: Frizzled-4 and CELSR-3, from mammalian cells. The fraction enriched in transmembrane proteins using TM-PEK is directly compatible with enzyme assays, non-denaturing gel electrophoresis, 1- and 2-D SDS-PAGE, MS analysis, Western blotting, immunoprecipitation and ELISA. Unlike many alternatives, TM-PEK extraction procedure does not require sonication, extended rigorous vortexing, ultracentrifugation, or incubation of samples at elevated temperatures--thus minimizing the risk of post-extraction degradation or modifications.

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

Lashuel, Hilal A.; Aljabari, Bayan; Sigurdsson, Einar M.

We demonstrate herein that human macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine expressed in the brain and not previously considered to be amyloidogenic, forms amyloid fibrils similar to those derived from the disease associated amyloidogenic proteins {beta}-amyloid and {alpha}-synuclein. Acid denaturing conditions were found to readily induce MIF to undergo amyloid fibril formation. MIF aggregates to form amyloid-like structures with a morphology that is highly dependent on pH. The mechanism of MIF amyloid formation was probed by electron microscopy, turbidity, Thioflavin T binding, circular dichroism spectroscopy, and analytical ultracentrifugation. The fibrillar structures formed by MIF bind Congo red andmore » exhibit the characteristic green birefringence under polarized light. These results are consistent with the notion that amyloid fibril formation is not an exclusive property of a select group of amyloidogenic proteins, and contribute to a better understanding of the factors which govern protein conformational changes and amyloid fibril formation in vivo.« less

Biophysical characterization of soluble Pseudomonas syringae ice nucleation protein InaZ fragments.

PubMed

Han, Yu Jin; Song, HyoJin; Lee, Chang Woo; Ly, Nguyễn Hoàng; Joo, Sang-Woo; Lee, Jun Hyuck; Kim, Soon-Jong; Park, SangYoun

2017-01-01

Ice nucleation protein (INP) with its functional domain consisting of multiple 48-residue repeat units effectively induces super-cooled water into ice. Circular dichroism and infrared deconvolution analyses on a soluble 240-residue fragment of Pseudomonas syringae InaZ (InaZ240) containing five 48-residue repeat units indicated that it is mostly composed of β-sheet and random coil. Analytical ultracentrifugation suggested that InaZ240 behaves as a monomer of an elongated ellipsoid. However, InaZ240 showed only minimum ice binding compared to anti-freeze proteins. Other P. syringae InaZ proteins with more 48-residue repeat units were made, in which the largest soluble fragment obtainable was an InaZ with twelve 48-residue repeat units. Size-exclusion chromatography analyses further suggested that the overall shape of the expressed InaZ fragments is pH-dependent, which becomes compact as the numbers of 48-residue repeat unit increase. Copyright © 2016 Elsevier B.V. All rights reserved.

Localization of organ-specific antigens in the nervous system of the rat.

PubMed

Weinrauder, H; Lach, B

1977-08-16

Localization of organ-specific brain antigens in the central nervous system of the rat has been studied by means of indirect immunofluorescence. Rabbit antiserum against homogenate of rat brain, previously absorbed with normal serum and homogenates of rat organs (kidney, liver, spleen), reacted with the water-soluble antigens of rat brain prepared by extraction with phosphate buffer (pH 7.3) and ultracentrifugation at 50 000 X g to give one band in the immunodiffusion test and 2--3 precipitation arcs in immunoelectrophoresis. There was also a positive reaction with peripheral nerve. The antigen was detectable in all regions of the CNS. Cells with distinct cytoplasmic immunofluorescence were most frequently observed in cerebellar white matter, pons, cerebellar pedunculi, longitudinal tracts of the brain stem. Positive immunofluorecence reaction has appeared in the outer plexiform layer and granular layer of the retina, satelite cells of the spinal root ganglia and Schwann cells. A similar reaction was observed in human, mouse and guinea pig brain slices. Both the morphological and immunochemical reactions are indicative of glial localization of this antigen.

In situ investigation of complex BaSO4 fiber generation in the presence of sodium polyacrylate. 1. Kinetics and solution analysis.

PubMed

Wang, Tongxin; Cölfen, Helmut

2006-10-10

Simple solution analysis of the formation mechanism of complex BaSO(4) fiber bundles in the presence of polyacrylate sodium salt, via a bioinspired approach, is reported. Titration of the polyacrylate solution with Ba(2+) revealed complex formation and the optimum ratio of Ba(2+) to polyacrylate for a slow polymer-controlled mineralization process. This is a much simpler and faster method to determine the appropriate additive/mineral concentration pairs as opposed to more common crystallization experiments in which the additive/mineral concentration is varied. Time-dependent pH measurements were carried out to determine the concentration of solution species from which BaSO(4) supersaturation throughout the fiber formation process can be calculated and the second-order kinetics of the Ba(2+) concentration in solution can be identified. Conductivity measurements, pH measurements, and analytical ultracentrifugation revealed the first formed species to be Ba-polyacrylate complexes. A combination of the solution analysis results and optical microscopic images allows a detailed picture of the complex precipitation and self-organization process, a particle-mediated process involving mesoscopic transformations, to be revealed.

Fractionation of Exosomes and DNA using Size-Based Separation at the Nanoscale

NASA Astrophysics Data System (ADS)

Wunsch, Benjamin; Smith, Joshua; Wang, Chao; Gifford, Stacey; Brink, Markus; Bruce, Robert; Solovitzky, Gustavo; Austin, Robert; Astier, Yann

Exosomes, a key target of ``liquid biopsies'', are nano-vesicles found in nearly all biological fluids. Exosomes are secreted by eukaryotic and prokaryotic cells alike, and contain information about their originating cells, including surface proteins, cytoplasmic proteins, and nucleic acids. One challenge in studying exosome morphology is the difficulty of sorting exosomes by size and surface markers. Common separation techniques for exosomes include ultracentrifugation and ultrafiltration, for preparation of large volume samples, but these techniques often show contamination and significant heterogeneity between preparations. To date, deterministic lateral displacement (DLD) pillar arrays in silicon have proven an efficient technology to sort, separate, and enrich micron-scale particles including human parasites, eukaryotic cells, blood cells, and circulating tumor cells in blood; however, the DLD technology has never been translated to the true nanoscale, where it could function on bio-colloids such as exosomes. We have fabricated nanoscale DLD (nanoDLD) arrays capable of rapidly sorting colloids down to 20 nm in continuous flow, and demonstrated size sorting of individual exosome vesicles and dsDNA polymers, opening the potential for on-chip biomolecule separation and diagnosti

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

PubMed Central

Chen, Chihchen; Lin, Bo-Ren; Hsu, Min-Yen; Cheng, Chao-Min

2015-01-01

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain nucleic acid and protein cargo and are increasingly recognized as a means of intercellular communication utilized by both eukaryote and prokaryote cells. However, due to their small size, current protocols for isolation of EVs are often time consuming, cumbersome, and require large sample volumes and expensive equipment, such as an ultracentrifuge. To address these limitations, we developed a paper-based immunoaffinity platform for separating subgroups of EVs that is easy, efficient, and requires sample volumes as low as 10 μl. Biological samples can be pipetted directly onto paper test zones that have been chemically modified with capture molecules that have high affinity to specific EV surface markers. We validate the assay by using scanning electron microscopy (SEM), paper-based enzyme-linked immunosorbent assays (P-ELISA), and transcriptome analysis. These paper-based devices will enable the study of EVs in the clinic and the research setting to help advance our understanding of EV functions in health and disease. PMID:25867034

Small-angle X-ray scattering reveals the solution structure of the full-length DNA gyrase a subunit.

PubMed

Costenaro, Lionel; Grossmann, J Günter; Ebel, Christine; Maxwell, Anthony

2005-02-01

DNA gyrase is the topoisomerase uniquely able to actively introduce negative supercoils into DNA. Vital in all bacteria, but absent in humans, this enzyme is a successful target for antibacterial drugs. From biophysical experiments in solution, we report the low-resolution structure of the full-length A subunit (GyrA). Analytical ultracentrifugation shows that GyrA is dimeric, but nonglobular. Ab initio modeling from small-angle X-ray scattering allows us to retrieve the molecular envelope of GyrA and thereby the organization of its domains. The available crystallographic structure of the amino-terminal domain (GyrA59) forms a dimeric core, and two additional pear-shaped densities closely flank it in an unexpected position. Each accommodates very well a carboxyl-terminal domain (GyrA-CTD) built from a homologous crystallographic structure. The uniqueness of gyrase is due to the ability of the GyrA-CTDs to wrap DNA. Their position within the GyrA structure strongly suggests a large conformation change of the enzyme upon DNA binding.

Analysis of lipoprotein profiles of healthy cats by gel-permeation high-performance liquid chromatography

PubMed Central

MIZUTANI, Hisashi; SAKO, Toshinori; OKUDA, Hiroko; ARAI, Nobuaki; KURIYAMA, Koji; MORI, Akihiro; YOSHIMURA, Itaru; KOYAMA, Hidekazu

2016-01-01

Density gradient ultracentrifugation (DGUC) and gel electrophoresis are conventionally used to obtain lipoprotein profiles of animals. We recently applied high-performance liquid chromatography with a gel permeation column (GP-HPLC) and an on-line dual enzymatic system to dogs for lipoprotein profile analysis. We compared the GP-HPLC with DGUC as a method to obtain a feline lipoprotein profile. The lipoprotein profiles showed large and small peaks, which corresponded to high-density lipoprotein (HDL) and low-density lipoprotein (LDL), respectively, whereas very low-density lipoprotein (VLDL) and chylomicron (CM) were only marginally detected. This profile was very similar to that of dogs reported previously. Healthy cats also had a small amount of cholesterol-rich particles distinct from the normal LDL or HDL profile. There was no difference in lipoprotein profiles between the sexes, but males had a significantly larger LDL particle size (P=0.015). This study shows the feasibility of GP-HPLC for obtaining accurate lipoprotein profiles with small sample volumes and provides valuable reference data for healthy cats that should facilitate diagnoses. PMID:27170431

The characterization of exosome from blood plasma of patients with colorectal cancer

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

Yunusova, N. V., E-mail: Bochkarevanv@oncology.tomsk.ru; Siberian State Medical University, Moskovsky Trakt 2, Tomsk, 634050; Tamkovich, S. N., E-mail: s.tamk@niboch.nsc.ru

Exosomes are extracellular membrane structures involved in many physiological and pathological processes including cancerogenesis and metastasis. The clarification of the criteria for exosome isolating and identifying is the purpose of this study. Exosome samples from the plasma of patients with colorectal cancer and healthy donors were examined using transmission electron microscopy and flow cytometry in accordance with the minimum requirements of “International Society for Extracellular Vesicles”. The choice of the method for isolation of exosomes from the blood plasma by ultrafiltration and ultracentrifugation allowed obtaining highly purified samples of exosomes, in which all the structural components were clearly seen. Themore » results obtained with flow cytometry suggest that exosomes of blood plasma from patients with colorectal cancer can be produced by epithelial cells. Moreover, cells produce different types of exosomes, which correspond to different mechanisms in sorting macromolecules in the membrane of multivesicular bodies. Determination of significant differences in the expression of specific exosomal proteins from colorectal cancer patients compared to healthy donors suggests a high diagnostic potential significance of circulating exosomes.« less

Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions

PubMed Central

Zhao, Huaying; Fu, Yan; Glasser, Carla; Andrade Alba, Eric J; Mayer, Mark L; Patterson, George; Schuck, Peter

2016-01-01

The dynamic assembly of multi-protein complexes underlies fundamental processes in cell biology. A mechanistic understanding of assemblies requires accurate measurement of their stoichiometry, affinity and cooperativity, and frequently consideration of multiple co-existing complexes. Sedimentation velocity analytical ultracentrifugation equipped with fluorescence detection (FDS-SV) allows the characterization of protein complexes free in solution with high size resolution, at concentrations in the nanomolar and picomolar range. Here, we extend the capabilities of FDS-SV with a single excitation wavelength from single-component to multi-component detection using photoswitchable fluorescent proteins (psFPs). We exploit their characteristic quantum yield of photo-switching to imprint spatio-temporal modulations onto the sedimentation signal that reveal different psFP-tagged protein components in the mixture. This novel approach facilitates studies of heterogeneous multi-protein complexes at orders of magnitude lower concentrations and for higher-affinity systems than previously possible. Using this technique we studied high-affinity interactions between the amino-terminal domains of GluA2 and GluA3 AMPA receptors. DOI: http://dx.doi.org/10.7554/eLife.17812.001 PMID:27436096

From synthetic coiled coils to functional proteins: automated design of a receptor for the calmodulin-binding domain of calcineurin.

PubMed

Ghirlanda, G; Lear, J D; Lombardi, A; DeGrado, W F

1998-08-14

A series of synthetic receptors capable of binding to the calmodulin-binding domain of calcineurin (CN393-414) was designed, synthesized and characterized. The design was accomplished by docking CN393-414 against a two-helix receptor, using an idealized three-stranded coiled coil as a starting geometry. The sequence of the receptor was chosen using a side-chain re-packing program, which employed a genetic algorithm to select potential binders from a total of 7.5x10(6) possible sequences. A total of 25 receptors were prepared, representing 13 sequences predicted by the algorithm as well as 12 related sequences that were not predicted. The receptors were characterized by CD spectroscopy, analytical ultracentrifugation, and binding assays. The receptors predicted by the algorithm bound CN393-414 with apparent dissociation constants ranging from 0.2 microM to >50 microM. Many of the receptors that were not predicted by the algorithm also bound to CN393-414. Methods to circumvent this problem and to improve the automated design of functional proteins are discussed. Copyright 1998 Academic Press

An apparatus for sequentially combining microvolumes of reagents by infrasonic mixing.

PubMed

Camien, M N; Warner, R C

1984-05-01

A method employing high-speed infrasonic mixing for obtaining timed samples for following the progress of a moderately rapid chemical reaction is described. Drops of 10 to 50 microliter each of two reagents are mixed to initiate the reaction, followed, after a measured time interval, by mixing with a drop of a third reagent to quench the reaction. The method was developed for measuring the rate of denaturation of covalently closed, circular DNA in NaOH at several temperatures. For this purpose the timed samples were analyzed by analytical ultracentrifugation. The apparatus was tested by determination of the rate of hydrolysis of 2,4-dinitrophenyl acetate in an alkaline buffer. The important characteristics of the method are (i) it requires very small volumes of sample and reagents; (ii) the components of the reaction mixture are pre-equilibrated and mixed with no transfer outside the prescribed constant temperature environment; (iii) the mixing is very rapid; and (iv) satisfactorily precise measurements of relatively short time intervals (approximately 2 sec minimum) between sequential mixings of the components are readily obtainable.

Formation of graded vanadium oxide (V-O compound) under strong gravitational field

NASA Astrophysics Data System (ADS)

Khandaker, Jahirul Islam; Tokuda, Makoto; Ogata, Yudai; Januszko, Kamila; Nishiyama, Tadao; Yoshiasa, Akira; Mashimo, Tsutomu

2015-05-01

Sedimentation of atoms induced under strong gravitational field gives a tool for controlling elemental compositions in condensed matter. We performed a strong-gravity experiment (0.397 × 106 G at 400 °C for 24 h) on a V2O5 polycrystal using the high-temperature ultracentrifuge to examine the composition change and further the structure change. The graded composition structure of V and O was formed along gravity direction, where V increases and O decreases with gravity. It was found by the x-ray diffraction and Raman scattering method that VO2 and V2O3 phases appeared and the amounts increased, while one of the V2O5 phase decreased gradually along gravity direction. The X-ray absorption near edge structure spectra analysis identified the chemical valency decrease (+5 to +3). The UV-Vis absorption spectroscopy addressed the shifting in center of major absorption peak to longer wavelength (red shift) with the increase in gravitational field. The tail absorption peak (band gap 2.09 eV) at strong gravity region in the graded structure showed transparent conductive oxide.

Properties of cupric ions in benzylamine oxidase from pig plasma as studied by magnetic-resonance and kinetic methods.

PubMed Central

Barker, R; Boden, N; Cayley, G; Charlton, S C; Henson, R; Holmes, M C; Kelly, I D; Knowles, P F

1979-01-01

Benzylamine oxidase from pig plasma has been studied by a variety of chemical and physical techniques. 1. Analytical ultracentrifugation, gel electrophoresis and isoelectric-focusing studies suggest that the enzyme is composed of two subunits with closely similar primary structures. 2. E.s.r. and n.m.r. measurements show that the enzyme contains two well-separated (greater than 0.6 nm) Cu2+ ions at chemically distinct sites. Each Cu2+ ion is coordinated by two water molecules, one 'axial' and the other 'equatorial'. Both water molecules undergo fast exchange (10(5)--10(8) s-1) with solvent and are deprotonated in the pH range 8--9, but only the equatorial water molecule is displaced by the inhibitors N3- and CN-. 3. Kinetic and e.s.r. measurements show that azide and cyanide compete against O2 binding and also make the two Cu2+ sites identical. It is concluded that Cu2+ must participate in the re-oxidation of reduced enzyme by molecular O2. PMID:218560

Extracellular Vesicles Present in Human Ovarian Tumor Microenvironments Induce a Phosphatidylserine Dependent Arrest in the T Cell Signaling Cascade

PubMed Central

Kelleher, Raymond J.; Balu-Iyer, Sathy; Loyall, Jenni; Sacca, Anthony J.; Shenoy, Gautam N.; Peng, Peng; Iyer, Vandana; Fathallah, Anas M.; Berenson, Charles S.; Wallace, Paul K.; Tario, Joseph; Odunsi, Kunle; Bankert, Richard B.

2015-01-01

The identification of immunosuppressive factors within human tumor microenvironments, and the ability to block these factors, would be expected to enhance patients’ anti-tumor immune responses. We previously established that an unidentified factor, or factors, present in ovarian tumor ascites fluids reversibly inhibited the activation of T cells by arresting the T cell signaling cascade. Ultracentrifugation of the tumor ascites fluid has now revealed a pellet that contains small extracellular vesicles (EV) with an average diameter of 80nm. The T cell arrest was determined to be causally linked to phosphatidylserine (PS) that is present on the outer leaflet of the vesicle bilayer, as a depletion of PS expressing EV or a blockade of PS with anti-PS antibody significantly inhibits the vesicle induced signaling arrest. The inhibitory EV were also isolated from solid tumor tissues. The presence of immune suppressive vesicles in the microenvironments of ovarian tumors and our ability to block their inhibition of T cell function represent a potential therapeutic target for patients with ovarian cancer. PMID:26112921

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

Yi, Jae-Sung; Choo, Hyo-Jung; Cho, Bong-Rae

Lipid rafts are plasma membrane platforms mediating signal transduction pathways for cellular proliferation, differentiation and apoptosis. Here, we show that membrane fluidity was increased in HeLa cells following treatment with ginsenoside Rh2 (Rh2), as determined by cell staining with carboxy-laurdan (C-laurdan), a two-photon dye designed for measuring membrane hydrophobicity. In the presence of Rh2, caveolin-1 appeared in non-raft fractions after sucrose gradient ultracentrifugation. In addition, caveolin-1 and GM1, lipid raft landmarkers, were internalized within cells after exposure to Rh2, indicating that Rh2 might disrupt lipid rafts. Since cholesterol overloading, which fortifies lipid rafts, prevented an increase in Rh2-induced membrane fluidity,more » caveolin-1 internalization and apoptosis, lipid rafts appear to be essential for Rh2-induced apoptosis. Moreover, Rh2-induced Fas oligomerization was abolished following cholesterol overloading, and Rh2-induced apoptosis was inhibited following treatment with siRNA for Fas. This result suggests that Rh2 is a novel lipid raft disruptor leading to Fas oligomerization and apoptosis.« less

Crystal Structure of Chicken γS-Crystallin Reveals Lattice Contacts with Implications for Function in the Lens and the Evolution of the βγ-Crystallins.

PubMed

Sagar, Vatsala; Chaturvedi, Sumit K; Schuck, Peter; Wistow, Graeme

2017-07-05

Previous attempts to crystallize mammalian γS-crystallin were unsuccessful. Native L16 chicken γS crystallized avidly while the Q16 mutant did not. The X-ray structure for chicken γS at 2.3 Å resolution shows the canonical structure of the superfamily plus a well-ordered N arm aligned with a β sheet of a neighboring N domain. L16 is also in a lattice contact, partially shielded from solvent. Unexpectedly, the major lattice contact matches a conserved interface (QR) in the multimeric β-crystallins. QR shows little conservation of residue contacts, except for one between symmetry-related tyrosines, but molecular dipoles for the proteins with QR show striking similarities while other γ-crystallins differ. In γS, QR has few hydrophobic contacts and features a thin layer of tightly bound water. The free energy of QR is slightly repulsive and analytical ultracentrifugation confirms no dimerization in solution. The lattice contacts suggest how γ-crystallins allow close packing without aggregation in the crowded environment of the lens. Published by Elsevier Ltd.

Monomerization of viral entry inhibitor griffithsin elucidates the relationship between multivalent binding to carbohydrates and anti-HIV activity

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

Moulaei, Tinoush; Shenoy, Shilpa R.; Giomarelli, Barbara

2010-10-28

Mutations were introduced to the domain-swapped homodimer of the antiviral lectin griffithsin (GRFT). Whereas several single and double mutants remained dimeric, insertion of either two or four amino acids at the dimerization interface resulted in a monomeric form of the protein (mGRFT). Monomeric character of the modified proteins was confirmed by sedimentation equilibrium ultracentrifugation and by their high resolution X-ray crystal structures, whereas their binding to carbohydrates was assessed by isothermal titration calorimetry. Cell-based antiviral activity assays utilizing different variants of mGRFT indicated that the monomeric form of the lectin had greatly reduced activity against HIV-1, suggesting that the antiviralmore » activity of GRFT stems from crosslinking and aggregation of viral particles via multivalent interactions between GRFT and oligosaccharides present on HIV envelope glycoproteins. Atomic resolution crystal structure of a complex between mGRFT and nonamannoside revealed that a single mGRFT molecule binds to two different nonamannoside molecules through all three carbohydrate-binding sites present on the monomer.« less

Density contrast sedimentation velocity for the determination of protein partial-specific volumes.

PubMed

Brown, Patrick H; Balbo, Andrea; Zhao, Huaying; Ebel, Christine; Schuck, Peter

2011-01-01

The partial-specific volume of proteins is an important thermodynamic parameter required for the interpretation of data in several biophysical disciplines. Building on recent advances in the use of density variation sedimentation velocity analytical ultracentrifugation for the determination of macromolecular partial-specific volumes, we have explored a direct global modeling approach describing the sedimentation boundaries in different solvents with a joint differential sedimentation coefficient distribution. This takes full advantage of the influence of different macromolecular buoyancy on both the spread and the velocity of the sedimentation boundary. It should lend itself well to the study of interacting macromolecules and/or heterogeneous samples in microgram quantities. Model applications to three protein samples studied in either H(2)O, or isotopically enriched H(2) (18)O mixtures, indicate that partial-specific volumes can be determined with a statistical precision of better than 0.5%, provided signal/noise ratios of 50-100 can be achieved in the measurement of the macromolecular sedimentation velocity profiles. The approach is implemented in the global modeling software SEDPHAT.

Production of infectious ferret hepatitis E virus in a human hepatocarcinoma cell line PLC/PRF/5.

PubMed

Li, Tian-Cheng; Yoshizaki, Sayaka; Yang, Tingting; Kataoka, Michiyo; Nakamura, Tomofumi; Ami, Yasushi; Yuriko, Suzaki; Takeda, Naokazu; Wakita, Takaji

2016-02-02

A strain of ferret hepatitis E virus (HEV), sF4370, isolated from an imported ferret was used to inoculate a human hepatocarcinoma cell line, PLC/PRF/5. The virus genome and capsid protein were detected in the cell culture supernatant. Immunofluorescence microscopy indicated that the capsid protein was located in the cytoplasm. The virus particles were purified from the culture supernatant by sucrose gradient ultracentrifugation. The capsid protein with molecular mass of ∼72 kDa was detected in fractions with density of 1.150-1.162 g/cm(3), and particles of ferret HEV was associated with cell membrane. The virus recovered from the supernatant was serially passaged with PLC/PRF/5 cells and had the ability to infect ferrets by oral inoculation, indicating that the ferret HEV grown in PLC/PRF/5 was infectious. The establishment of ferret HEV cell culture system might be useful to understand the life cycle, mechanism of infection and replication of ferret HEV. Copyright © 2015 Elsevier B.V. All rights reserved.

Toward the establishment of standardized in vitro tests for lipid-based formulations, part 1: method parameterization and comparison of in vitro digestion profiles across a range of representative formulations.

PubMed

Williams, Hywel D; Sassene, Philip; Kleberg, Karen; Bakala-N'Goma, Jean-Claude; Calderone, Marilyn; Jannin, Vincent; Igonin, Annabel; Partheil, Anette; Marchaud, Delphine; Jule, Eduardo; Vertommen, Jan; Maio, Mario; Blundell, Ross; Benameur, Hassan; Carrière, Frédéric; Müllertz, Anette; Porter, Christopher J H; Pouton, Colin W

2012-09-01

The Lipid Formulation Classification System Consortium is an industry-academia collaboration, established to develop standardized in vitro methods for the assessment of lipid-based formulations (LBFs). In this first publication, baseline conditions for the conduct of digestion tests are suggested and a series of eight model LBFs are described to probe test performance across different formulation types. Digestion experiments were performed in vitro using a pH-stat apparatus and danazol employed as a model poorly water-soluble drug. LBF digestion (rate and extent) and drug solubilization patterns on digestion were examined. To evaluate cross-site reproducibility, experiments were conducted at two sites and highly consistent results were obtained. In a further refinement, bench-top centrifugation was explored as a higher throughput approach to separation of the products of digestion (and compared with ultracentrifugation), and conditions under which this method was acceptable were defined. Drug solubilization was highly dependent on LBF composition, but poorly correlated with simple performance indicators such as dispersion efficiency, confirming the utility of the digestion model as a means of formulation differentiation. Copyright © 2012 Wiley Periodicals, Inc.

The characterization of exosome from blood plasma of patients with colorectal cancer

NASA Astrophysics Data System (ADS)

Yunusova, N. V.; Tamkovich, S. N.; Stakheeva, M. N.; Afanas'ev, S. G.; Frolova, A. Y.; Kondakova, I. V.

2016-08-01

Exosomes are extracellular membrane structures involved in many physiological and pathological processes including cancerogenesis and metastasis. The clarification of the criteria for exosome isolating and identifying is the purpose of this study. Exosome samples from the plasma of patients with colorectal cancer and healthy donors were examined using transmission electron microscopy and flow cytometry in accordance with the minimum requirements of "International Society for Extracellular Vesicles". The choice of the method for isolation of exosomes from the blood plasma by ultrafiltration and ultracentrifugation allowed obtaining highly purified samples of exosomes, in which all the structural components were clearly seen. The results obtained with flow cytometry suggest that exosomes of blood plasma from patients with colorectal cancer can be produced by epithelial cells. Moreover, cells produce different types of exosomes, which correspond to different mechanisms in sorting macromolecules in the membrane of multivesicular bodies. Determination of significant differences in the expression of specific exosomal proteins from colorectal cancer patients compared to healthy donors suggests a high diagnostic potential significance of circulating exosomes.

Micromethod for phosphonoformate inhibition assay of hepatitis B viral DNA polymerase.

PubMed

Lin, H J; Wu, P C; Lai, C L; Chak, W

1984-04-01

A micromethod for the specific measurement of hepatitis B viral DNA polymerase in serum is presented, based on the phosphonoformate inhibition assay (J Med Virol 12: 61-70, 1983). In the micromethod, sample volume is reduced to 120 microL and the ultracentrifugation step is eliminated. The method allows good discrimination between serum infected with hepatitis B virus and uninfected serum. The cutoff value for rate of nucleotide incorporation, based on assays of 41 serum specimens negative for hepatitis B serological markers, was about 15 nU/L (90th percentile). Serum containing hepatitis B surface and antigens exhibited rates of phosphonoformate-inhibitive nucleotide incorporation of 150 (SD 150) nU/L, with an upper 90th percentile range of 17 to 667 nU/L (n = 41). The micromethod makes use of commercially available [32P]dCTP (specific activity about 7000 kCi/mol). 125I-labeled dCTP was found to be unsuitable for this assay. Human DNA polymerases in serum are detected by this method but are excluded from the phosphonoformate-inhibitive fraction.

In vitro synthesis of cellulose II from a cytoplasmic membrane fraction of Acetobacter xylinum

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

Bureau, T.E.; Brown, R.M. Jr.

1987-10-01

The cytoplasmic and outer membranes of Acetobacter xylinum were isolated by discontinuous sucrose density ultracentrifugation. Both lysozyme and trypsin were required for efficient crude membrane separation. Primary dehydrogenases and NADH oxidase were used as cytoplasmic membrane markers, and 2-keto-3-deoxyoctulosonic acid was used to identify the outer membranes. Cellulose synthetase activity was assayed as the conversion of radioactivity from UDP-(/sup 14/C)glucose into an alkali-insoluble ..beta..-1,4-D-(/sup 14/C)glucan. This activity was predominantly found in the cytoplasmic membrane. The cellulose nature of the product was demonstrated by (i) enzymatic hydrolysis followed by TLC, (ii) methylation analysis followed by TLC, and (iii) GC/MS. Further, themore » weight-average and number-average degree of polymerization of the in vitro product, determined by high-performance gel permeation chromatography, were 4820 and 5270, respectively. In addition, x-ray diffraction analysis indicated that the in vitro product is cellulose II, which is in contrast to the in vivo product--namely, cellulose I.« less

Flavonol Activation Defines an Unanticipated Ligand-Binding Site in the Kinase-RNase Domain of IRE1

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

Wiseman, R. Luke; Zhang, Yuhong; Lee, Kenneth P.K.

2010-08-18

Signaling in the most conserved branch of the endoplasmic reticulum (ER) unfolded protein response (UPR) is initiated by sequence-specific cleavage of the HAC1/XBP1 mRNA by the ER stress-induced kinase-endonuclease IRE1. We have discovered that the flavonol quercetin activates yeast IRE1's RNase and potentiates activation by ADP, a natural activating ligand that engages the IRE1 nucleotide-binding cleft. Enzyme kinetics and the structure of a cocrystal of IRE1 complexed with ADP and quercetin reveal engagement by quercetin of an unanticipated ligand-binding pocket at the dimer interface of IRE1's kinase extension nuclease (KEN) domain. Analytical ultracentrifugation and crosslinking studies support the preeminence ofmore » enhanced dimer formation in quercetin's mechanism of action. These findings hint at the existence of endogenous cytoplasmic ligands that may function alongside stress signals from the ER lumen to modulate IRE1 activity and at the potential for the development of drugs that modify UPR signaling from this unanticipated site.« less

Enzymatic dehalogenation of pentachlorophenol by extracts from Arthrobacter sp. strain ATCC 33790.

PubMed Central

Schenk, T; Müller, R; Mörsberger, F; Otto, M K; Lingens, F

1989-01-01

Arthrobacter sp. strain ATCC 33790 was grown with pentachlorophenol (PCP) as the sole source of carbon and energy. Crude extracts, which were prepared by disruption of the bacteria with a French pressure cell, showed no dehalogenating activity with PCP as the substrate. After sucrose density ultracentrifugation of the crude extract at 145,000 x g, various layers were found in the gradient. One yellow layer showed enzymatic conversion of PCP. One chloride ion was released per molecule of PCP. The product of the enzymatic conversion was tetrachlorohydroquinone. NADPH and oxygen were essential for this reaction. EDTA stimulated the enzymatic activity by 67%. The optimum pH for the enzyme activity was 7.5, and the temperature optimum was 25 degrees C. Enzymatic activity was also detected with 2,4,5-trichlorophenol, 2,3,4-trichlorophenol, 2,4,6-trichlorophenol, and 2,3,4,5-tetrachlorophenol as substrates, whereas 3,4,5-trichlorophenol, 2,4-dichlorophenol, 3,4-dichlorophenol, and 4-chlorophenol did not serve as substrates. PMID:2793827

STUDIES ON THE CHEMICAL NATURE OF THE SUBSTANCE INDUCING TRANSFORMATION OF PNEUMOCOCCAL TYPES

PubMed Central

Avery, Oswald T.; MacLeod, Colin M.; McCarty, Maclyn

1944-01-01

1. From Type III pneumococci a biologically active fraction has been isolated in highly purified form which in exceedingly minute amounts is capable under appropriate cultural conditions of inducing the transformation of unencapsulated R variants of Pneumococcus Type II into fully encapsulated cells of the same specific type as that of the heat-killed microorganisms from which the inducing material was recovered. 2. Methods for the isolation and purification of the active transforming material are described. 3. The data obtained by chemical, enzymatic, and serological analyses together with the results of preliminary studies by electrophoresis, ultracentrifugation, and ultraviolet spectroscopy indicate that, within the limits of the methods, the active fraction contains no demonstrable protein, unbound lipid, or serologically reactive polysaccharide and consists principally, if not solely, of a highly polymerized, viscous form of desoxyribonucleic acid. 4. Evidence is presented that the chemically induced alterations in cellular structure and function are predictable, type-specific, and transmissible in series. The various hypotheses that have been advanced concerning the nature of these changes are reviewed. PMID:19871359

Nano-structure of the laminin γ-1 short arm reveals an extended and curved multidomain assembly.

PubMed

Patel, Trushar R; Morris, Gordon A; Zwolanek, Daniela; Keene, Douglas R; Li, Jianhua; Harding, Stephen E; Koch, Manuel; Stetefeld, Jörg

2010-09-01

Laminins are multidomain glycoproteins that play important roles in development and maintenance of the extracellular matrix via their numerous interactions with other proteins. Several receptors for the laminin short arms revealed their importance in network formation and intercellular signaling. However, both the detailed structure of the laminin γ-1 short arm and its organization within the complexes is poorly understood due to the complexity of the molecule and the lack of a high-resolution structure. The presented data provide the first subatomic resolution structure for the laminin γ-1 short arm in solution. This was achieved using an integrated approach that combined a number of complementary biophysical techniques such as small angle X-ray scattering (SAXS), analytical ultracentrifugation, dynamic light scattering and electron microscopy. As a result of this study, we have obtained a significantly improved model for the laminin γ-1 short arm that represents a major step forward in molecular understanding of laminin-mediated complex formations. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

Density Contrast Sedimentation Velocity for the Determination of Protein Partial-Specific Volumes

PubMed Central

Brown, Patrick H.; Balbo, Andrea; Zhao, Huaying; Ebel, Christine; Schuck, Peter

2011-01-01

The partial-specific volume of proteins is an important thermodynamic parameter required for the interpretation of data in several biophysical disciplines. Building on recent advances in the use of density variation sedimentation velocity analytical ultracentrifugation for the determination of macromolecular partial-specific volumes, we have explored a direct global modeling approach describing the sedimentation boundaries in different solvents with a joint differential sedimentation coefficient distribution. This takes full advantage of the influence of different macromolecular buoyancy on both the spread and the velocity of the sedimentation boundary. It should lend itself well to the study of interacting macromolecules and/or heterogeneous samples in microgram quantities. Model applications to three protein samples studied in either H2O, or isotopically enriched H2 18O mixtures, indicate that partial-specific volumes can be determined with a statistical precision of better than 0.5%, provided signal/noise ratios of 50–100 can be achieved in the measurement of the macromolecular sedimentation velocity profiles. The approach is implemented in the global modeling software SEDPHAT. PMID:22028836

Guidance to Achieve Accurate Aggregate Quantitation in Biopharmaceuticals by SV-AUC.

PubMed

Arthur, Kelly K; Kendrick, Brent S; Gabrielson, John P

2015-01-01

The levels and types of aggregates present in protein biopharmaceuticals must be assessed during all stages of product development, manufacturing, and storage of the finished product. Routine monitoring of aggregate levels in biopharmaceuticals is typically achieved by size exclusion chromatography (SEC) due to its high precision, speed, robustness, and simplicity to operate. However, SEC is error prone and requires careful method development to ensure accuracy of reported aggregate levels. Sedimentation velocity analytical ultracentrifugation (SV-AUC) is an orthogonal technique that can be used to measure protein aggregation without many of the potential inaccuracies of SEC. In this chapter, we discuss applications of SV-AUC during biopharmaceutical development and how characteristics of the technique make it better suited for some applications than others. We then discuss the elements of a comprehensive analytical control strategy for SV-AUC. Successful implementation of these analytical control elements ensures that SV-AUC provides continued value over the long time frames necessary to bring biopharmaceuticals to market. © 2015 Elsevier Inc. All rights reserved.

Extra-domain B in oncofetal fibronectin structurally promotes fibrillar head-to-tail dimerization of extracellular matrix protein.

PubMed

Schiefner, André; Gebauer, Michaela; Skerra, Arne

2012-05-18

The type III extra-domain B (ED-B) is specifically spliced into fibronectin (Fn) during embryogenesis and neoangiogenesis, including many cancers. The x-ray structure of the recombinant four-domain fragment Fn(III)7B89 reveals a tightly associated, extended head-to-tail dimer, which is stabilized via pair-wise shape and charge complementarity. A tendency toward ED-B-dependent dimer formation in solution was supported by size exclusion chromatography and analytical ultracentrifugation. When amending the model with the known three-dimensional structure of the Fn(III)10 domain, its RGD loop as well as the adhesion synergy region in Fn(III)9-10 become displayed on the same face of the dimer; this should allow simultaneous binding of at least two integrins and, thus, receptor clustering on the cell surface and intracellular signaling. Insertion of ED-B appears to stabilize overall head-to-tail dimerization of two separate Fn chains, which, together with alternating homodimer formation via disulfide bridges at the C-terminal Fn tail, should lead to the known macromolecular fibril formation.

Evidence of Intertube Excitons Observed in the Raman Resonance Excitation Profiles of (6 , 5) -Enriched SWCNT Bundles

NASA Astrophysics Data System (ADS)

Simpson, J. R.; Hight Walker, A. R.; Roslyak, O.; Haroz, E.; Telg, H.; Duque, J. G.; Crochet, J. J.; Piryatinkski, A.; Doorn, S. K.

Understanding the photophysics of exciton behavior in single wall carbon nanotube (SWCNT) bundles remains important for opto-electronic device applications. We report resonance Raman spectroscopy (RRS) measurements on (6 , 5) -enriched SWCNTs, dispersed in aqueous solutions and separated using density gradient ultracentrifugation into fractions of increasing bundle size. Near-IR to UV absorption spectroscopy demonstrates a redshift and broadening of the main excitonic transitions with bundling. A continuously tunable dye laser coupled to a triple-grating spectrometer affords measurement of Raman resonance excitation profiles (REPs) over a range of wavelengths, (505 to 585) nm, covering the (6 , 5) -E22S excitation. REPs of both the radial breathing mode (RBM) and GLO+reveal a redshifting and broadening of the (6 , 5) E22S transition energy with increasing bundle size. Most interestingly, we observe an additional peak in both the RBM and GLO+REPs of bundled SWCNTs, which is shifted lower in energy than the main E22S and is anomalously narrow. We attribute this additional peak to a transverse, intertube exciton.

New Feature Observed in the Raman Resonance Excitation Profiles of (6 , 5) -Enriched, Selectively Bundled SWCNTs

NASA Astrophysics Data System (ADS)

Hight Walker, A. R.; Simpson, J. R.; Roslyak, O.; Haroz, E.; Telg, H.; Duque, J. G.; Crochet, J. J.; Piryatinski, A.; Doorn, S. K.

Understanding the photophysics of exciton behavior in single wall carbon nanotube (SWCNT) bundles remains important for opto-electronic device applications. We report resonance Raman spectroscopy (RRS) measurements on (6 , 5) -enriched SWCNTs, dispersed in aqueous solutions and separated using density gradient ultracentrifugation into fractions of increasing bundling. Near-IR to UV absorption spectroscopy shows a redshift and broadening of the main excitonic transitions with increasing bundling. A continuously tunable dye laser coupled to a triple-grating spectrometer affords measurement of Raman resonance excitation profiles (REPs) over a range of wavelengths covering the (6 , 5) -E22 range (505 to 585) nm. REPs of both the radial breathing mode (RBM) and G-band reveal a redshifting and broadening of the (6 , 5) E22 transition energy with increasing bundling. Additionally, we observe an unexpected peak in the REP of bundled SWCNTs, which is shifted lower in energy than the main E22 and is anomalously narrow. We compare these observations to a theoretical model that examines the origin of this peak in relation to bundle polarization-enhanced exciton response.

Secreted Progranulin Is a Homodimer and Is Not a Component of High Density Lipoproteins (HDL)*

PubMed Central

Nguyen, Andrew D.; Nguyen, Thi A.; Cenik, Basar; Yu, Gang; Herz, Joachim; Walther, Tobias C.; Davidson, W. Sean; Farese, Robert V.

2013-01-01

Progranulin is a secreted glycoprotein, and the GRN gene is mutated in some cases of frontotemporal dementia. Progranulin has also been implicated in cell growth, wound healing, inflammation, and cancer. We investigated the molecular nature of secreted progranulin and provide evidence that progranulin exists as a homodimer. Although recombinant progranulin has a molecular mass of ∼85 kDa by SDS-PAGE, it elutes in fractions corresponding to ∼170–180 kDa by gel-filtration chromatography. Additionally, recombinant progranulin can be intermolecularly cross-linked, yielding a complex corresponding to a dimer (∼180 kDa), and progranulins containing different epitope tags physically interact. In plasma, progranulin similarly forms complexes of ∼180–190 kDa. Although progranulin partially co-fractionated with high density lipoproteins (HDL) by gel-filtration chromatography, we found no evidence that progranulin in mouse or human plasma is a component of HDL either by ultracentrifugation or by lipid binding assays. We conclude that circulating progranulin exists as a dimer and is not likely a component of HDL. PMID:23364791

Secreted progranulin is a homodimer and is not a component of high density lipoproteins (HDL).

PubMed

Nguyen, Andrew D; Nguyen, Thi A; Cenik, Basar; Yu, Gang; Herz, Joachim; Walther, Tobias C; Davidson, W Sean; Farese, Robert V

2013-03-22

Progranulin is a secreted glycoprotein, and the GRN gene is mutated in some cases of frontotemporal dementia. Progranulin has also been implicated in cell growth, wound healing, inflammation, and cancer. We investigated the molecular nature of secreted progranulin and provide evidence that progranulin exists as a homodimer. Although recombinant progranulin has a molecular mass of ∼85 kDa by SDS-PAGE, it elutes in fractions corresponding to ∼170-180 kDa by gel-filtration chromatography. Additionally, recombinant progranulin can be intermolecularly cross-linked, yielding a complex corresponding to a dimer (∼180 kDa), and progranulins containing different epitope tags physically interact. In plasma, progranulin similarly forms complexes of ∼180-190 kDa. Although progranulin partially co-fractionated with high density lipoproteins (HDL) by gel-filtration chromatography, we found no evidence that progranulin in mouse or human plasma is a component of HDL either by ultracentrifugation or by lipid binding assays. We conclude that circulating progranulin exists as a dimer and is not likely a component of HDL.

Characterization of a hyperthermostable Fe-superoxide dismutase from hot spring.

PubMed

He, Yong-Zhi; Fan, Ke-Qiang; Jia, Cui-Juan; Wang, Zhi-Jun; Pan, Wu-Bin; Huang, Li; Yang, Ke-Qian; Dong, Zhi-Yang

2007-05-01

A new gene encoding a thermostable Fe-superoxide dismutase (tcSOD) was identified from a metagenomic library prepared from a hot spring sample. The open reading frame of tcSOD encoded a 211 amino acid protein. The recombinant protein was overexpressed in Escherichia coli and confirmed to be a Fe-SOD with a specific activity of 1,890 U/mg using the pyrogallol method. The enzyme was highly stable at 80 degrees C and retained 50% activity after heat treatment at 95 degrees C for 2 h. It showed striking stability across a wide pH span from 4 to 11. The native form of the enzyme was determined as a homotetramer by analytical ultracentrifugation and gradient native polyacrylamide gel electrophoresis. Fe(2+) was found to be important to SOD activity and to the stability of tcSOD dimer. Comparative modeling analyses of tcSOD tetramer indicate that its high thermostability is mainly due to the presence of a large number of intersubunit ion pairs and hydrogen bonds and to a decrease in solvent accessible hydrophobic surfaces.

Separation and quantification of monoclonal-antibody aggregates by hollow-fiber-flow field-flow fractionation.

PubMed

Fukuda, Jun; Iwura, Takafumi; Yanagihara, Shigehiro; Kano, Kenji

2014-10-01

Hollow-fiber-flow field-flow fractionation (HF5) separates protein molecules on the basis of the difference in the diffusion coefficient, and can evaluate the aggregation ratio of proteins. However, HF5 is still a minor technique because information on the separation conditions is limited. We examined in detail the effect of different settings, including the main-flow rate, the cross-flow rate, the focus point, the injection amount, and the ionic strength of the mobile phase, on fractographic characteristics. On the basis of the results, we proposed optimized conditions of the HF5 method for quantification of monoclonal antibody in sample solutions. The HF5 method was qualified regarding the precision, accuracy, linearity of the main peak, and quantitation limit. In addition, the HF5 method was applied to non-heated Mab A and heat-induced-antibody-aggregate-containing samples to evaluate the aggregation ratio and the distribution extent. The separation performance was comparable with or better than that of conventional methods including analytical ultracentrifugation-sedimentation velocity and asymmetric-flow field-flow fractionation.

Determination of relative amounts of ribosome and subunits in Escherichia coli using asymmetrical flow field-flow fractionation.

PubMed

Nilsson, M; Birnbaum, S; Wahlund, K G

1996-10-15

The 30S and 50S subunits and the 70S ribosome of Escherichia coli were separated in 6 minutes by using asymmetrical flow field-flow fractionation (FFF). The total analysis time for determination of the relative amounts of ribosomes and free subunits in a preparation from a cell suspension was 8 min. The method can detect a change in the mass fraction of ribosomes if it exceeds approx, 10%. The separation is based on differences in diffusion coefficients, i.e., hydrodynamic diameters, and these can be determined from observed retention times. The hydrodynamic diameters were in good agreement with literature values obtained from electron microscopy. The mass fraction of ribosomes changed as a function of the magnesium ion concentration which confirms previous knowledge and shows the accuracy of the method. The method appears as an alternative to ultracentrifugation analysis and avoids some of its drawbacks and artefacts. An obvious application can be the optimisation of cell design in metabolic engineering in order to maximise translation and protein production.

Self-assembled nanocages based on the coiled coil bundle motif

NASA Astrophysics Data System (ADS)

Sinha, Nairiti; Villegas, Jose; Saven, Jeffery; Kiick, Kristi; Pochan, Darrin

Computational design of coiled coil peptide bundles that undergo solution phase self-assembly presents a diverse toolbox for engineering new materials with tunable and pre-determined nanostructures that can have various end applications such as in drug delivery, biomineralization and electronics. Self-assembled cages are especially advantageous as the cage geometry provides three distinct functional sites: the interior, the exterior and the solvent-cage interface. In this poster, syntheses and characterization of a peptide cage based on computationally designed homotetrameric coiled coil bundles as building blocks is discussed. Techniques such as Transmission Electron Microscopy (TEM), Small-Angle Neutron Scattering (SANS) and Analytical Ultracentrifugation (AUC) are employed to characterize the size, shape and molecular weight of the self-assembled peptide cages under different pH and temperature conditions. Various self-assembly pathways such as dialysis and thermal quenching are shown to have a significant impact on the final structure of these peptides in solution. Comparison of results with the target cage design can be used to iteratively improve the peptide design and provide greater understanding of its interactions and folding.

Electron microscopic studies of bacteriophage M13 DNA replication. [Escherichia coli

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

Allison, D.P.; Ganesan, A.T.; Olson, A.C.

Intracellular forms of M13 phage DNA isolated after infection of Escherichia coli with wild-type phage have been studied by electron microscopy and ultracentrifugation. The data indicate the involvement of rolling-circle intermediates in single-stranded DNA synthesis. In addition to single-stranded, circular DNA, we observed covalently closed and nicked replicative-form (RF) DNAs, dimer RF DNAs, concatenated RF DNAs, RF DNAs with single-stranded tails (sigma, rolling circles), and, occasionally, RF DNAs with theta structures. The tails in sigma molecules are always single stranded and are never longer than the DNA from mature phage; the proportion of sigma to other RF molecules does notmore » change significantly with time after infection. The origin of single-stranded DNA synthesis has been mapped by electron microscopy at a unique location on RF DNA by use of partial denaturation mapping and restriction endonuclease digestion. This location is between gene IV and gene II, and synthesis proceeds in a counterclockwise direction on the conventional genetic map.« less

Plasma rotation by electric and magnetic fields in a discharge cylinder

NASA Technical Reports Server (NTRS)

Wilhelm, H. E.; Hong, S. H.

1977-01-01

A theoretical model for an electric discharge consisting of a spatially diverging plasma sustained electrically between a small ring cathode and a larger ring anode in a cylindrical chamber with an axial magnetic field is developed to study the rotation of the discharge plasma in the crossed electric and magnetic fields. The associated boundary-value problem for the coupled partial differential equations which describe the electric potential and the plasma velocity fields is solved in closed form. The electric field, current density, and velocity distributions are discussed in terms of the Hartmann number and the Hall coefficient. As a result of Lorentz forces, the plasma rotates with speeds as high as 1 million cm/sec around its axis of symmetry at typical conditions. As an application, it is noted that rotating discharges of this type could be used to develop a high-density plasma-ultracentrifuge driven by j x B forces, in which the lighter (heavier) ion and atom components would be enriched in (off) the center of the discharge cylinder.

Development of candidate reference materials for the measurement of lead in bone

PubMed Central

Hetter, Katherine M.; Bellis, David J.; Geraghty, Ciaran; Todd, Andrew C.; Parsons, Patrick J.

2010-01-01

The production of modest quantities of candidate bone lead (Pb) reference materials is described, and an optimized production procedure is presented. The reference materials were developed to enable an assessment of the interlaboratory agreement of laboratories measuring Pb in bone; method validation; and for calibration of solid sampling techniques such as laser ablation ICP-MS. Long bones obtained from Pb-dosed and undosed animals were selected to produce four different pools of a candidate powdered bone reference material. The Pb concentrations of these pools reflect both environmental and occupational exposure levels in humans. The animal bones were harvested post mortem, cleaned, defatted, and broken into pieces using the brittle fracture technique at liquid nitrogen temperature. The bone pieces were then ground in a knife mill to produce fragments of 2-mm size. These were further ground in an ultra-centrifugal mill, resulting in finely powdered bone material that was homogenized and then sampled-scooped into vials. Testing for contamination and homogeneity was performed via instrumental methods of analysis. PMID:18421443

Heparin affinity purification of extracellular vesicles

PubMed Central

Balaj, Leonora; Atai, Nadia A.; Chen, Weilin; Mu, Dakai; Tannous, Bakhos A.; Breakefield, Xandra O.; Skog, Johan; Maguire, Casey A.

2015-01-01

Extracellular vesicles (EVs) are lipid membrane vesicles released by cells. They carry active biomolecules including DNA, RNA, and protein which can be transferred to recipient cells. Isolation and purification of EVs from culture cell media and biofluids is still a major challenge. The most widely used isolation method is ultracentrifugation (UC) which requires expensive equipment and only partially purifies EVs. Previously we have shown that heparin blocks EV uptake in cells, supporting a direct EV-heparin interaction. Here we show that EVs can be purified from cell culture media and human plasma using ultrafiltration (UF) followed by heparin-affinity beads. UF/heparin-purified EVs from cell culture displayed the EV marker Alix, contained a diverse RNA profile, had lower levels of protein contamination, and were functional at binding to and uptake into cells. RNA yield was similar for EVs isolated by UC. We were able to detect mRNAs in plasma samples with comparable levels to UC samples. In conclusion, we have discovered a simple, scalable, and effective method to purify EVs taking advantage of their heparin affinity. PMID:25988257

Ribonucleic artefacts: are some extracellular RNA discoveries driven by cell culture medium components?

PubMed

Tosar, Juan Pablo; Cayota, Alfonso; Eitan, Erez; Halushka, Marc K; Witwer, Kenneth W

2017-01-01

In a recently published study, Anna Krichevsky and colleagues raise the important question of whether results of in vitro extracellular RNA (exRNA) studies, including extracellular vesicle (EV) investigations, are confounded by the presence of RNA in cell culture medium components such as foetal bovine serum (FBS). The answer, according to their data, is a resounding "yes". Even after lengthy ultracentrifugation to remove bovine EVs from FBS, the majority of exRNA in FBS remained. Although technical factors may affect the degree of depletion, residual EVs and exRNA in FBS could influence the conclusions of in vitro studies: certainly, for secreted RNA, and possibly also for cell-associated RNA. In this commentary, we critically examine some of the literature in this field, including a recent study from some of the authors of this piece, in light of the Wei et al. study and explore how cell culture-derived RNAs may affect what we think we know about EV RNAs. These findings hold particular consequence as the field moves towards a deeper understanding of EV-RNA associations and potential functions.

Exosome separation using microfluidic systems: size-based, immunoaffinity-based and dynamic methodologies.

PubMed

Yang, Fang; Liao, Xiangzhi; Tian, Yuan; Li, Guiying

2017-04-01

Exosomes, nanovesicles secreted by most types of cells, exist in virtually all bodily fluids. Their rich nucleic acid and protein content make them potentially valuable biomarkers for noninvasive molecular diagnostics. They also show promise, after further development, to serve as a drug delivery system. Unfortunately, existing exosome separation technologies, such as ultracentrifugation and methods incorporating magnetic beads, are time-consuming, laborious and separate only exosomes of low purity. Thus, a more effective separation method is highly desirable. Microfluidic platforms are ideal tools for exosome separation, since they enable fast, cost-efficient, portable and precise processing of nanoparticles and small volumes of liquid samples. Recently, several microfluidic-based exosome separation technologies have been studied. In this article, the advantages of the most recent technologies, as well as their limitations, challenges and potential uses in novel microfluidic exosome separation and collection applications is reviewed. This review outlines the uses of new powerful microfluidic exosome detection tools for biologists and clinicians, as well as exosome separation tools for microfluidic engineers. Current challenges of exosome separation methodologies are also described, in order to highlight areas for future research and development. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Structure of the Metal Transporter Tp34 and its Affinity for Divalent Metal Ions

NASA Astrophysics Data System (ADS)

Knutsen, Gregory; Deka, Ranjit; Brautigam, Chad; Tomchick, Diana; Machius, Mischa; Norgard, Michael

2007-10-01

Tp34 is periplasmic membrane protein of the nonculitvatable spirochete Treponema pallidum, the pathogen of syphillis. It was proposed that Tp34 is a divalent metal transporter, but the identity of the preferred metal ion(s) was unclear. In this study we investigated the ability of divalent metal ions to induce rTp34 dimerization using hydrodynamic techniques and determine the crystal structure of metal bound forms. Using analytical ultracentrifugation sedimentation velocity experiments, we determined that cobalt is superior to nickel at inducing the dimerization of rTp34. rTp34 was crystallized and selected crystals were incubated at a pH 7.5 with CuSO4 and NiSO4. Diffraction experiments were conducted and the processed electron density maps showed that copper was bound to the major metal binding site as well as to three additional minor binding sites. By contrast nickel was only bound to the major metal binding site in one monomer and to three additional minor sites. These results along with previous findings support evidence of Tp34 being involved with metal transport and/or iron utilization.

The contribution of photosynthetic pigments to the development of biochemical separation methods: 1900-1980.

PubMed

Albertsson, Per-Ake

2003-01-01

The role of photosynthetic pigments in the development of separation methods in biochemistry during the period 1900-1980 is described beginning with M. Tswett who introduced separation of chlorophylls and carotenoids on columns and coined the term chromatography in 1906. In Uppsala, T. Svedberg developed the ultracentrifuge in the 1920s. A. Tiselius improved electrophoresis in the 1930s and developed chromatography of proteins in the 1940s and 1950s. Others of 'The Uppsala school in separation science' include J. Porath, P. Flodin and S. Hjertén who further developed various gel chromatographic methods. Hjertén introduced free zone electrophoresis in narrow tubes, a forerunner of capillary electrophoresis. Two proteins, phycoerythrin and phycocyanin, were used as test substances in all these methodological studies. Aqueous two-phase partitioning as a separation method was introduced in 1956 by the author. In this work, chloroplast particles were used, and the method was applied for the separation and purification of intact chloroplasts, inside-out thylakoid vesicles and plasma membranes. My research was carried out in cooperation with G. Blomquist, G. Johansson, C. Larsson, B. Andersson and H.-E. Akerlund during a 20-year period, 1960-1980.

Laboratory production in vivo of infectious human papillomavirus type 11.

PubMed Central

Kreider, J W; Howett, M K; Leure-Dupree, A E; Zaino, R J; Weber, J A

1987-01-01

Human papillomaviruses (HPV) induce among patients natural lesions which produce small amounts of virus. Infection of human cell cultures does not lead to the multiplication of virus, which also does not replicate in experimental animals. We have developed a unique system for the laboratory production of HPV type 11 (HPV-11). Fragments of human neonatal foreskin were infected with an extract of naturally occurring human vulvar condylomata and grafted beneath the renal capsule of athymic mice. Later (3 to 5 months), condylomatous cysts developed from those grafts. Nuclei of koilocytotic cells contained large amounts of capsid antigen and intranuclear virions. The experimentally induced condylomata were homogenized, and the virions were extracted and used to infect another generation of human foreskin grafts in athymic mice. The HPV-11 DNA content and infectivity of the natural and experimental condylomata were similar. Extracts of experimental condylomata were subjected to differential ultracentrifugation and sedimentation in CsCl density gradients. A single, opalescent band was visible at a density of 1.34 g/ml. It contained HPV virions with HPV-11 DNA. This report is the first demonstration of the laboratory production of an HPV. Images PMID:3027386

A Method for Isolation of Extracellular Vesicles and Characterization of Exosomes from Brain Extracellular Space.

PubMed

Pérez-González, Rocío; Gauthier, Sebastien A; Kumar, Asok; Saito, Mitsuo; Saito, Mariko; Levy, Efrat

2017-01-01

Extracellular vesicles (EV), including exosomes, secreted vesicles of endocytic origin, and microvesicles derived from the plasma membrane, have been widely isolated and characterized from conditioned culture media and bodily fluids. The difficulty in isolating EV from tissues, however, has hindered their study in vivo. Here, we describe a novel method designed to isolate EV and characterize exosomes from the extracellular space of brain tissues. The purification of EV is achieved by gentle dissociation of the tissue to free the brain extracellular space, followed by sequential low-speed centrifugations, filtration, and ultracentrifugations. To further purify EV from other extracellular components, they are separated on a sucrose step gradient. Characterization of the sucrose step gradient fractions by electron microscopy demonstrates that this method yields pure EV preparations free of large vesicles, subcellular organelles, or debris. The level of EV secretion and content are determined by assays for acetylcholinesterase activity and total protein estimation, and exosomal identification and protein content are analyzed by Western blot and immuno-electron microscopy. Additionally, we present here a method to delipidate EV in order to improve the resolution of downstream electrophoretic analysis of EV proteins.

[Cardiovascular risk study in patients with renin-angiotensin system blockade by means of the proteone of circulating extracellular vesicles].

PubMed

de la Cuesta, F; Baldan-Martin, M; Mourino-Alvarez, L; Sastre-Oliva, T; Alvarez-Llamas, G; Gonzalez-Calero, L; Ruiz-Hurtado, G; Segura, J; Vivanco, F; Ruilope, L M; Barderas, M G

2016-01-01

Extracellular vesicles (EVs) are released to the bloodstream by certain cell types due to transport, activation and cell death processes. Blood count of EVs from platelet and endothelial origin has been proved to be a cardiovascular risk biomarker. Thus, EVs proteome might reflect the underlying cellular processes in hypertensive patients with albuminuria. Protein content of circulating EVs was analyzed by liquid chromatography coupled to mass spectrometry. EVs were isolated by an ultracentrifugation protocol optimized in order to avoid contamination by blood plasma proteins. Purity of the isolated fraction was verified by electronic and confocal microscopy, and by flow cytometry. We hereby show a method to isolate circulating EVs from hypertensive patients with/without albuminuria with high yield and purity. Besides, we provide a reference proteome of the EVs of these patients, composed of 2,463 proteins, and prove that the proteins carried by these vesicles are associated with crucial processes involved in the inherent cardiovascular risk. The proteome of circulating EVs is an interesting source of indicators in the evaluation of cardiovascular risk in hypertensive patients with renin-angiotensin system blockage. Copyright © 2015 SEHLELHA. Published by Elsevier España, S.L.U. All rights reserved.

(NZ)CH...O contacts assist crystallization of a ParB-like nuclease.

PubMed

Shaw, Neil; Cheng, Chongyun; Tempel, Wolfram; Chang, Jessie; Ng, Joseph; Wang, Xin-Yu; Perrett, Sarah; Rose, John; Rao, Zihe; Wang, Bi-Cheng; Liu, Zhi-Jie

2007-07-07

The major bottleneck for determination of 3 D structures of proteins using X-rays is the production of diffraction quality crystals. Often proteins are subjected to chemical modification to improve the chances of crystallization Here, we report the successful crystallization of a nuclease employing a reductive methylation protocol. The key to crystallization was the successful introduction of 44 new cohesive (NZ) CH...O contacts (3.2-3.7 A) by the addition of 2 methyl groups to the side chain amine nitrogen (NZ) of 9 lysine residues of the nuclease. The new contacts dramatically altered the crystallization properties of the protein, resulting in crystals that diffracted to 1.2 A resolution. Analytical ultracentrifugation analysis and thermodynamics results revealed a more compact protein structure with better solvent exclusion of buried Trp residues in the folded state of the methylated protein, assisting crystallization. In this study, introduction of novel cohesive (NZ)CH...O contacts by reductive methylation resulted in the crystallization of a protein that had previously resisted crystallization in spite of extensive purification and crystallization space screening. Introduction of (NZ)CH...O contacts could provide a solution to crystallization problems for a broad range of protein targets.

Plasma lipoprotein and apolipoprotein distribution as a function of density in the rainbow trout (Salmo gairdneri).

PubMed Central

Babin, P J

1987-01-01

I have previously described [Babin (1987) J. Biol. Chem. 262, 4290-4296] the apolipoprotein composition of the major classes of trout plasma lipoproteins. The present work describes the use of an isopycnic density gradient centrifugation procedure and sequential flotation ultracentrifugation to show: (1) the presence of intermediate density lipoproteins (IDL) in the plasma, between 1.015 and 1.040 g/ml; (2) the existence of a single type of Mr 240,000 apoB-like in the low density lipoproteins (LDL, 1.040 less than p less than 1.085 g/ml); (3) the presence of apoA-I-like (Mr 25,000) in the densest LDL; (4) the adequacy of 1.085 g/ml as a cutoff between the LDL and high density lipoproteins (HDL); (5) the accumulation of Mr 55,000 and 76,000 apolipoproteins and apoA-like apolipoproteins in the 1.21 g/ml infranatant. The fractionation of trout lipoprotein spectrum thus furnishes the distribution of the different lipoprotein classes and leads to the description of the constituent apolipoproteins, which account for about 36% of circulating plasma proteins in this species. Images Fig. 2. Fig. 3. PMID:3689318

Unveiling massive numbers of cancer-related urinary-microRNA candidates via nanowires

PubMed Central

Yasui, Takao; Yanagida, Takeshi; Ito, Satoru; Konakade, Yuki; Takeshita, Daiki; Naganawa, Tsuyoshi; Nagashima, Kazuki; Shimada, Taisuke; Kaji, Noritada; Nakamura, Yuta; Thiodorus, Ivan Adiyasa; He, Yong; Rahong, Sakon; Kanai, Masaki; Yukawa, Hiroshi; Ochiya, Takahiro; Kawai, Tomoji; Baba, Yoshinobu

2017-01-01

Analyzing microRNAs (miRNAs) within urine extracellular vesicles (EVs) is important for realizing miRNA-based, simple, and noninvasive early disease diagnoses and timely medical checkups. However, the inherent difficulty in collecting dilute concentrations of EVs (<0.01 volume %) from urine has hindered the development of these diagnoses and medical checkups. We propose a device composed of nanowires anchored into a microfluidic substrate. This device enables EV collections at high efficiency and in situ extractions of various miRNAs of different sequences (around 1000 types) that significantly exceed the number of species being extracted by the conventional ultracentrifugation method. The mechanical stability of nanowires anchored into substrates during buffer flow and the electrostatic collection of EVs onto the nanowires are the two key mechanisms that ensure the success of the proposed device. In addition, we use our methodology to identify urinary miRNAs that could potentially serve as biomarkers for cancer not only for urologic malignancies (bladder and prostate) but also for nonurologic ones (lung, pancreas, and liver). The present device concept will provide a foundation for work toward the long-term goal of urine-based early diagnoses and medical checkups for cancer. PMID:29291244

The International Society for Extracellular Vesicles launches the first massive open online course on extracellular vesicles.

PubMed

Lässer, Cecilia; Théry, Clotilde; Buzás, Edit I; Mathivanan, Suresh; Zhao, Weian; Gho, Yong Song; Lötvall, Jan

2016-01-01

The International Society for Extracellular Vesicles (ISEV) has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs). This course, "Basics of Extracellular Vesicles," uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field covering the nomenclature and history of EVs. Module 2 focuses on the biogenesis and uptake mechanisms of EVs, as well as their RNA, protein and lipid cargo. Module 3 covers the collection and processing of cell culture media and body fluids such as blood, breast milk, cerebrospinal fluid and urine prior to isolation of EVs. Modules 4 and 5 present different isolation methods and characterisation techniques utilised in the EV field. Here, differential ultracentrifugation, size-exclusion chromatography, density gradient centrifugation, kit-based precipitation, electron microscopy, cryo-electron microscopy, flow cytometry, atomic-force microscopy and nanoparticle-tracking analysis are covered. This first massive open online course (MOOC) on EVs was launched on 15 August 2016 at the platform "Coursera" and is free of charge.

Thermally assisted acoustofluidic separation of extracellular vesicles from cells

NASA Astrophysics Data System (ADS)

Mirtaheri, Elnaz; Dolatmoradi, Ata; Pimentel, Krystine; Bhansali, Shekhar; El-Zahab, Bilal

2018-02-01

Extracellular vesicles (EVs) have been gaining increasing attention given their role in communicating information between cells. Composition-based isolation of EVs is particularly of high significance as the proteomic and lipidomic characterization of their cargo could provide valuable clues to the role of EVs in mediating the biology of various conditions. This has, however, proved to be challenging as EVs, despite their abundance, are very small and difficult to be differentiated from the other constituents of host media. In addition, currently available methods like ultracentrifugation and filtration are cumbersome and capable of achieving mostly size-based separations. In this work, we demonstrate the possibility of separating submicron EV-like vesicles from cancer cells using a thermally-assisted acoustophoretic device. In a system composed of MCF-7 breast cancer cells spiked with two different types of same-size vesicles, composition-based isolation of vesicles was shown to be realizable through opposite focusing of the system's components at the node and antinodes of the overlaid ultrasonic standing wave. By proper choice of temperature in the microchannel, we were able to achieve separations with purities exceeding 93%. Furthermore, cells recovered from the channel were shown to be viable after the separation.

Diffusion phenomenon at the interface of Cu-brass under a strong gravitational field

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

Ogata, Yudai; Tokuda, Makoto; Januszko, Kamila

2015-03-28

To investigate diffusion phenomenon at the interface between Cu and brass under a strong gravitational field generated by ultracentrifuge apparatus, we performed gravity experiments on samples prepared by electroplating with interfaces normal and parallel to the direction of gravity. For the parallel-mode sample, for which sedimentation cannot occur thorough the interface, the concentration change was significant within the lower gravity region; many pores were observed in this region. Many vacancies arising from crystal strain due to the strong gravitational field moved into the lower gravity region, and enhanced the atoms mobilities. For the two normal-mode samples, which have interface normalmore » to the direction of gravity, the composition gradient of the brass-on-Cu sample was steeper than that for Cu-on-brass. This showed that the atoms of denser Cu diffuse in the direction of gravity, whereas Zn atoms diffuse in the opposite direction by sedimentation. The interdiffusion coefficients became higher in the Cu-on-brass sample, and became lower in the brass-on-Cu sample. This rise may be related to the behavior of the vacancies.« less

Robust peptide bundles designed computationally

NASA Astrophysics Data System (ADS)

Haider, Michael; Zhang, Huixi Violet; Kiick, Kristi; Saven, Jeffery; Pochan, Darrin

Peptides are ideal candidates for the design and controlled assembly of nanoscale materials due to their potential to assemble with atomistic precision as in biological systems. Unlike other work utilizing natural proteins and structural motifs, this effort is completely de novo in order to build arbitrary structures with desired size for the specific placement and separation of functional groups. We have successfully computationally designed soluble, coiled coil, peptide, tetramer bundles which are robust and stable. Using circular dichroism we demonstrated the thermal stability of these bundles as well as confirmed their alpha helical and coiled coil nature. The stability of these bundles arises from the computational design of the coiled coil interior core residues. The coiled coil tetramer was confirmed to be the dominant species by analytical ultra-centrifugation sedimentation studies. We also established how these bundles behave in solution using small angle neutron scattering. The form factor of the bundles is well represented by a cylinder model and their behavior at high concentrations is modeled using a structure factor for aggregates of the cylinders. All of these experiments support our claim that the designed coiled coil bundles were achieved in solution. NSF DMREF 1234161.

Generation of phase II in vitro metabolites using homogenized horse liver.

PubMed

Wong, Jenny K Y; Chan, George H M; Leung, David K K; Tang, Francis P W; Wan, Terence S M

2016-02-01

The successful use of homogenized horse liver for the generation of phase I in vitro metabolites has been previously reported by the authors' laboratory. Prior to the use of homogenized liver, the authors' laboratory had been using mainly horse liver microsomes for carrying out equine in vitro metabolism studies. Homogenized horse liver has shown significant advantages over liver microsomes for in vitro metabolism studies as the procedures are much quicker and have higher capability for generating more in vitro metabolites. In this study, the use of homogenized liver has been extended to the generation of phase II in vitro metabolites (glucuronide and/or sulfate conjugates) using 17β-estradiol, morphine, and boldenone undecylenate as model substrates. It was observed that phase II metabolites could also be generated even without the addition of cofactors. To the authors' knowledge, this is the first report of the successful use of homogenized horse liver for the generation of phase II metabolites. It also demonstrates the ease with which both phase I and phase II metabolites can now be generated in vitro simply by using homogenized liver without the need for ultracentrifuges or tedious preparation steps. Copyright © 2015 John Wiley & Sons, Ltd.

Altered Exosomal RNA Profiles in Bronchoalveolar Lavage from Lung Transplants with Acute Rejection.

PubMed

Gregson, Aric L; Hoji, Aki; Injean, Patil; Poynter, Steven T; Briones, Claudia; Palchevskiy, Vyacheslav; Weigt, S Sam; Shino, Michael Y; Derhovanessian, Ariss; Sayah, David; Saggar, Rajan; Ross, David; Ardehali, Abbas; Lynch, Joseph P; Belperio, John A

2015-12-15

The mechanism by which acute allograft rejection leads to chronic rejection remains poorly understood despite its common occurrence. Exosomes, membrane vesicles released from cells within the lung allograft, contain a diverse array of biomolecules that closely reflect the biologic state of the cell and tissue from which they are released. Exosome transcriptomes may provide a better understanding of the rejection process. Furthermore, biomarkers originating from this transcriptome could provide timely and sensitive detection of acute cellular rejection (AR), reducing the incidence of severe AR and chronic lung allograft dysfunction and improving outcomes. To provide an in-depth analysis of the bronchoalveolar lavage fluid exosomal shuttle RNA population after lung transplantation and evaluate for differential expression between acute AR and quiescence. Serial bronchoalveolar lavage specimens were ultracentrifuged to obtain the exosomal pellet for RNA extraction, on which RNA-Seq was performed. AR demonstrates an intense inflammatory environment, skewed toward both innate and adaptive immune responses. Novel, potential upstream regulators identified offer potential therapeutic targets. Our findings validate bronchoalveolar lavage fluid exosomal shuttle RNA as a source for understanding the pathophysiology of AR and for biomarker discovery in lung transplantation.

Synthesis of recombinant human parainfluenza virus 1 and 3 nucleocapsid proteins in yeast Saccharomyces cerevisiae.

PubMed

Juozapaitis, Mindaugas; Zvirbliene, Aurelija; Kucinskaite, Indre; Sezaite, Indre; Slibinskas, Rimantas; Coiras, Mayte; de Ory Manchon, Fernando; López-Huertas, María Rosa; Pérez-Breña, Pilar; Staniulis, Juozas; Narkeviciute, Irena; Sasnauskas, Kestutis

2008-05-01

Human parainfluenza virus types 1 and 3 (HPIV1 and HPIV3, respectively), members of the virus family Paramyxoviridae, are common causes of lower respiratory tract infections in infants, young children, the immunocompromised, the chronically ill, and the elderly. In order to synthesize recombinant HPIV1 and HPIV3 nucleocapsid proteins, the coding sequences were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of recombinant virus nucleocapsid proteins expression (20-24 mg l(-1) of yeast culture) was obtained. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid proteins, characteristic for other paramyxoviruses. These structures contained host RNA, which was resistant to RNase treatment. The nucleocapsid proteins were stable in yeast and were easily purified by caesium chloride gradient ultracentrifugation. Therefore, this system proved to be simple, efficient and cost-effective, suitable for high-level production of parainfluenza virus nucleocapsids as nucleocapsid-like particles. When used as coating antigens in an indirect ELISA, the recombinant N proteins reacted with sera of patients infected with HPIV1 or 3. Serological assays to detect HPIV-specific antibodies could be designed on this basis.

Generation of henipavirus nucleocapsid proteins in yeast Saccharomyces cerevisiae.

PubMed

Juozapaitis, Mindaugas; Serva, Andrius; Zvirbliene, Aurelija; Slibinskas, Rimantas; Staniulis, Juozas; Sasnauskas, Kestutis; Shiell, Brian J; Wang, Lin-Fa; Michalski, Wojtek P

2007-03-01

Hendra and Nipah viruses are newly emerged, zoonotic viruses and their genomes have nucleotide and predicted amino acid homologies placing them in the family Paramyxoviridae. Currently these viruses are classified in the new genus Henipavirus, within the subfamily Paramyxovirinae, family Paramyxoviridae. The genes encoding HeV and NiV nucleocapsid proteins were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of expression of these proteins (18-20 mg l(-1) of yeast culture) was obtained. Mass spectrometric analysis confirmed the primary structure of both proteins with 92% sequence coverage obtained using MS/MS analysis. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid proteins, characteristic for other paramyxoviruses. The nucleocapsid proteins revealed stability in yeast and can be easily purified by cesium chloride gradient ultracentrifugation. HeV nucleocapsid protein was detected by sera derived from fruit bats, humans, horses infected with HeV, and NiV nucleocapsid protein was immunodetected with sera from, fruit bats, humans and pigs. The development of an efficient and cost-effective system for generation of henipavirus nucleocapsid proteins might help to improve reagents for diagnosis of viruses.

[Ag115S34(SCH2C6H4 tBu)47(dpph)6]: synthesis, crystal structure and NMR investigations of a soluble silver chalcogenide nanocluster† †Dedicated to Evamarie Hey-Hawkins on the occasion of her 60th birthday. ‡ ‡Electronic supplementary information (ESI) available: CCDC 1507868. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6sc04578b Click here for additional data file. Click here for additional data file.

PubMed Central

Fuhr, Olaf; Breitung, Ben; Kiran Chakravadhanula, Venkata Sei; Guthausen, Gisela; Hennrich, Frank; Yu, Wen; Kappes, Manfred M.; Roesky, Peter W.

2017-01-01

With the aim to synthesize soluble cluster molecules, the silver salt of (4-(tert-butyl)phenyl)methanethiol [AgSCH2C6H4 tBu] was applied as a suitable precursor for the formation of a nanoscale silver sulfide cluster. In the presence of 1,6-(diphenylphosphino)hexane (dpph), the 115 nuclear silver cluster [Ag115S34(SCH2C6H4 tBu)47(dpph)6] was obtained. The molecular structure of this compound was elucidated by single crystal X-ray analysis and fully characterized by spectroscopic techniques. In contrast to most of the previously published cluster compounds with more than a hundred heavy atoms, this nanoscale inorganic molecule is soluble in organic solvents, which allowed a comprehensive investigation in solution by UV-Vis spectroscopy and one- and two-dimensional NMR spectroscopy including 31P/109Ag-HSQC and DOSY experiments. These are the first heteronuclear NMR investigations on coinage metal chalcogenides. They give some first insight into the behavior of nanoscale silver sulfide clusters in solution. Additionally, molecular weight determinations were performed by 2D analytical ultracentrifugation and HR-TEM investigations confirm the presence of size-homogeneous nanoparticles present in solution. PMID:28507679

[Ag115S34(SCH2C6H4 t Bu)47(dpph)6]: synthesis, crystal structure and NMR investigations of a soluble silver chalcogenide nanocluster.

PubMed

Bestgen, Sebastian; Fuhr, Olaf; Breitung, Ben; Kiran Chakravadhanula, Venkata Sei; Guthausen, Gisela; Hennrich, Frank; Yu, Wen; Kappes, Manfred M; Roesky, Peter W; Fenske, Dieter

2017-03-01

With the aim to synthesize soluble cluster molecules, the silver salt of (4-( tert -butyl)phenyl)methanethiol [AgSCH 2 C 6 H 4 t Bu] was applied as a suitable precursor for the formation of a nanoscale silver sulfide cluster. In the presence of 1,6-(diphenylphosphino)hexane (dpph), the 115 nuclear silver cluster [Ag 115 S 34 (SCH 2 C 6 H 4 t Bu) 47 (dpph) 6 ] was obtained. The molecular structure of this compound was elucidated by single crystal X-ray analysis and fully characterized by spectroscopic techniques. In contrast to most of the previously published cluster compounds with more than a hundred heavy atoms, this nanoscale inorganic molecule is soluble in organic solvents, which allowed a comprehensive investigation in solution by UV-Vis spectroscopy and one- and two-dimensional NMR spectroscopy including 31 P/ 109 Ag-HSQC and DOSY experiments. These are the first heteronuclear NMR investigations on coinage metal chalcogenides. They give some first insight into the behavior of nanoscale silver sulfide clusters in solution. Additionally, molecular weight determinations were performed by 2D analytical ultracentrifugation and HR-TEM investigations confirm the presence of size-homogeneous nanoparticles present in solution.

[Expression, crystallization and crystallographic study of the 1st IgV domain of human CD96].

PubMed

Jiang, Wenjing; Zhang, Shuijun; Yan, Jinghua; Guo, Ning

2013-05-01

CD96 (Tactile) is an adhesion receptor expressed mainly on activated T cells, NK cells. As a family member of the immunoglobulin-like cell receptor, CD96 consists of three immunoglobulin-like domains (V1, V2/C and C) in the extracellular region. Recent studies have shown that the 1st IgV domain of CD96 (CD96V1) plays an essential role in cell adhesion and NK cell-mediated killing. In this study, the 1st IgV domain of human CD96 (hCD96V1) was cloned and expressed in Escherichia coli (BL21). The soluble protein was obtained by refolding of the hCD96V1 inclusion bodies. From analytical ultracentrifugation, we could predict that CD96 V1 maily exists as dimer with approximate molecular weight of 26.9 kDa. The protein was then successfully crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to 1.9 angstrom resolution and belonged to space group P21, with unit-cell parameters a = 35.1, b = 69.5, c = 49.6A, alpha=gamma=90 degrees, beta=105.4 degrees.

Isolation and separation of physicochemically distinct fimbrial types expressed on a single culture of Escherichia coli O7:K1:H6.

PubMed Central

Karch, H; Leying, H; Büscher, K H; Kroll, H P; Opferkuch, W

1985-01-01

The fimbrial (pili) profile of a single strain of Escherichia coli O7:K1:H6 (WF96) was evaluated. Fimbriae were isolated by sucrose density gradient ultracentrifugation, purified from flagellae by the use of 0.4% sodium dodecyl sulfate (SDS), and separated into distinct fimbrial types. Analysis of the purified WF96 fimbriae by SDS-polyacrylamide gel electrophoresis revealed two polypeptide bands with molecular weights of 16,000 and 21,000. Treatment of the fimbrial mixture with saturated guanidine hydrochloride resulted in the appearance of a third band with a molecular weight of 19,500. The relative susceptibilities of the WF96 fimbrial types to disrupting chemicals (octyl-glucoside, urea, SDS, and guanidine hydrochloride) were assessed by exposure of the fimbrial mixture to each agent, separation of the depolymerized fimbriae from intact fimbriae by gel filtration on Sepharose CL-4B, and identification of the disaggregated fimbrial types by SDS-polyacrylamide gel electrophoresis of column fractions. The physicochemical heterogeneity of the three fimbrial types coexpressed on WF96 was exploited to develop a method for separation of individual fimbriae. Images PMID:2857155

Extracting archaeal populations from iron oxidizing systems

NASA Astrophysics Data System (ADS)

Whitmore, L. M.; Hutchison, J.; Chrisler, W.; Jay, Z.; Moran, J.; Inskeep, W.; Kreuzer, H.

2013-12-01

Unique environments in Yellowstone National Park offer exceptional conditions for studying microorganisms in extreme and constrained systems. However, samples from some extreme systems often contain inorganic components that pose complications during microbial and molecular analysis. Several archaeal species are found in acidic, geothermal ferric-oxyhydroxide mats; these species have been shown to adhere to mineral surfaces in flocculated colonies. For optimal microbial analysis, (microscopy, flow cytometry, genomic extractions, proteomic analysis, stable isotope analysis, and others), improved techniques are needed to better facilitate cell detachment and separation from mineral surfaces. As a requirement, these techniques must preserve cell structure while simultaneously minimizing organic carryover to downstream analysis. Several methods have been developed for removing sediments from mixed prokaryotic populations, including ultra-centrifugation, nycodenz gradient, sucrose cushions, and cell straining. In this study we conduct a comparative analysis of mechanisms used to detach archaeal cell populations from the mineral interface. Specifically, we evaluated mechanical and chemical approaches for cell separation and homogenization. Methods were compared using confocal microscopy, flow cytometry analyses, and real-time PCR detection. The methodology and approaches identified will be used to optimize biomass collection from environmental specimens or isolates grown with solid phases.

Crystal structures of penicillin-binding protein 3 (PBP3) from methicillin-resistant Staphylococcus aureus in the apo and cefotaxime-bound forms.

PubMed

Yoshida, Hisashi; Kawai, Fumihiro; Obayashi, Eiji; Akashi, Satoko; Roper, David I; Tame, Jeremy R H; Park, Sam-Yong

2012-10-26

Staphylococcus aureus is a widespread Gram-positive opportunistic pathogen, and a methicillin-resistant form (MRSA) is particularly difficult to treat clinically. We have solved two crystal structures of penicillin-binding protein (PBP) 3 (PBP3) from MRSA, the apo form and a complex with the β-lactam antibiotic cefotaxime, and used electrospray mass spectrometry to measure its sensitivity to a variety of penicillin derivatives. PBP3 is a class B PBP, possessing an N-terminal non-penicillin-binding domain, sometimes called a dimerization domain, and a C-terminal transpeptidase domain. The model shows a different orientation of its two domains compared to earlier models of other class B PBPs and a novel, larger N-domain. Consistent with the nomenclature of "dimerization domain", the N-terminal region forms an apparently tight interaction with a neighboring molecule related by a 2-fold symmetry axis in the crystal structure. This dimer form is predicted to be highly stable in solution by the PISA server, but mass spectrometry and analytical ultracentrifugation provide unequivocal evidence that the protein is a monomer in solution. Copyright © 2012 Elsevier Ltd. All rights reserved.

Altered Exosomal RNA Profiles in Bronchoalveolar Lavage from Lung Transplants with Acute Rejection

PubMed Central

Hoji, Aki; Injean, Patil; Poynter, Steven T.; Briones, Claudia; Palchevskiy, Vyacheslav; Sam Weigt, S.; Shino, Michael Y.; Derhovanessian, Ariss; Saggar, Rajan; Ross, David; Ardehali, Abbas; Lynch, Joseph P.; Belperio, John A.

2015-01-01

Rationale: The mechanism by which acute allograft rejection leads to chronic rejection remains poorly understood despite its common occurrence. Exosomes, membrane vesicles released from cells within the lung allograft, contain a diverse array of biomolecules that closely reflect the biologic state of the cell and tissue from which they are released. Exosome transcriptomes may provide a better understanding of the rejection process. Furthermore, biomarkers originating from this transcriptome could provide timely and sensitive detection of acute cellular rejection (AR), reducing the incidence of severe AR and chronic lung allograft dysfunction and improving outcomes. Objectives: To provide an in-depth analysis of the bronchoalveolar lavage fluid exosomal shuttle RNA population after lung transplantation and evaluate for differential expression between acute AR and quiescence. Methods: Serial bronchoalveolar lavage specimens were ultracentrifuged to obtain the exosomal pellet for RNA extraction, on which RNA-Seq was performed. Measurements and Main Results: AR demonstrates an intense inflammatory environment, skewed toward both innate and adaptive immune responses. Novel, potential upstream regulators identified offer potential therapeutic targets. Conclusions: Our findings validate bronchoalveolar lavage fluid exosomal shuttle RNA as a source for understanding the pathophysiology of AR and for biomarker discovery in lung transplantation. PMID:26308930

Proteomics analysis of latex from Hevea brasiliensis (clone RRIM 600).

PubMed

Habib, Mohd Afiq Hazlami; Yuen, Gan Chee; Othman, Fazilah; Zainudin, Nurul Nabilah; Latiff, Aishah Abdul; Ismail, Mohd Nazri

2017-04-01

The natural rubber latex extracted from the bark of Hevea brasiliensis plays various important roles in today's modern society. Following ultracentrifugation, the latex can be separated into 3 layers: C-serum, lutoids, and rubber particles. Previous studies have shown that a large number of proteins are present in these 3 layers. However, a complete proteome for this important plant is still unavailable. Protein sequences have been recently translated from the completed draft genome database of H. brasiliensis, leading to the creation of annotated protein databases of the following H. brasiliensis biosynthetic pathways: photosynthesis, latex allergens, rubberwood formation, latex biosynthesis, and disease resistance. This research was conducted to identify the proteins contained within the latex by way of de novo sequencing from mass spectral data obtained from the 3 layers of the latex. Peptides from these proteins were fragmented using collision-induced dissociation, higher-energy collisional dissociation, and electron-transfer dissociation activation methods. A large percentage of proteins from the biosynthetic pathways (63% to 100%) were successfully identified. In addition, a total of 1839 unique proteins were identified from the whole translated draft genome database (AnnHBM).

[Thyroid proteins in endemic goitre and their relationship to the intrathyroidal thyroid hormone concentration].

PubMed

Platzer, S; Groebner, P; Hausen, A; Obendorf, L; Riccabona, G

1980-02-01

According to several reports we suspected that the pathogenesis of endemic goitre cannot be explained by iodine deficiency only, but that other--partially endogenous--goitrogenic factors must be present. We therefore studied 16 cases of "euthyroid" endemic goitre from the endemic goitre area of the province of Bolzano in Italy. After fractionation of tissue homogenates, T 4 and T 3 were measured by RIA and the I concentration was also termined. Thyroglobulin and its fractions were measured by ultracentrifuge procedures after assessment of the total protein concentration. Evaluation of the present results suggests that an insufficient synthesis of thyroglobulin in the examined goitres induces an inadequate adaptation of the organism to iodine deficiency, which, in turn, decreases the thyroid hormone concentration in thyroid tissue and enhances goitrogenesis. Considering the normal iodine content of the examined tissues, there obviously seems to be two intrathyroidal iodine pools, one of which supplies the body with thyroid hormones under pituitary stimulation even though its thyroglobulin pool is reduced, while a significant amount of the thyroidal iodine pool is bound in metabolically inert protein molecules and therefore increases the goitrogenic effect of iodine deficiency.

Effect of carrageenan addition on the rennet-induced gelation of skim milk.

PubMed

Wang, Fang; Zhang, Wei; Ren, Fazheng

2016-09-01

Carrageenan (CG) (κ-CG, ι-CG and λ-CG) was added to skim milk and the rennet-induced aggregation was studied. Caseinomacropeptide (CMP) release, diffusing wave spectroscopy (DWS) and rheology were used to follow the structural dynamics of casein micelles during gelation. The influence of carrageenan on the nature of protein interactions in the gels was investigated using a combination of ultracentrifugation and specific dissociating agents. For the recombined samples containing κ-CG and low concentrations of ι-CG and λ-CG, the CMP release was slowed down; however, the development of DWS and rheological parameters was similar to that of the control sample, and the increase in the incorporation of proteins through calcium bridges and hydrophobic interactions may be the most likely contributors. For the recombined samples containing high concentrations of ι-CG and λ-CG, other factors may impede the gel formation process. High concentrations of ι-CG and λ-CG strongly interfered with the rennet-induced aggregation, interrupted the interaction of caseins and therefore may contribute to good quality of low-fat cheese. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Copper absorption from human milk, cow's milk, and infant formulas using a suckling rat model

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

Loennerdal, B.B.; Bell, J.G.; Keen, C.L.

1985-11-01

Since copper deficiency is known to occur during infancy, it becomes important to assess copper uptake from various infant diets. The authors have investigated the uptake of copper from human milk, cow's milk, cow's milk formulas, cereal/milk formula and soy formula, compensating for the decay of /sup 64/Cu and using the suckling rat as a model. Radiocopper was added to the diet in trace amounts. Ultracentrifugation, ultrafiltration, and gel filtration were used to show that the added /sup 64/Cu bound to milk fractions and individual binding compounds in a manner analogous to the distribution of native copper, thus validating themore » use of extrinsically labeled diets. Labeled diets were intubated into 14-day-old suckling rats. Animals were killed after 6 h and tissues removed and counted. Liver copper uptake was 25% from human milk, 23% from cow's milk formula, 18% from cow's milk, 17% from premature (cow's milk based) infant formula, 17% from cereal/milk formula and 10% from soy formula. These results show that the rat pup model may provide a rapid, inexpensive, and sensitive method to assay bioavailability of copper from infant foods.« less

Purification and characterization of a β-amylase from soya beans

PubMed Central

Gertler, A.; Birk, Yehudith

1965-01-01

1. β-Amylase obtained by acidic extraction of soya-bean flour was purified by ammonium sulphate precipitation, followed by chromatography on calcium phosphate, diethylaminoethylcellulose, Sephadex G-25 and carboxymethylcellulose. 2. The homogeneity of the pure enzyme was established by criteria such as ultracentrifugation and electrophoresis on paper and in polyacrylamide gel. 3. The pure enzyme had a nitrogen content of 16·3%, its extinction coefficient, E1%1cm., at 280mμ was 17·3 and its specific activity/mg. of enzyme was 880 amylase units. 4. The molecular weight of the pure enzyme was determined as 61700 and its isoelectric point was pH5·85. 5. Preliminary examinations indicated that glutamic acid formed the N-terminus and glycine the C-terminus. 6. The amino acid content of the pure enzyme was established, one molecule consisting of 617 amino acid residues. 7. The pH optimum for pure soya-bean β-amylase is in the range 5–6. Pretreatment of the enzyme at pH3–5 decreases enzyme activity, whereas at pH6–9 it is not affected. ImagesFig. 2.Fig. 3. PMID:14342495

Partial purification of a toxin found in hamsters with antibiotic-associated colitis. Reversible binding of the toxin by cholestyramine.

PubMed

Humphrey, C D; Condon, C W; Cantey, J R; Pittman, F E

1979-03-01

A toxin with cytotoxic and enterotoxic activities was isolated from cecal contents of hamsters receiving lincomycin. The toxin was partially purified by ultracentrifugation, ultrafiltration, (NH4)2SO4 precipitation, and gel filtration. Cytotoxic activity, assayed on monolayers of HeLa cells, was restricted to material that eluted in the molecular weight range of 107,000 +/- 6,000 daltons. Cytotoxicity of crude AAC toxin could be demonstrated at concentrations as low as 0.04 microgram/ml. The toxin was heat labile (55 degrees-60 degrees C for 0.5 hr) and sensitive to trypsinization, acidification at pH 3, or alkalinization at pH 9. Cytotoxic activity was inhibited by Clostridium sordellii antitoxin. Enterotoxic activity of the crude toxin and the cytotoxic fraction from gel filtration was demonstrated by fluid secretion in ligated rabbit ileal loops. Studies were done in vitro with cholestyramine resin, vancomycin, or gentamicin to determine if the toxin was bound or denatured by these drugs. It was demonstrated that cholestyramine bound the toxin, significantly reducing its cytotoxicity. Reversible binding of the cytotoxic material was demonstrated by salt gradient elution. Neither vancomycin nor gentamicin had any effect on the in vitro cytotoxic activity of the toxin.

Geographical trends of PFAS in cod livers along the Norwegian coast

PubMed Central

Valdersnes, Stig; Nilsen, Bente M.; Breivik, Joar F.; Borge, Asbjørn; Maage, Amund

2017-01-01

The level of perfluorinated alkyl substances (PFAS) was determined in North East Arctic cod (Gadus morhua) liver samples from 15 Norwegian fjords and harbors. Five harbors in the eastern part of Norway, six harbors in the western part and four harbours in the northern part. A total of 200 samples were analyzed for 16 PFAS. Determination of PFAS were carried out by LC-MS/MS following sample clean up by solid phase extraction and ultracentrifugation. The predominating PFAS was PFOS, which was found to be higher than the level of quantification (1.5 μg kg-1 wet weight) in 72% of the samples. The highest level of PFOS found was 21.8 μg kg-1 wet weight in a sample from Kragerø in the eastern part of Norway. A significantly higher level of PFOS was found in the eastern fjords and harbors compared to fjords and harbors in the western and northern part of Norway. Within the northern fjords and harbors elevated PFOS levels were found in Narvik, which may indicate a local source there. Variations in PFOS of the cod livers thus reflect differences in levels of pollution between the areas. PMID:28531177

Geographical trends of PFAS in cod livers along the Norwegian coast.

PubMed

Valdersnes, Stig; Nilsen, Bente M; Breivik, Joar F; Borge, Asbjørn; Maage, Amund

2017-01-01

The level of perfluorinated alkyl substances (PFAS) was determined in North East Arctic cod (Gadus morhua) liver samples from 15 Norwegian fjords and harbors. Five harbors in the eastern part of Norway, six harbors in the western part and four harbours in the northern part. A total of 200 samples were analyzed for 16 PFAS. Determination of PFAS were carried out by LC-MS/MS following sample clean up by solid phase extraction and ultracentrifugation. The predominating PFAS was PFOS, which was found to be higher than the level of quantification (1.5 μg kg-1 wet weight) in 72% of the samples. The highest level of PFOS found was 21.8 μg kg-1 wet weight in a sample from Kragerø in the eastern part of Norway. A significantly higher level of PFOS was found in the eastern fjords and harbors compared to fjords and harbors in the western and northern part of Norway. Within the northern fjords and harbors elevated PFOS levels were found in Narvik, which may indicate a local source there. Variations in PFOS of the cod livers thus reflect differences in levels of pollution between the areas.

Binding mode of cytochalasin B to F-actin is altered by lateral binding of regulatory proteins.

PubMed

Suzuki, N; Mihashi, K

1991-01-01

The binding of cytochalasin B (CB) to F-actin was studied using a trace amount of [3H]-cytochalasin B. F-Actin-bound CB was separated from free CB by ultracentrifugation and the amount of F-actin-bound CB was determined by comparing the radioactivity both in the supernatant and in the precipitate. A filament of pure F-actin possessed one high-affinity binding site for CB (Kd = 5.0 nM) at the B-end. When the filament was bound to native tropomyosin (complex of tropomyosin and troponin), two low-affinity binding sites for CB (Kd = 230 nM) were created, while the high-affinity binding site was reserved (Kd = 3.4 nM). It was concluded that the creation of low-affinity binding sites was primarily due to binding of tropomyosin to F-actin, as judged from the following two observations: (1) a filament of F-actin/tropomyosin complex possessed one high-affinity binding site (Kd = 3.9 nM) plus two low-affinity binding sites (Kd = 550 nM); (2) the Ca2(+)-receptive state of troponin C in F-actin/native tropomyosin complex did not affect CB binding.

High-density lipoprotein apolipoproteins in urine: I. Characterization in normal subjects and in patients with proteinuria.

PubMed

Gomo, Z A; Henderson, L O; Myrick, J E

1988-09-01

A high-resolution two-dimensional electrophoretic method for protein, with silver staining, has been used to characterize and identify urinary high-density-lipoprotein apolipoproteins (HDL-Apos) and their isoforms in healthy subjects and in patients with kidney disease. Analytical techniques based on both molecular mass and ultracentrifugal flotation properties were used to isolate urinary lipoprotein particles with characteristics identical to those of HDL in plasma. HDL-Apos identified in urine of normal subjects and patients with glomerular proteinuria were Apos A-I, A-II, and C. Five isoforms of Apo A-I were present. Immunostaining of electroblotted proteins further confirmed the presence of HDL-Apos in urine. Creatinine clearance rate was decreased in the patients with proteinuria, and ranged from 32.5 to 40 mL/min. Concentrations of cholesterol and triglycerides in serum were greater in the patients' group, whereas mean HDL-cholesterol (0.68, SD 0.10 mmol/L) and Apo A-I (0.953, SD 0.095 g/L) were significantly (each P less than 0.01) lower. Results of this study suggest that measurement of urinary Apo A-I will reflect excretion of HDL in urine.

New, rapid method to measure dissolved silver concentration in silver nanoparticle suspensions by aggregation combined with centrifugation

NASA Astrophysics Data System (ADS)

Dong, Feng; Valsami-Jones, Eugenia; Kreft, Jan-Ulrich

2016-09-01

It is unclear whether the antimicrobial activities of silver nanoparticles (AgNPs) are exclusively mediated by the release of silver ions (Ag+) or, instead, are due to combined nanoparticle and silver ion effects. Therefore, it is essential to quantify dissolved Ag in nanosilver suspensions for investigations of nanoparticle toxicity. We developed a method to measure dissolved Ag in Ag+/AgNPs mixtures by combining aggregation of AgNPs with centrifugation. We also describe the reproducible synthesis of stable, uncoated AgNPs. Uncoated AgNPs were quickly aggregated by 2 mM Ca2+, forming large clusters that could be sedimented in a low-speed centrifuge. At 20,100g, the sedimentation time of AgNPs was markedly reduced to 30 min due to Ca2+-mediated aggregation, confirmed by the measurements of Ag content in supernatants with graphite furnace atomic absorption spectrometry. No AgNPs were detected in the supernatant by UV-Vis absorption spectra after centrifuging the aggregates. Our approach provides a convenient and inexpensive way to separate dissolved Ag from AgNPs, avoiding long ultracentrifugation times or Ag+ adsorption to ultrafiltration membranes.

Identification of boric acid as a novel chemoattractant and elucidation of its chemoreceptor in Ralstonia pseudosolanacearum Ps29.

PubMed

Hida, Akiko; Oku, Shota; Nakashimada, Yutaka; Tajima, Takahisa; Kato, Junichi

2017-08-17

Chemotaxis enables bacteria to move toward more favorable environmental conditions. We observed chemotaxis toward boric acid by Ralstonia pseudosolanacearum Ps29. At higher concentrations, the chemotactic response of R. pseudosolanacearum toward boric acid was comparable to or higher than that toward L-malate, indicating that boric acid is a strong attractant for R. pseudosolanacearum. Chemotaxis assays under different pH conditions suggested that R. pseudosolanacearum recognizes B(OH) 3 (or B(OH 3 ) + B(OH) 4 - ) but not B(OH) 4 - alone. Our previous study revealed that R. pseudosolanacearum Ps29 harbors homologs of all 22R. pseudosolanacearum GMI1000 mcp genes. Screening of 22 mcp single-deletion mutants identified the RS_RS17100 homolog as the boric acid chemoreceptor, which was designated McpB. The McpB ligand-binding domain (LBD) was purified in order to characterize its binding to boric acid. Using isothermal titration calorimetry, we demonstrated that boric acid binds directly to the McpB LBD with a K D (dissociation constant) of 5.4 µM. Analytical ultracentrifugation studies revealed that the McpB LBD is present as a dimer that recognizes one boric acid molecule.

Purification of circular DNA using benzoylated naphthoylated DEAE-cellulose.

PubMed

Gamper, H; Lehman, N; Piette, J; Hearst, J E

1985-04-01

Un-nicked circular DNA can be separated from protein, RNA, and other DNA in a simple three-step protocol consisting of exonuclease III digestion, extraction with benzoylated naphthoylated DEAE-cellulose (BND cellulose) in 1 M NaCl, and alcohol precipitation of the remaining supercoiled DNA. Exonuclease III treatment introduces single-stranded regions into contaminating linear and nicked circular DNA. This DNA, together with most RNA and protein, is adsorbed onto BND cellulose leaving form I DNA in solution. The protocol can be used to purify analytical as well as preparative amounts of supercoiled DNA. This procedure is a substitute for cesium chloride-ethidium bromide gradient ultracentrifugation and gives a comparable yield of pure form I DNA. Other classes of DNA can be isolated by changing the pretreatment step. Selective digestion of linear DNA with lambda exonuclease permits the isolation of both nicked circular and supercoiled DNA while brief heat-induced or alkali-induced denaturation leads to the recovery of rapidly reannealing DNA. In large-scale purifications, the basic protocol is usually preceded by one or more BND cellulose extractions in 1 M NaCl to remove contaminants absorbing UV or inhibiting exonuclease III.

Hydroxypropyl-ß-cyclodextrin as a membrane protectant during freeze-drying of hydrogenated and non-hydrogenated liposomes and molecule-in-cyclodextrin-in- liposomes: Application to trans-anethole.

PubMed

Gharib, Riham; Greige-Gerges, Hélène; Fourmentin, Sophie; Charcosset, Catherine

2018-11-30

The effect of hydrogenation of phospholipids on the characteristics of freeze-dried liposomes was investigated using hydroxypropyl-ß-cyclodextrin (HP-ß-CD) as membrane protectant. The ethanol-injection method was applied to prepare liposomes using hydrogenated (Phospholopion-90H and 80H) and non-hydrogenated phospholipids (Lipoid-S100) in combination with cholesterol. Various liposomal formulations were tested: conventional liposomes (CL) and HP-ß-CD-loaded liposomes (CDL). Liposome suspensions were concentrated by ultracentrifugation; the pellets were reconstituted in water or CD solution and the dispersions were characterized for their size, polydispersity index and zeta potential. Results demonstrated that HP-ß-CD protected only the hydrogenated batches (CL and CDL) during freeze-drying. Moreover, the presence of HP-ß-CD in the aqueous phase of CDL protected them during freeze-drying. Freeze-dried CL and CDL made of phospholipon-90H loading anethole were demonstrated to be physically stable upon reconstitution in HP-ß-CD solutions, and are able to retain anethole after 6 months of storage at 4 °C thereby making them valuable for food applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Multimeric species in equilibrium in detergent-solubilized Na,K-ATPase.

PubMed

Yoneda, Juliana Sakamoto; Scanavachi, Gustavo; Sebinelli, Heitor Gobbi; Borges, Júlio Cesar; Barbosa, Leandro R S; Ciancaglini, Pietro; Itri, Rosangela

2016-08-01

In this work, we find an equilibrium between different Na,K-ATPase (NKA) oligomeric species solubilized in a non-ionic detergent C12E8 by means of Dynamic Light Scattering (DLS), Analytical Ultracentrifugation (AUC), Small Angle X-ray Scattering (SAXS), Spectrophotometry (absorption at 280/350nm) and enzymatic activity assay. The NKA sample after chromatography purification presented seven different populations as identified by AUC, with monomers and tetramers amounting to ∼55% of the total protein mass in solution. These two species constituted less than 40% of the total protein mass after increasing the NKA concentration. Removal of higher-order oligomer/aggregate species from the NKA solution using 220nm-pore filter resulted in an increase of the specific enzymatic activity. Nevertheless, the enzyme forms new large aggregates over an elapsed time of 20h. The results thus point out that C12E8-solubilized NKA is in a dynamic equilibrium of monomers, tetramers and high-order oligomers/subunit aggregates. These latter have low or null activity. High amount of detergent leads to the dissociation of NKA into smaller aggregates with no enzymatic activity. Copyright © 2016. Published by Elsevier B.V.

A method for the purification of bacterial flagellin that allows simple upscaling.

PubMed

Hiriart, Yanina; Errea, Agustina; González Maciel, Dolores; Lopez, Juan Carlos; Rumbo, Martin

2012-01-01

There is a growing interest in enterobacterial flagellins that may result in a demand to produce flagellin on an industrial scale for possible applications as an adjuvant, immunomodulatory agent or vaccine antigen. Traditionally, small-scale production of flagellin has occurred in the laboratory by flagellar shearing of bacterial surfaces and subsequent ultracentrifugation. The main drawback of this method is the need to use low-agitation cultures to avoid the loss of flagella due to shearing during culture. In the present work, we describe a scalable protocol for the production of flagellin with higher yields than traditional laboratory-scale protocols. The use of cross-flow filtration to concentrate bacterial cultures combines extensive shearing of flagella with a reduction in volume, greatly simplifying downstream processing. This technique also allows the use of highly-agitated culture conditions because any sheared flagella are retained in the bacterial concentrate. Flagella obtained with this procedure showed in vivo and in vitro innate activating capacities similar to those of flagella produced at laboratory scale. This procedure is flexible, allowing an increase in production scale, an enhancement of flagellin yield and no requirement for expensive equipment.

CRADA with Beckman Instruments and Pacific Northwest National Laboratory (PNL-013): Development and commercialization of the Unsaturated Flow Apparatus (UFA) using characterization of aridisols

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

Wright, J.; Conca, J.

1996-10-01

The objective of this Cooperative Research and Development Agreement (CRADA) was to develop and commercialize a technology conceived by scientists at Pacific Northwest National Laboratory (PNNL) and manufactured by Beckman Instruments, Inc. (Beckman), and to apply this technology to the characterization of and soils. The technology is the Unsaturated Flow Apparatus (UFA). The UFA provides a highly efficient method of direct, rapid measurement of hydraulic conductivity and other flow properties according to Darcy-Buckingham principles because the operator controls both the fluid driving force, using an ultracentrifuge, and the flow into the sample while it is spinning, with a rotating sealmore » assembly. The concept of using centrifugation to significantly decrease the time needed, from years or months to days, for study of subsurface transport, particularly under unsaturated conditions, was conceived by James Conca, Ph.D., and Judith Wright, Ph.D., in 1986. The prototype UFA was developed in 1988 because there was a need to rapidly and accurately determine transport parameters in soils, sediments, and rocks for the Grout Waste Disposal Program. Transport parameters are critical to modeling outcomes for site-specific solutions to environmental remediation and waste disposal problems.« less

Identification of the trypanocidal factor in normal human serum: high density lipoprotein.

PubMed Central

Rifkin, M R

1978-01-01

The differentiation of Trypanosoma brucei from T. rhodesiense, the causative agent of human sleeping sickness, depends on their relative sensitivities to the cytotoxic effects of normal human serum. The molecule responsible for the specific lysis of T. brucei has now been isolated. Serum lipoproteins were fractionated and purified by ultracentrifugal flotation and chromatography on Bio-Gel A-5m. Trypanocidal activity was recovered in the high density lipoprotein fraction (density, 1.063-1.216 g/ml). Contamination by other serum proteins was checked by crossed immunoelectrophoresis and sodium dodecyl sulfate/acrylamide gel electrophoresis. Only a trace of beta-lipoprotein was found. The trypanocidal activity of pure human high density lipoprotein was identical to that of unfractionated serum when the following were tested: (i) time course of in vitro lysis of T. bruceli; (ii) in vivo destruction of T. brucei; (iii) relative resistance of T. rhodesiense to lysis. Rat or rabbit high density lipoprotein had no trypanocidal activity. Identification of the trypanocidal factor as high density lipoprotein was confirmed by the finding that serum from patients with Tangier disease, an autosomal recessive disorder characterized by a severe deficiency of high density lipoprotein, had no trypanocidal activity. Images PMID:210461

Purification and biochemical characterization of asclepain c I from the latex of Asclepias curassavica L.

PubMed

Liggieri, Constanza; Arribére, M Cecilia; Trejo, Sebastián A; Canals, Francesc; Avilés, Francesc X; Priolo, Nora S

2004-08-01

In this work we report the isolation, purification and characterization of a new protease from latex of Asclepias curassavica L. Crude extract (CE) was obtained by gathering latex on 0.1 M citric-phosphate buffer with EDTA and cysteine with subsequent ultracentrifugation. Proteolytic assays were made on casein or azocasein as substrates. Caseinolytic activity was completely inhibited by E-64. Stability at different temperatures, optimum pH and ionic strength were evaluated by measuring the residual caseinolytic activity at different times after the incubation. CE showed the highest caseinolytic activity at pH 8.5 in the presence of 12 mM cysteine. CE was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by SDS-PAGE, were isolated. The major purified protease (asclepain cI) showed a molecular mass of 23.2 kDa by mass spectrometry and a pI higher than 9.3. The N-terminal sequence showed a high similarity with those of other plant cysteine proteinases. When assayed on N-alpha-CBZ-aminoacid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative. Determinations of kinetic parameter (km and Kcat) were performed with PFLNA.

An integrated double-filtration microfluidic device for isolation, enrichment and quantification of urinary extracellular vesicles for detection of bladder cancer

PubMed Central

Liang, Li-Guo; Kong, Meng-Qi; Zhou, Sherry; Sheng, Ye-Feng; Wang, Ping; Yu, Tao; Inci, Fatih; Kuo, Winston Patrick; Li, Lan-Juan; Demirci, Utkan; Wang, ShuQi

2017-01-01

Extracellular vesicles (EVs), including exosomes and microvesicles, are present in a variety of bodily fluids, and the concentration of these sub-cellular vesicles and their associated biomarkers (proteins, nucleic acids, and lipids) can be used to aid clinical diagnosis. Although ultracentrifugation is commonly used for isolation of EVs, it is highly time-consuming, labor-intensive and instrument-dependent for both research laboratories and clinical settings. Here, we developed an integrated double-filtration microfluidic device that isolated and enriched EVs with a size range of 30–200 nm from urine, and subsequently quantified the EVs via a microchip ELISA. Our results showed that the concentration of urinary EVs was significantly elevated in bladder cancer patients (n = 16) compared to healthy controls (n = 8). Receiver operating characteristic (ROC) analysis demonstrated that this integrated EV double-filtration device had a sensitivity of 81.3% at a specificity of 90% (16 bladder cancer patients and 8 healthy controls). Thus, this integrated device has great potential to be used in conjunction with urine cytology and cystoscopy to improve clinical diagnosis of bladder cancer in clinics and at point-of-care (POC) settings. PMID:28436447

Molecular Simulations of Mutually Exclusive Folding in a Two-Domain Protein Switch

PubMed Central

Mills, Brandon M.; Chong, Lillian T.

2011-01-01

A major challenge with testing designs of protein conformational switches is the need for experimental probes that can independently monitor their individual protein domains. One way to circumvent this issue is to use a molecular simulation approach in which each domain can be directly observed. Here we report what we believe to be the first molecular simulations of mutually exclusive folding in an engineered two-domain protein switch, providing a direct view of how folding of one protein drives unfolding of the other in a barnase-ubiquitin fusion protein. These simulations successfully capture the experimental effects of interdomain linker length and ligand binding on the extent of unfolding in the less stable domain. In addition, the effect of linker length on the potential for oligomerization, which eliminates switch activity, is in qualitative agreement with analytical ultracentrifugation experiments. We also perform what we believe to be the first study of protein unfolding via progressive localized compression. Finally, we are able to explore the kinetics of mutually exclusive folding by determining the effect of linker length on rates of unfolding and refolding of each protein domain. Our results demonstrate that molecular simulations can provide seemingly novel biological insights on the behavior of individual protein domains, thereby aiding in the rational design of bifunctional switches. PMID:21281591

Proteomic analysis and comparison of the biopsy and autopsy specimen of human brain temporal lobe.

PubMed

He, Sizhi; Wang, Qingsong; He, Jintang; Pu, Hai; Yang, Wei; Ji, Jianguo

2006-09-01

The proteomic study on human temporal lobe can help us to understand the physiological function of CNS in normal as well as in pathological state. Proteomic tools are potent for the assessment of protein stability post mortem. In this pilot study, the human temporal lobe biopsy specimen with chronic pharmacoresistant temporal lobe epilepsy (TLE) and autopsy specimen in control were separated by 2-DE. Using MALDI-TOF-MS and MS/MS, 375 protein spots were identified which were the products of 267 genes. Six down-regulated and 23 up-regulated protein spots in the autopsy specimen were ascertained after the gel image analysis with the ImageMaster software. A number of proteins that include neurotransmitter metabolic and glycolytic enzymes, cytoprotective proteins and cytoskeleton were found decreased while the precursor of apolipoprotein A-I increased in the TLE brain. We tried several methods to prepare the protein samples and found that DNase and RNase treatment, ultracentrifugation and Amersham clean-up kit purification can improve gel separation quality. This work optimized the sample preparation method and constructed a primary protein database of human temporal lobe and found some proteins with remarkable level change probably involved in the post-mortem process and chronic pharmacoresistant TLE pathogenesis.

Serum Paraoxonase 1 Activity Is Associated with Fatty Acid Composition of High Density Lipoprotein

PubMed Central

Boshtam, Maryam; Pourfarzam, Morteza; Ani, Mohsen; Naderi, Gholam Ali; Basati, Gholam; Mansourian, Marjan; Dinani, Narges Jafari; Asgary, Seddigheh; Abdi, Soheila

2013-01-01

Introduction. Cardioprotective effect of high density lipoprotein (HDL) is, in part, dependent on its related enzyme, paraoxonase 1 (PON1). Fatty acid composition of HDL could affect its size and structure. On the other hand, PON1 activity is directly related to the structure of HDL. This study was designed to investigate the association between serum PON1 activity and fatty acid composition of HDL in healthy men. Methods. One hundred and forty healthy men participated in this research. HDL was separated by sequential ultracentrifugation, and its fatty acid composition was analyzed by gas chromatography. PON1 activity was measured spectrophotometrically using paraxon as substrate. Results. Serum PON1 activity was directly correlated with the amount of stearic acid and dihomo-gamma-linolenic acid (DGLA). PON1/HDL-C was directly correlated with the amount of miristic acid, stearic acid, and DGLA and was inversely correlated with total amount of ω6 fatty acids of HDL. Conclusion. The fatty acid composition of HDL could affect the activity of its associated enzyme, PON1. As dietary fats are the major determinants of serum lipids and lipoprotein composition, consuming some special dietary fatty acids may improve the activity of PON1 and thereby have beneficial effects on health. PMID:24167374

Serum paraoxonase 1 activity is associated with fatty acid composition of high density lipoprotein.

PubMed

Boshtam, Maryam; Razavi, Amirnader Emami; Pourfarzam, Morteza; Ani, Mohsen; Naderi, Gholam Ali; Basati, Gholam; Mansourian, Marjan; Dinani, Narges Jafari; Asgary, Seddigheh; Abdi, Soheila

2013-01-01

Cardioprotective effect of high density lipoprotein (HDL) is, in part, dependent on its related enzyme, paraoxonase 1 (PON1). Fatty acid composition of HDL could affect its size and structure. On the other hand, PON1 activity is directly related to the structure of HDL. This study was designed to investigate the association between serum PON1 activity and fatty acid composition of HDL in healthy men. One hundred and forty healthy men participated in this research. HDL was separated by sequential ultracentrifugation, and its fatty acid composition was analyzed by gas chromatography. PON1 activity was measured spectrophotometrically using paraxon as substrate. Serum PON1 activity was directly correlated with the amount of stearic acid and dihomo-gamma-linolenic acid (DGLA). PON1/HDL-C was directly correlated with the amount of miristic acid, stearic acid, and DGLA and was inversely correlated with total amount of ω 6 fatty acids of HDL. The fatty acid composition of HDL could affect the activity of its associated enzyme, PON1. As dietary fats are the major determinants of serum lipids and lipoprotein composition, consuming some special dietary fatty acids may improve the activity of PON1 and thereby have beneficial effects on health.

Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma.

PubMed

Kalra, Hina; Adda, Christopher G; Liem, Michael; Ang, Ching-Seng; Mechler, Adam; Simpson, Richard J; Hulett, Mark D; Mathivanan, Suresh

2013-11-01

Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including blood. Recent studies have highlighted the critical application of exosomes as personalized targeted drug delivery vehicles and as reservoirs of disease biomarkers. While these research applications have created significant interest and can be translated into practice, the stability of exosomes needs to be assessed and exosome isolation protocols from blood plasma need to be optimized. To optimize methods to isolate exosomes from blood plasma, we performed a comparative evaluation of three exosome isolation techniques (differential centrifugation coupled with ultracentrifugation, epithelial cell adhesion molecule immunoaffinity pull-down, and OptiPrep(TM) density gradient separation) using normal human plasma. Based on MS, Western blotting and microscopy results, we found that the OptiPrep(TM) density gradient method was superior in isolating pure exosomal populations, devoid of highly abundant plasma proteins. In addition, we assessed the stability of exosomes in plasma over 90 days under various storage conditions. Western blotting analysis using the exosomal marker, TSG101, revealed that exosomes are stable for 90 days. Interestingly, in the context of cellular uptake, the isolated exosomes were able to fuse with target cells revealing that they were indeed biologically active. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A glycoconjugate of Haemophilus influenzae Type b capsular polysaccharide with tetanus toxoid protein: hydrodynamic properties mainly influenced by the carbohydrate.

PubMed

Abdelhameed, Ali Saber; Adams, Gary G; Morris, Gordon A; Almutairi, Fahad M; Duvivier, Pierre; Conrath, Karel; Harding, Stephen E

2016-02-26

Three important physical properties which may affect the performance of glycoconjugate vaccines against serious disease are molar mass (molecular weight), heterogeneity (polydispersity), and conformational flexibility in solution. The dilute solution behaviour of native and activated capsular polyribosylribitol (PRP) polysaccharides extracted from Haemophilus influenzae type b (Hib), and the corresponding glycoconjugate made by conjugating this with the tetanus toxoid (TT) protein have been characterized and compared using a combination of sedimentation equilibrium and sedimentation velocity in the analytical ultracentrifuge with viscometry. The weight average molar mass of the activated material was considerably reduced (Mw ~ 0.24 × 10(6) g.mol(-1)) compared to the native (Mw ~ 1.2 × 10(6) g.mol(-1)). Conjugation with the TT protein yielded large polydisperse structures (of Mw ~ 7.4 × 10(6) g.mol(-1)), but which retained the high degree of flexibility of the native and activated polysaccharide, with frictional ratio, intrinsic viscosity, sedimentation conformation zoning behaviour and persistence length all commensurate with highly flexible coil behaviour and unlike the previously characterised tetanus toxoid protein (slightly extended and hydrodynamically compact structure with an aspect ratio of ~3). This non-protein like behaviour clearly indicates that it is the carbohydrate component which mainly influences the physical behaviour of the glycoconjugate in solution.

Structural studies of RNA-protein complexes: A hybrid approach involving hydrodynamics, scattering, and computational methods.

PubMed

Patel, Trushar R; Chojnowski, Grzegorz; Astha; Koul, Amit; McKenna, Sean A; Bujnicki, Janusz M

2017-04-15

The diverse functional cellular roles played by ribonucleic acids (RNA) have emphasized the need to develop rapid and accurate methodologies to elucidate the relationship between the structure and function of RNA. Structural biology tools such as X-ray crystallography and Nuclear Magnetic Resonance are highly useful methods to obtain atomic-level resolution models of macromolecules. However, both methods have sample, time, and technical limitations that prevent their application to a number of macromolecules of interest. An emerging alternative to high-resolution structural techniques is to employ a hybrid approach that combines low-resolution shape information about macromolecules and their complexes from experimental hydrodynamic (e.g. analytical ultracentrifugation) and solution scattering measurements (e.g., solution X-ray or neutron scattering), with computational modeling to obtain atomic-level models. While promising, scattering methods rely on aggregation-free, monodispersed preparations and therefore the careful development of a quality control pipeline is fundamental to an unbiased and reliable structural determination. This review article describes hydrodynamic techniques that are highly valuable for homogeneity studies, scattering techniques useful to study the low-resolution shape, and strategies for computational modeling to obtain high-resolution 3D structural models of RNAs, proteins, and RNA-protein complexes. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Purification, characterization and crystallization of the human 80S ribosome

PubMed Central

Khatter, Heena; Myasnikov, Alexander G.; Mastio, Leslie; Billas, Isabelle M. L.; Birck, Catherine; Stella, Stefano; Klaholz, Bruno P.

2014-01-01

Ribosomes are key macromolecular protein synthesis machineries in the cell. Human ribosomes have so far not been studied to atomic resolution because of their particularly complex structure as compared with other eukaryotic or prokaryotic ribosomes, and they are difficult to prepare to high homogeneity, which is a key requisite for high-resolution structural work. We established a purification protocol for human 80S ribosomes isolated from HeLa cells that allows obtaining large quantities of homogenous samples as characterized by biophysical methods using analytical ultracentrifugation and multiangle laser light scattering. Samples prepared under different conditions were characterized by direct single particle imaging using cryo electron microscopy, which helped optimizing the preparation protocol. From a small data set, a 3D reconstruction at subnanometric resolution was obtained showing all prominent structural features of the human ribosome, and revealing a salt concentration dependence of the presence of the exit site tRNA, which we show is critical for obtaining crystals. With these well-characterized samples first human 80S ribosome crystals were obtained from several crystallization conditions in capillaries and sitting drops, which diffract to 26 Å resolution at cryo temperatures and for which the crystallographic parameters were determined, paving the way for future high-resolution work. PMID:24452798

Synthesis, characterization, and evaluation of pluronic-based β-cyclodextrin polyrotaxanes for mobilization of accumulated cholesterol from Niemann-Pick type C fibroblasts.

PubMed

Collins, Christopher J; McCauliff, Leslie A; Hyun, Seok-Hee; Zhang, Zhaorui; Paul, Lake N; Kulkarni, Aditya; Zick, Klaus; Wirth, Mary; Storch, Judith; Thompson, David H

2013-05-14

Several lines of evidence suggest that β-cyclodextrin (β-CD) derivatives initiate the efflux of accumulated, unesterified cholesterol from the late endosomal/lysosomal compartment in Niemann Pick C (NPC) disease models. Unfortunately, repeated injections or continuous infusions of current β-CD therapies are required to sustain suppression of symptoms and prolong life. In an effort to make CD treatment a more viable option by boosting efficacy and improving pharmacokinetics, a library of Pluronic surfactant-based β-CD polyrotaxanes has been developed using biocompatible poly(ethylene glycol) (PEG)-polypropylene glycol (PPG)-PEG triblock copolymers. These compounds carry multiple copies of β-CD as shown by (1)H NMR, 2D nuclear Overhouser effect spectroscopy, gel permeation chromatography/multiangle light scattering, analytical ultracentrifugation analysis, matrix assisted laser desorption/ionization mass spectrometry, and diffusion-ordered spectroscopy. Analyses of free β-cyclodextrin contamination in the compounds were made by reverse phase high pressure liquid chromatography and hydrophilic interaction liquid chromatography. Dethreading kinetics were studied by reverse phase high pressure liquid chromatography, UV/vis, and (1)H NMR analysis. Filipin staining studies using npc2(-/-) fibroblasts show significant reversal of cholesterol accumulation after treatment with polyrotaxane compounds. The rate and efficacy of reversal is similar to that achieved by equivalent amounts of monomeric β-CD alone.

pyr RNA binding to the Bacillus caldolyticus PyrR attenuation protein. Characterization and regulation by uridine and guanosine nucleotides

PubMed Central

Jørgensen, Casper Møller; Fields, Christopher J.; Chander, Preethi; Watt, Desmond; Burgner, John W.; Smith, Janet L.; Switzer, Robert L.

2011-01-01

Summary The PyrR protein regulates expression of pyrimidine biosynthetic (pyr) genes in many bacteria. PyrR binds to specific sites in the 5’ leader RNA of target operons and favors attenuation of transcription. Filter binding and gel mobility assays were used to characterize the binding of PyrR from Bacillus caldolyticus to RNA sequences (binding loops) from the three attenuation regions of the B. caldolyticus pyr operon. Binding of PyrR to the three binding loops and modulation of RNA binding by nucleotides was similar for all three RNAs. Apparent dissociation constants at 0° C ranged from 0.13 to 0.87 nM in the absence of effectors; dissociation constants were decreased by 3 to 12 fold by uridine nucleotides and increased by 40 to 200 fold by guanosine nucleotides. The binding data suggest that pyr operon expression is regulated by the ratio of intracellular uridine nucleotides to guanosine nucleotides; the effects of nucleoside addition to the growth medium on aspartate transcarbamylase (pyrB) levels in B. subtilis cells in vivo supported this conclusion. Analytical ultracentrifugation established that RNA binds to dimeric PyrR, even though the tetrameric form of unbound PyrR predominates in solution at the concentrations studied. PMID:18190533

Nanosilver conductive ink: A case study for evaluating the potential risk of nanotechnology under hypothetical use scenarios.

PubMed

Martin, David P; Melby, Nicolas L; Jordan, Shinita M; Bednar, Anthony J; Kennedy, Alan J; Negrete, Maria E; Chappell, Mark A; Poda, Aimee R

2016-11-01

Engineered nanomaterials (ENMs) are being incorporated into a variety of consumer products due to unique properties that offer a variety of advantages over bulk materials. Understanding of the nano-specific risk associated with nano-enabled technologies, however, continues to lag behind research and development, registration with regulators, and commercialization. One example of a nano-enabled technology is nanosilver ink, which can be used in commercial ink-jet printers for the development of low-cost printable electronics. This investigation utilizes a tiered EHS framework to evaluate the potential nano-specific release, exposure and hazard associated with typical use of both nanosilver ink and printed circuits. The framework guides determination of the potential for ENM release from both forms of the technology in simulated use scenarios, including spilling of the ink, aqueous release (washing) from the circuits and UV light exposure. The as-supplied ink merits nano-specific consideration based on the presence of nanoparticles and their persistence in environmentally-relevant media. The material released from the printed circuits upon aqueous exposure was characterized by a number of analysis techniques, including ultracentrifugation and single particle ICP-MS, and the results suggest that a vast majority of the material was ionic in nature and nano-specific regulatory scrutiny may be less relevant. Published by Elsevier Ltd.

The use of analytical sedimentation velocity to extract thermodynamic linkage

PubMed Central

Cole, James L.; Correia, John J.; Stafford, Walter F.

2011-01-01

For 25 years, the Gibbs Conference on Biothermodynamics has focused on the use of thermodynamics to extract information about the mechanism and regulation of biological processes. This includes the determination of equilibrium constants for macromolecular interactions by high precision physical measurements. These approaches further reveal thermodynamic linkages to ligand binding events. Analytical ultracentrifugation has been a fundamental technique in the determination of macromolecular reaction stoichiometry and energetics for 85 years. This approach is highly amenable to the extraction of thermodynamic couplings to small molecule binding in the overall reaction pathway. In the 1980’s this approach was extended to the use of sedimentation velocity techniques, primarily by the analysis of tubulin-drug interactions by Na and Timasheff. This transport method necessarily incorporates the complexity of both hydrodynamic and thermodynamic nonideality. The advent of modern computational methods in the last 20 years has subsequently made the analysis of sedimentation velocity data for interacting systems more robust and rigorous. Here we review three examples where sedimentation velocity has been useful at extracting thermodynamic information about reaction stoichiometry and energetics. Approaches to extract linkage to small molecule binding and the influence of hydrodynamic nonideality are emphasized. These methods are shown to also apply to the collection of fluorescence data with the new Aviv FDS. PMID:21703752

A modified experimental setup for sedimentation equilibrium experiments with gels. Part 2: Technical developments.

PubMed

Cölfen, H; Borchard, W

1994-06-01

This part of the paper trilogy describes technical developments for an efficient experimental setup to investigate gels with equilibrium analytical ultracentrifugation. New 10-channel centerpieces for the Schlieren optics, a new programmable multiplexer, a modified Schlieren optical system, and a photo pickup with impulse transformer are introduced as major developments. Also, some new centerpieces suitable for equilibrium experiments with solutions using the Rayleigh interference and the uv-absorption optics are presented. These centerpieces allow the investigation of 10, 12, or even 26 samples per centerpiece. The problem to find suitable materials for cell centerpieces and windows in the case of adhering samples is discussed for the system gelatin/water. A phase volume calculation for circular sample channels as a correction for the case of broadened menisci is presented. The method described allows an accurate measurement of up to 70 samples simultaneously in an equilibrium experiment if the 8-hole rotor presented in part 1 of the trilogy is used. The number of samples is sufficient to characterize a gel/solvent system in the experimentally accessible range under identical conditions, which is not possible by means of any of the methods known before. All parts described are also applicable for the investigation of solutions.

The International Society for Extracellular Vesicles launches the first massive open online course on extracellular vesicles

PubMed Central

Lässer, Cecilia; Théry, Clotilde; Buzás, Edit I.; Mathivanan, Suresh; Zhao, Weian; Gho, Yong Song; Lötvall, Jan

2016-01-01

The International Society for Extracellular Vesicles (ISEV) has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs). This course, “Basics of Extracellular Vesicles,” uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field covering the nomenclature and history of EVs. Module 2 focuses on the biogenesis and uptake mechanisms of EVs, as well as their RNA, protein and lipid cargo. Module 3 covers the collection and processing of cell culture media and body fluids such as blood, breast milk, cerebrospinal fluid and urine prior to isolation of EVs. Modules 4 and 5 present different isolation methods and characterisation techniques utilised in the EV field. Here, differential ultracentrifugation, size-exclusion chromatography, density gradient centrifugation, kit-based precipitation, electron microscopy, cryo-electron microscopy, flow cytometry, atomic-force microscopy and nanoparticle-tracking analysis are covered. This first massive open online course (MOOC) on EVs was launched on 15 August 2016 at the platform “Coursera” and is free of charge. PMID:27989272

Incorporating beta-turns and a turn mimetic out of context in loop 1 of the WW domain affords cooperatively folded beta-sheets.

PubMed

Kaul, R; Angeles, A R; Jäger, M; Powers, E T; Kelly, J W

2001-06-06

To probe the conformational requirements of loop 1 in the Pin1 WW domain, the residues at the i + 2 and i + 3 positions of a beta-turn within this loop were replaced by dPro-Gly and Asn-Gly, which are known to prefer the conformations required at the i + 1 and i + 2 positions of type II' and type I' beta-turns. Conformational specificity or lack thereof was further examined by incorporating into the i + 2 and i + 3 positions a non-alpha-amino acid-based beta-turn mimetic (4-(2'-aminoethyl)-6-dibenzofuran propionic acid residue, 1), which was designed to replace the i + 1 and i + 2 positions of beta-turns. All these Pin WW variants are monomeric and folded as discerned by analytical ultracentrifugation, NMR, and CD. They exhibit cooperative two-state transitions and display thermodynamic stability within 0.5 kcal/mol of the wild-type WW domain, demonstrating that the acquisition of native structure and stability does not require a specific sequence and, by extension, conformation within loop 1. However, it could be that these loop 1 mutations alter the kinetics of antiparallel beta-sheet folding, which will be addressed by subsequent kinetic studies.

An Easy Method for Plant Polysome Profiling.

PubMed

Lecampion, Cécile; Floris, Maïna; Fantino, Jean Raphaël; Robaglia, Christophe; Laloi, Christophe

2016-08-28

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

Ferredoxin-thioredoxin reductase, an iron-sulfur enzyme linking light to enzyme regulation in oxygenic photosynthesis: purification and properties of the enzyme from C3, C4, and cyanobacterial species.

PubMed

Droux, M; Jacquot, J P; Miginac-Maslow, M; Gadal, P; Huet, J C; Crawford, N A; Yee, B C; Buchanan, B B

1987-02-01

Ferredoxin-thioredoxin reductase (FTR), an enzyme involved in the light regulation of chloroplast enzymes, was purified to homogeneity from leaves of spinach (a C3 plant) and corn (a C4 plant) and from cells of a cyanobacterium (Nostoc muscorum). The enzyme is a yellowish brown iron-sulfur protein, containing four nonheme iron and labile sulfide groups, that catalyzes the activation of NADP-malate dehydrogenase and fructose 1,6-bisphosphatase in the presence of ferredoxin and of thioredoxin m and f, respectively. FTR is synonymous with the protein earlier called ferralterin. FTR showed an Mr of about 30,000 (determined by sedimentation equilibrium ultracentrifugation, amino acid composition, gel filtration, and gradient gel electrophoresis) and was composed of two dissimilar subunits (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). One of the FTR subunits from each source was similar both in Mr (about 13,000) and immunological properties, while the other subunit (of variable molecular weight) was characteristic of a particular organism. The similar subunit contained a disulfide group that was rapidly reduced by a dithiol (dithiothreitol) but not by monothiols (2-mercaptoethanol or reduced glutathione). Homogeneous FTR formed a tight noncovalent complex with ferredoxin on affinity columns. The basis for the structural variation in the different FTR enzymes remains to be determined.

Characterization of the Group A Streptococcus Mga Virulence Regulator Reveals a Role for the C-terminal Region in Oligomerization and Transcriptional Activation

PubMed Central

Hondorp, Elise R.; Hou, Sherry C.; Hempstead, Andrew D.; Hause, Lara L.; Beckett, Dorothy M.; McIver, Kevin S.

2012-01-01

The Group A Streptococcus (GAS) is a strict human pathogen that causes a broad spectrum of illnesses. One of the key regulators of virulence in GAS is the transcriptional activator Mga, which coordinates the early stages of infection. Although the targets of Mga have been well characterized, basic biochemical analyses have been limited due to difficulties in obtaining purified protein. In this study, high-level purification of soluble Mga was achieved, enabling the first detailed characterization of the protein. Fluorescence titrations coupled with filter-binding assays indicate that Mga binds cognate DNA with nanomolar affinity. Gel filtration analyses, analytical ultracentrifugation, and co-immunoprecipitation experiments demonstrate that Mga forms oligomers in solution. Moreover, the ability of the protein to oligomerize in solution was found to correlate with transcriptional activation; DNA binding appears to be necessary but insufficient for full activity. Truncation analyses reveal that the uncharacterized C-terminal region of Mga, possessing similarity to phosphotransferase system EIIB proteins, plays a critical role in oligomerization and in vivo activity. Mga from a divergent serotype was found to behave similarly, suggesting that this study describes a general mechanism for Mga regulation of target virulence genes within GAS and provides insight into related regulators in other Gram-positive pathogens. PMID:22468267

Sedimentation Velocity Analysis of Large Oligomeric Chromatin Complexes Using Interference Detection.

PubMed

Rogge, Ryan A; Hansen, Jeffrey C

2015-01-01

Sedimentation velocity experiments measure the transport of molecules in solution under centrifugal force. Here, we describe a method for monitoring the sedimentation of very large biological molecular assemblies using the interference optical systems of the analytical ultracentrifuge. The mass, partial-specific volume, and shape of macromolecules in solution affect their sedimentation rates as reflected in the sedimentation coefficient. The sedimentation coefficient is obtained by measuring the solute concentration as a function of radial distance during centrifugation. Monitoring the concentration can be accomplished using interference optics, absorbance optics, or the fluorescence detection system, each with inherent advantages. The interference optical system captures data much faster than these other optical systems, allowing for sedimentation velocity analysis of extremely large macromolecular complexes that sediment rapidly at very low rotor speeds. Supramolecular oligomeric complexes produced by self-association of 12-mer chromatin fibers are used to illustrate the advantages of the interference optics. Using interference optics, we show that chromatin fibers self-associate at physiological divalent salt concentrations to form structures that sediment between 10,000 and 350,000S. The method for characterizing chromatin oligomers described in this chapter will be generally useful for characterization of any biological structures that are too large to be studied by the absorbance optical system. © 2015 Elsevier Inc. All rights reserved.

Intraluminal proteome and peptidome of human urinary extracellular vesicles.

PubMed

Liu, Xinyu; Chinello, Clizia; Musante, Luca; Cazzaniga, Marta; Tataruch, Dorota; Calzaferri, Giulio; James Smith, Andrew; De Sio, Gabriele; Magni, Fulvio; Zou, Hequn; Holthofer, Harry

2015-06-01

Urinary extracellular vesicles (UEVs) are a novel source for disease biomarker discovery. However, Tamm-Horsfall protein (THP) is still a challenge for proteomic analysis since it can inhibit detection of low-abundance proteins. Here, we introduce a new approach that does not involve an ultracentrifugation step to enrich vesicles and that reduces the amount of THP to manageable levels. UEVs were dialyzed and ultrafiltered after reduction and alkylation. The retained fraction was digested with trypsin to reduce the remaining THP and incubated with deoxycholate (DOC). The internal peptidome and internal proteome were analyzed by LC-ESI-MS. A total of 942 different proteins and 3115 unique endogenous peptide fragments deriving from 973 different protein isoforms were identified. Around 82% of the key endosomal sorting complex required for transport components of UEVs generation could be detected from the intraluminal content. Our UEVs preparation protocol provides a simplified way to investigate the intraluminal proteome and peptidome, in particular the subpopulation of UEVs of the trypsin-resistant class of exosomes (positive for tumor susceptibility gene101) and eliminates the majority of interfering proteins such as THP. This method allows the possibility to study endoproteome and endopeptidome of UEVs, thus greatly facilitating biomarker discovery. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Purification of Drug Loaded PLGA Nanoparticles Prepared by Emulsification Solvent Evaporation Using Stirred Cell Ultrafiltration Technique.

PubMed

Paswan, Suresh K; Saini, T R

2017-12-01

The emulsifiers in an exceedingly higher level are used in the preparation of drug loaded polymeric nanoparticles prepared by emulsification solvent evaporation method. This creates great problem to the formulator due to their serious toxicities when it is to be administered by parenteral route. The final product is therefore required to be freed from the used surfactants by the conventional purification techniques which is a cumbersome job. The solvent resistant stirred cell ultrafiltration unit (Millipore) was used in this study using polyethersulfone ultrafiltration membrane (Biomax®) having pore size of NMWL 300 KDa as the membrane filter. The purification efficiency of this technique was compared with the conventional centrifugation technique. The flow rate of ultrafiltration was optimized for removal of surfactant (polyvinyl alcohol) impurities to the acceptable levels in 1-3.5 h from the nanoparticle dispersion of tamoxifen prepared by emulsification solvent evaporation method. The present investigations demonstrate the application of solvent resistant stirred cell ultrafiltration technique for removal of toxic impurities of surfactant (PVA) from the polymeric drug nanoparticles (tamoxifen) prepared by emulsification solvent evaporation method. This technique offers added benefit of producing more concentrated nanoparticles dispersion without causing significant particle size growth which is observed in other purification techniques, e.g., centrifugation and ultracentrifugation.

A method for detergent-free isolation of membrane proteins in their local lipid environment.

PubMed

Lee, Sarah C; Knowles, Tim J; Postis, Vincent L G; Jamshad, Mohammed; Parslow, Rosemary A; Lin, Yu-Pin; Goldman, Adrian; Sridhar, Pooja; Overduin, Michael; Muench, Stephen P; Dafforn, Timothy R

2016-07-01

Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1-2 h); investigate SMALP protein purity by SDS-PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (∼2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins.

Structures and ice-binding faces of the alanine-rich type I antifreeze proteins.

PubMed

Patel, Shruti N; Graether, Steffen P

2010-04-01

Antifreeze proteins (AFPs) protect cold-blooded organisms from the damage caused by freezing through their ability to inhibit ice growth. The type I AFP family, found in several fish species, contains proteins that have a high alanine content (>60% of the sequence) and structures that are almost all alpha-helical. We examine the structure of the type I AFP isoforms HPLC6 from winter flounder, shorthorn sculpin 3, and the winter flounder hyperactive type I AFP. The HPLC6 isoform structure consists of a single alpha-helix that is 37 residues long, whereas the shorthorn sculpin 3 isoform consists of two helical regions separated by a kink. The high-resolution structure of the hyperactive type I AFP has yet to be determined, but circular dichroism data and analytical ultracentrifugation suggest that the 195 residue protein is a side-by-side dimer of two alpha-helices. The alanine-rich ice-binding faces of HPLC6 and hyperactive type I AFP are discussed, and we propose that the ice-binding face of the shorthorn sculpin 3 AFP contains Ala14, Ala19, and Ala25. We also propose that the denaturation of hyperactive type I AFP at room temperature is explained by the stabilization of the dimerization interface through hydrogen bonds.

A polygalacturonase localized in the Golgi apparatus in Pisum sativum.

PubMed

Ohashi, Takao; Jinno, Jun; Inoue, Yoshiyuki; Ito, Shoko; Fujiyama, Kazuhito; Ishimizu, Takeshi

2017-09-01

Pectin is a plant cell wall constituent that is mainly composed of polygalacturonic acid (PGA), a linear α1,4-d-galacturonic acid (GalUA) backbone. Polygalacturonase (PG) hydrolyzes the α1,4-linkages in PGA. Nearly all plant PGs identified thus far are secreted as soluble proteins. Here we describe the microsomal PG activity in pea (Pisum sativum) epicotyls and present biochemical evidence that it was localized to the Golgi apparatus, where pectins are biosynthesized. The microsomal PG was purified, and it was enzymatically characterized. The purified enzyme showed maximum activity towards pyridylaminated oligogalacturonic acids with six degrees of polymerization (PA-GalUA6), with a Km value of 11 μM for PA-GalUA6. The substrate preference of the enzyme was complementary to that of PGA synthase. The main PG activity in microsomes was detected in the Golgi fraction by sucrose density gradient ultracentrifugation. The activity of the microsomal PG was lower in rapidly growing epicotyls, in contrast to the high expression of PGA synthase. The role of this PG in the regulation of pectin biosynthesis or plant growth is discussed. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Protective effects of circulating microvesicles derived from myocardial ischemic rats on apoptosis of cardiomyocytes in myocardial ischemia/reperfusion injury.

PubMed

Wang, Yao; Wei, Su; Wang, Yi-Lu; Liu, Miao; Shang, Man; Zhang, Qi; Wu, Yan-Na; Liu, Ming-Lin; Song, Jun-Qiu; Liu, Yan-Xia

2017-08-15

To investigate the effects of circulating microvesicles derived from myocardial ischemia (I-MVs) on apoptosis in myocardial ischemia/reperfusion (I/R) injury in rats. I-MVs from rats undergoing myocardial left anterior descending (LAD) coronary artery ligation were isolated by ultracentrifugation from circulating blood and characterized by flow cytometry. I-MVs were administered intravenously (4.8 mg/kg) at 5 min before reperfusion procedure in I/R injury model which was induced by 30-min of ischemia and 120-min of reperfusion of LAD in rats. Treatment with I-MVssignificantly reduced the size of myocardial infarction, the activities of serum CK-MB and LDH, and the number of apoptotic cardiomyocytes. The activities of caspase 3, caspase 9 and caspase 12 in myocardium were also decreased significantly with I-MVs treatment. Moreover, the expression of Bax was decreased but Bcl-2 was increased. The expression of glucose regulated protein 78 (GRP78), sarco/endoplasmic reticulum Ca 2+ -ATPase 2 (SERCA2) and phosphorylated phospholamban (p-PLB) were increased after being treated with I-MVs. I-MVs could protect hearts from I/R injury in rats through SERCA2 and p-PLB of calcium regulatory proteins to alleviate intrinsic myocardial apoptosis including mitochondrial and endoplasmic reticulum pathways.

The study on the entrapment efficiency and in vitro release of puerarin submicron emulsion.

PubMed

Yue, Peng-Fei; Lu, Xiu-Yun; Zhang, Zeng-Zhu; Yuan, Hai-Long; Zhu, Wei-Feng; Zheng, Qin; Yang, Ming

2009-01-01

The entrapment efficiency (EE) and release in vitro are very important physicochemical characteristics of puerarin submicron emulsion (SME). In this paper, the performance of ultrafiltration (UF), ultracentrifugation (UC), and microdialysis (MD) for determining the EE of SME were evaluated, respectively. The release study in vitro of puerarin from SME was studied by using MD and pressure UF technology. The EE of SME was 86.5%, 72.8%, and 55.8% as determined by MD, UF, and UC, respectively. MD was not suitable for EE measurements of puerarin submicron oil droplet, which could only determine the total EE of submicron oil droplet and liposomes micelles, but it could be applied to determine the amount of free drug in SMEs. Although UC was the fastest and simplest to use, its results were the least reliable. UF was still the relatively accurate method for EE determination of puerarin SME. The release of puerarin SME could be evaluated by using MD and pressure UF, but MD seemed to be more suitable for the release study of puerarin emulsion. The drug release from puerarin SME at three drug concentrations was initially rapid, but reached a plateau value within 30 min. Drug release of puerarin from the SME occurred via burst release.

Proteomic analysis and cross species comparison of casein fractions from the milk of dairy animals

PubMed Central

Wang, Xiaxia; Zhao, Xiaowei; Huang, Dongwei; Pan, Xiaocheng; Qi, Yunxia; Yang, Yongxin; Zhao, Huiling; Cheng, Guanglong

2017-01-01

Casein micelles contribute to the physicochemical properties of milk and may also influence its functionality. At present, however, there is an incomplete understanding of the casein micelle associated proteins and its diversity among the milk obtained from different species. Therefore, milk samples were collected from seven dairy animals groups, casein fractions were prepared by ultracentrifugation and their constituent proteins were identified by liquid chromatography tandem mass spectrometry. A total of 193 distinct proteins were identified among all the casein micelle preparations. Protein interaction analysis indicated that caseins could interact with major whey proteins, including β-lactoglobulin, α-lactalbumin, lactoferrin, and serum albumin, and then whey proteins interacted with other proteins. Pathway analysis found that the peroxisome proliferator-activated receptor signaling pathway is shared among the studied animals. Additionally, galactose metabolism pathway is also found to be commonly involved for proteins derived from camel and horse milk. According to the similarity of casein micelle proteomes, two major sample clusters were classified into ruminant animals (Holstein and Jersey cows, buffaloes, yaks, and goats) and non-ruminants (camels and horses). Our results provide new insights into the protein profile associated with casein micelles and the functionality of the casein micelle from the studied animals. PMID:28240229

The effect of ultrasound on casein micelle integrity.

PubMed

Chandrapala, J; Martin, G J O; Zisu, B; Kentish, S E; Ashokkumar, M

2012-12-01

Samples of fresh skim milk, reconstituted micellar casein, and casein powder were sonicated at 20 kHz to investigate the effect of ultrasonication. For fresh skim milk, the average size of the remaining fat globules was reduced by approximately 10 nm after 60 min of sonication; however, the size of the casein micelles was determined to be unchanged. A small increase in soluble whey protein and a corresponding decrease in viscosity also occurred within the first few minutes of sonication, which could be attributed to the breakup of casein-whey protein aggregates. No measurable changes in free casein content could be detected in ultracentrifuged skim milk samples sonicated for up to 60 min. A small, temporary decrease in pH resulted from sonication; however, no measurable change in soluble calcium concentration was observed. Therefore, casein micelles in fresh skim milk were stable during the exposure to ultrasonication. Similar results were obtained for reconstituted micellar casein, whereas larger viscosity changes were observed as whey protein content was increased. Controlled application of ultrasound can be usefully applied to reverse process-induced protein aggregation without affecting the native state of casein micelles. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Isolation and immunochemical characterization of fractions from membranes of Aspergillus fumigatus with protease activity.

PubMed

Piechura, J E; Kurup, V P; Daft, L J

1990-01-01

Two fractions exhibiting acid protease activity (AFPI and AFPII) were isolated by extraction of membrane vesicles of Aspergillus fumigatus with Triton X-100. These two fractions produced single bands in both polyacrylamide and sodium dodecyl sulfate polyacrylamide gel electrophoresis and showed apparent molecular weights of 73,000 and 43,000, respectively. Molecular weights determined by gel filtration in the absence and presence of Triton X-100 and sedimentation velocities in analytical ultracentrifugation indicated hydrophobic characteristics, since both fractions readily aggregated and complexed with Triton X-100; both exhibited elevated enzyme activities in the presence of Triton X-100. Carbohydrate content was 93% for AFPI and 85% for AFPII. The enzymatic fractions demonstrated different pH optima in the acid range as well as different temperature stabilities. Both protease fractions cross reacted in double immunodiffusion, while in crossed immunoelectrophoresis both demonstrated five precipitin peaks, each with similar patterns. AFPI demonstrated two additional precipitin peaks in crossed immunoelectrophoresis. As determined by crossed immunoaffinoelectrophoresis, the protease fractions demonstrated galactose and mannose residues. In biotin-avidin enzyme-linked immunosorbent assay both fractions reacted with allergic bronchopulmonary aspergillosis and aspergilloma sera. It can be concluded that two fractions with protease activity of A. fumigatus reported here may be of significance in Aspergillus-induced diseases.

Light activation of the LOV protein Vivid generates a rapidly exchanging dimer†‡

PubMed Central

Zoltowski, Brian D.; Crane, Brian R.

2009-01-01

The fungal photoreceptor Vivid (VVD) plays an important role in the adaptation of blue-light responses in Neurospora crassa. VVD, an FAD-binding LOV (Light, Oxygen, Voltage) protein, couples light-induced cysteinyl-adduct formation at the flavin ring to conformational changes in the N-terminal cap (Ncap) of the VVD PAS domain. Size-exclusion chromatography (SEC), equilibrium ultracentrifugation, and static and dynamic light scattering show that these conformational changes generate a rapidly exchanging VVD dimer, with an expanded hydrodynamic radius. A three-residue N-terminal β-turn that assumes two different conformations in a crystal structure of a VVD C71V variant is essential for light-state dimerization. Residue substitutions at a critical hinge between the Ncap and PAS core can inhibit or enhance dimerization, whereas a Tyr to Trp substitution at the Ncap-to-PAS interface stabilizes the light-state dimer. Cross-linking through engineered disulfides indicates that the light-state dimer differs considerably from the dark-state dimer found in VVD crystal structures. These results verify the role of Ncap conformational changes in gating the photic response of Neurospora crassa, and indicate that LOV:LOV homo or hetero dimerization may be a mechanism for regulating light-activated gene expression. PMID:18553928

Light activation of the LOV protein vivid generates a rapidly exchanging dimer.

PubMed

Zoltowski, Brian D; Crane, Brian R

2008-07-08

The fungal photoreceptor Vivid (VVD) plays an important role in the adaptation of blue-light responses in Neurospora crassa. VVD, an FAD-binding LOV (light, oxygen, voltage) protein, couples light-induced cysteinyl adduct formation at the flavin ring to conformational changes in the N-terminal cap (Ncap) of the VVD PAS domain. Size-exclusion chromatography (SEC), equilibrium ultracentrifugation, and static and dynamic light scattering show that these conformational changes generate a rapidly exchanging VVD dimer, with an expanded hydrodynamic radius. A three-residue N-terminal beta-turn that assumes two different conformations in a crystal structure of a VVD C71V variant is essential for light-state dimerization. Residue substitutions at a critical hinge between the Ncap and PAS core can inhibit or enhance dimerization, whereas a Tyr to Trp substitution at the Ncap-PAS interface stabilizes the light-state dimer. Cross-linking through engineered disulfides indicates that the light-state dimer differs considerably from the dark-state dimer found in VVD crystal structures. These results verify the role of Ncap conformational changes in gating the photic response of N. crassa and indicate that LOV-LOV homo- or heterodimerization may be a mechanism for regulating light-activated gene expression.

Fluorinated diglucose detergents for membrane-protein extraction.

PubMed

Boussambe, Gildas Nyame Mendendy; Guillet, Pierre; Mahler, Florian; Marconnet, Anaïs; Vargas, Carolyn; Cornut, Damien; Soulié, Marine; Ebel, Christine; Le Roy, Aline; Jawhari, Anass; Bonneté, Françoise; Keller, Sandro; Durand, Grégory

2018-05-29

Fluorinated surfactants have scarcely been explored for the direct extraction of proteins from membranes because fluorination is believed to abrogate detergency. However, we have recently shown that a commercially available fluorinated surfactant readily solubilizes lipid membranes, thereby suggesting that fluorination per se does not interfere with detergent activity. In this work, we developed new fluorinated surfactants that exhibit detergency in terms of both lipid-vesicle solubilization and membrane-protein extraction. The compounds made and tested contain two glucose moieties as polar headgroup, a hydrogenated thioether linker, and a perfluorinated alkyl tail with either 4, 6, or 8 carbon atoms. The physicochemical properties of the micelles formed by the three fluorinated surfactants were evaluated by NMR spectroscopy, surface tensiometry, isothermal titration calorimetry, dynamic light scattering, small-angle X-ray scattering, and analytical ultracentrifugation. At 25°C, micellization was mainly entropy-driven, and the CMC values were found to decrease with chain length of the fluorinated tail, whereas the aggregation number increased with chain length. Remarkably, all three surfactants were found to solubilize lipid vesicles and extract a broad range of proteins from Escherichiacoli membranes. These findings demonstrate, for the first time, that nonionic fluorinated surfactants could be further exploited for the direct extraction and solubilization of membrane proteins. Copyright © 2018. Published by Elsevier Inc.

In Situ Study on the Evolution of Multimodal Particle Size Distributions of ZnO Quantum Dots: Some General Rules for the Occurrence of Multimodalities.

PubMed

Schindler, Torben; Walter, Johannes; Peukert, Wolfgang; Segets, Doris; Unruh, Tobias

2015-12-10

Properties of small semiconductor nanoparticles (NPs) are strongly governed by their size. Precise characterization is a key requirement for tailored dispersities and thus for high-quality devices. Results of a careful analysis of particle size distributions (PSDs) of ZnO are presented combining advantages of UV/vis absorption spectroscopy, analytical ultracentrifugation, and small-angle X-ray scattering (SAXS). Our study reveals that careful cross-validation of these different methods is mandatory to end up with reliable resolution. PSDs of ZnO NPs are multimodal on a size range of 2-8 nm, a finding that is not yet sufficiently addressed. In the second part of our work the evolution of PSDs was studied using in situ SAXS. General principles for the appearance of multimodalities covering a temperature range between 15 and 45 °C were found which are solely determined by the aging state indicated by the size of the medium-sized fraction. Whenever this fraction exceeds a critical diameter, a new multimodality is identified, independent of the particular time-temperature combination. A fraction of larger particles aggregates first before a fraction of smaller particles is detected. Fixed multimodalities have not yet been addressed adequately and could only be evidenced due to careful size analysis.

Optimizing exosomal RNA isolation for RNA-Seq analyses of archival sera specimens.

PubMed

Prendergast, Emily N; de Souza Fonseca, Marcos Abraão; Dezem, Felipe Segato; Lester, Jenny; Karlan, Beth Y; Noushmehr, Houtan; Lin, Xianzhi; Lawrenson, Kate

2018-01-01

Exosomes are endosome-derived membrane vesicles that contain proteins, lipids, and nucleic acids. The exosomal transcriptome mediates intercellular communication, and represents an understudied reservoir of novel biomarkers for human diseases. Next-generation sequencing enables complex quantitative characterization of exosomal RNAs from diverse sources. However, detailed protocols describing exosome purification for preparation of exosomal RNA-sequence (RNA-Seq) libraries are lacking. Here we compared methods for isolation of exosomes and extraction of exosomal RNA from human cell-free serum, as well as strategies for attaining equal representation of samples within pooled RNA-Seq libraries. We compared commercial precipitation with ultracentrifugation for exosome purification and confirmed the presence of exosomes via both transmission electron microscopy and immunoblotting. Exosomal RNA extraction was compared using four different RNA purification methods. We determined the minimal starting volume of serum required for exosome preparation and showed that high quality exosomal RNA can be isolated from sera stored for over a decade. Finally, RNA-Seq libraries were successfully prepared with exosomal RNAs extracted from human cell-free serum, cataloguing both coding and non-coding exosomal transcripts. This method provides researchers with strategic options to prepare RNA-Seq libraries and compare RNA-Seq data quantitatively from minimal volumes of fresh and archival human cell-free serum for disease biomarker discovery.

Effect of alcohol on hepatic receptor of high density lipoproteins (HDL)

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

Lin, R.C.; Miller, B.M.

1991-03-11

Moderate alcohol intake has been shown to increase HDL cholesterol and proteins. The seemingly protective effect' of moderate alcohol drinking against cardiovascular diseases has been attributed to an increase in serum HDL. In this study, the authors show that a receptor for HDL is present in rat liver. Rat liver membrane was prepared by stepwise ultracentrifugation. Apo Al was iodinated using {sup 125}I-NaI and IODO-beads. HDL was labeled by incubating with {sup 125}I-apo Al then refloated be centrifugation. Binding of {sup 125}I-HDL to rat liver membrane reached equilibrium by 2-3 h and was saturable at 37C. The binding was inhibitedmore » 80% by excess unlabeled HDL, but was inhibited only 25% by excess LDL. It could also be inhibited by preincubating HDL with anti-apo Al or anti-apo E antisera but not with anti-apo AIV or control sera. The binding affinity of HDL to the liver membrane of rats fed alcohol for 5 wk was 50% that of their pair-fed controls. Thus a decrease in the binding of HDL to liver membrane due to alcohol-drinking may result in a slower clearance of HDL by the liver and consequently a higher HDL concentration in the serum.« less

Generation of Tioman virus nucleocapsid-like particles in yeast Saccharomyces cerevisiae.

PubMed

Petraityte, Rasa; Tamosiunas, Paulius L; Juozapaitis, Mindaugas; Zvirbliene, Aurelija; Sasnauskas, Kestutis; Shiell, Brian; Russell, Gail; Bingham, John; Michalski, Wojtek P

2009-10-01

Tioman virus (TioV) was isolated from a number of pooled urine samples of Tioman Island flying foxes (Pteropus hypomelanus) during the search for the reservoir host of Nipah virus. Studies have established TioV as a new virus in the family Paramyxoviridae. This novel paramyxovirus is antigenically related to Menangle virus that was isolated in Australia in 1997 during disease outbreak in pigs. TioV causes mild disease in pigs and has a predilection for lymphoid tissues. Recent serosurvey showed that 1.8% of Tioman Islanders had neutralizing antibodies against TioV, indicating probable past infection. For the development of convenient serological tests for this virus, recombinant TioV nucleocapsid (N) protein was expressed in the yeast Saccharomyces cerevisiae. High yields of recombinant TioV N protein were obtained. Electron microscopy demonstrated that purified recombinant N protein self-assembled into nucleocapsid-like particles which were identical in density and morphology to authentic nucleocapsids from paramyxovirus-infected cells. Different size nucleocapsid-like particles were stable and readily purified by CsCl gradient ultracentrifugation. Polyclonal sera raised in rabbits after immunization with recombinant TioV N protein reacted reliably with TioV infected tissues in immunohistochemistry tests. It confirmed that the antigenic properties of yeast derived TioV N protein are identical to authentic viral protein.

Synthesis and characterization of novel polymers from non-petroleum sources for use in enhanced oil recovery. Final report, July 1, 1983-June 30, 1984

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

Hogen-Esch, T.E.

1984-01-01

Accomplishments for the past year are discussed for grafting of acrylamide to: (1) starch and related polysaccharides; and (2) cellulose solutions. In grafting acrylamide to various polysaccharide substrates such as okra polysaccharide, yellow dextrin, waxy corn starch, potato amylose, gum arabic, the efficiency of Ce/sup 4 +/ as initiator was found to vary from 0.02 to 0.89, depending on reaction conditions. Okra polysaccharide was isolated, characterized, and evaluated for use in enhanced oil recovery. A series of experiments designed to increase the viscosifying power of certain polymers by chain extension techniques has also been conducted. Characterization of the polymers bymore » ultracentrifugation, size exclusion chromatography, membrane filtration, multi-cell equilibrium dialysis, and rheological studies has also been done. In grafting of acrylamide to cellulose solutions the following two courses were taken: (1) dissolution of cellulose in 70% aqueous zinc chloride, followed by Ce/sup 4 +/ initiated grafting of acrylamide, and (2) introduction of a 1,2-diol substituent onto the anhydroglucose units of the cellulose chain via dissolution of cellulose in concentrated aqueous NaOH, followed by treatment with glyceryl chlorohydrin. Considerable progress has been made via both approaches. 11 refs., 1 fig., 1 tab.« less

Olfactomedin-1 Has a V-shaped Disulfide-linked Tetrameric Structure*

PubMed Central

Pronker, Matti F.; Bos, Trusanne G. A. A.; Sharp, Thomas H.; Thies-Weesie, Dominique M. E.; Janssen, Bert J. C.

2015-01-01

Olfactomedin-1 (Olfm1; also known as noelin and pancortin) is a member of the olfactomedin domain-containing superfamily and a highly expressed neuronal glycoprotein important for nervous system development. It binds a number of secreted proteins and cell surface-bound receptors to induce cell signaling processes. Using a combined approach of x-ray crystallography, solution scattering, analytical ultracentrifugation, and electron microscopy we determined that full-length Olfm1 forms disulfide-linked tetramers with a distinctive V-shaped architecture. The base of the “V” is formed by two disulfide-linked dimeric N-terminal domains. Each of the two V legs consists of a parallel dimeric disulfide-linked coiled coil with a C-terminal β-propeller dimer at the tips. This agrees with our crystal structure of a C-terminal coiled-coil segment and β-propeller combination (Olfm1coil-Olf) that reveals a disulfide-linked dimeric arrangement with the β-propeller top faces in an outward exposed orientation. Similar to its family member myocilin, Olfm1 is stabilized by calcium. The dimer-of-dimers architecture suggests a role for Olfm1 in clustering receptors to regulate signaling and sheds light on the conformation of several other olfactomedin domain family members. PMID:25903135

Dimeric, trimeric and tetrameric complexes of immunoglobulin G fix complement.

PubMed Central

Wright, J K; Tschopp, J; Jaton, J C; Engel, J

1980-01-01

The binding of pure dimers, trimers and tetramers of randomly cross-linked non-immune rabbit immunoglobulin G to the first component and subcomponent of the complement system, C1 and C1q respectively, was studied. These oligomers possessed open linear structures. All three oligomers fixed complement with decreasing affinity in the order: tetramer, trimer, dimer. Complement fixation by dimeric immunoglobulin exhibited the strongest concentration-dependence. No clear distinction between a non-co-operative and a co-operative binding mechanism could be achieved, although the steepness of the complement-fixation curves for dimers and trimers was better reflected by the co-operative mechanism. Intrinsic binding constants were about 10(6)M-1 for dimers, 10(7)M-1 for trimers and 3 X 10(9)M-1 for tetramers, assuming non-co-operative binding. The data are consistent with a maximum valency of complement component C1 for immunoglobulin G protomers in the range 6-18. The binding of dimers to purified complement subcomponent C1q was demonstrated by sedimentation-velocity ultracentrifugation. Mild reduction of the complexes by dithioerythritol caused the immunoglobulin to revert to the monomeric state (S20,w = 6.2-6.5S) with concomitant loss of complement-fixing ability. Images Fig. 2. PMID:6985362

A Supercomplex Spanning the Inner and Outer Membranes Mediates the Biogenesis of β-Barrel Outer Membrane Proteins in Bacteria*

PubMed Central

Wang, Yan; Wang, Rui; Jin, Feng; Liu, Yang; Yu, Jiayu; Fu, Xinmiao; Chang, Zengyi

2016-01-01

β-barrel outer membrane proteins (OMPs) are ubiquitously present in Gram-negative bacteria, mitochondria and chloroplasts, and function in a variety of biological processes. The mechanism by which the hydrophobic nascent β-barrel OMPs are transported through the hydrophilic periplasmic space in bacterial cells remains elusive. Here, mainly via unnatural amino acid-mediated in vivo photo-crosslinking studies, we revealed that the primary periplasmic chaperone SurA interacts with nascent β-barrel OMPs largely via its N-domain but with β-barrel assembly machine protein BamA mainly via its satellite P2 domain, and that the nascent β-barrel OMPs interact with SurA via their N- and C-terminal regions. Additionally, via dual in vivo photo-crosslinking, we demonstrated the formation of a ternary complex involving β-barrel OMP, SurA, and BamA in cells. More importantly, we found that a supercomplex spanning the inner and outer membranes and involving the BamA, BamB, SurA, PpiD, SecY, SecE, and SecA proteins appears to exist in living cells, as revealed by a combined analyses of sucrose-gradient ultra-centrifugation, Blue native PAGE and mass spectrometry. We propose that this supercomplex integrates the translocation, transportation, and membrane insertion events for β-barrel OMP biogenesis. PMID:27298319

Development and validation of a rapid multi-class method for the confirmation of fourteen prohibited medicinal additives in pig and poultry compound feed by liquid chromatography-tandem mass spectrometry.

PubMed

Cronly, Mark; Behan, P; Foley, B; Malone, E; Earley, S; Gallagher, M; Shearan, P; Regan, L

2010-12-01

A confirmatory method has been developed to allow for the analysis of fourteen prohibited medicinal additives in pig and poultry compound feed. These compounds are prohibited for use as feed additives although some are still authorised for use in medicated feed. Feed samples are extracted by acetonitrile with addition of sodium sulfate. The extracts undergo a hexane wash to aid with sample purification. The extracts are then evaporated to dryness and reconstituted in initial mobile phase. The samples undergo an ultracentrifugation step prior to injection onto the LC-MS/MS system and are analysed in a run time of 26 min. The LC-MS/MS system is run in MRM mode with both positive and negative electrospray ionisation. The method was validated over three days and is capable of quantitatively analysing for metronidazole, dimetridazole, ronidazole, ipronidazole, chloramphenicol, sulfamethazine, dinitolimide, ethopabate, carbadox and clopidol. The method is also capable of qualitatively analysing for sulfadiazine, tylosin, virginiamycin and avilamycin. A level of 100 microg kg(-1) was used for validation purposes and the method is capable of analysing to this level for all the compounds. Validation criteria of trueness, precision, repeatability and reproducibility along with measurement uncertainty are calculated for all analytes. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis.

PubMed

de Sousa, Marylane; Manzo, Ricardo M; García, José L; Mammarella, Enrique J; Gonçalves, Luciana R B; Pessela, Benevides C

2017-12-06

l-Arabinose isomerase (EC 5.3.1.4) (l-AI) from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N -His-l-AI and C -His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics. Analytical ultracentrifugation revealed that C -His-l-AI was preferentially hexameric in solution, whereas N -His-l-AI was mainly monomeric. The specific activity of the N -His-l-AI at acidic pH was higher than that of C -His-l-AI and showed a maximum bioconversion yield of 26% at 50 °C for d-tagatose biosynthesis, with Km and Vmax parameters of 252 mM and 0.092 U mg -1 , respectively. However, C -His-l-AI was more active and stable at alkaline pH than N -His-l-AI. N -His-l-AI follows a Michaelis-Menten kinetic, whereas C -His-l-AI fitted to a sigmoidal saturation curve.

Selective binding and oligomerization of the murine granulocyte colony-stimulating factor receptor by a low molecular weight, nonpeptidyl ligand.

PubMed

Doyle, Michael L; Tian, Shin-Shay; Miller, Stephen G; Kessler, Linda; Baker, Audrey E; Brigham-Burke, Michael R; Dillon, Susan B; Duffy, Kevin J; Keenan, Richard M; Lehr, Ruth; Rosen, Jon; Schneeweis, Lumelle A; Trill, John; Young, Peter R; Luengo, Juan I; Lamb, Peter

2003-03-14

Granulocyte colony-stimulating factor regulates neutrophil production by binding to a specific receptor, the granulocyte colony-stimulating factor receptor, expressed on cells of the granulocytic lineage. Recombinant forms of granulocyte colony-stimulating factor are used clinically to treat neutropenias. As part of an effort to develop granulocyte colony-stimulating factor mimics with the potential for oral bioavailability, we previously identified a nonpeptidyl small molecule (SB-247464) that selectively activates murine granulocyte colony-stimulating factor signal transduction pathways and promotes neutrophil formation in vivo. To elucidate the mechanism of action of SB-247464, a series of cell-based and biochemical assays were performed. The activity of SB-247464 is strictly dependent on the presence of zinc ions. Titration microcalorimetry experiments using a soluble murine granulocyte colony-stimulating factor receptor construct show that SB-247464 binds to the extracellular domain of the receptor in a zinc ion-dependent manner. Analytical ultracentrifugation studies demonstrate that SB-247464 induces self-association of the N-terminal three-domain fragment in a manner that is consistent with dimerization. SB-247464 induces internalization of granulocyte colony-stimulating factor receptor on intact cells, consistent with a mechanism involving receptor oligomerization. These data show that small nonpeptidyl compounds are capable of selectively binding and inducing productive oligomerization of cytokine receptors.

Role of carbohydrate in multimeric structure of factor VIII/von Willebrand factor protein.

PubMed Central

Gralnick, H R; Williams, S B; Rick, M E

1983-01-01

The carbohydrate moiety of the factor VIII/von Willebrand (vW) factor protein is important in the expression of vW factor activity and the intravascular survival of the protein. Studies of normal human factor VIII/vW factor protein indicate that there is a requirement of a full complement of penultimate galactose for the maintenance of a normal multimeric structure. Release of penultimate galactose by beta-galactosidase or modification by galactose oxidase results in loss of the largest molecular weight multimers and increased numbers of intermediate and smaller multimers. In contrast, terminal galactose on the factor VIII/vW factor protein does not appear to play a significant role in the maintenance of the multimer organization. The abnormalities in multimeric structure and molecular size were demonstrated by NaDodSO4/polyacrylamide/agarose gel electrophoresis, NaDodSO4/glyoxyl-agarose electrophoresis, and sucrose density ultracentrifugation. These studies indicate that the penultimate galactose plays a role in the maintenance of the largest multimers of the factor VIII/vW factor protein. This may explain why, in some patients with variant forms of vW disease, a carbohydrate abnormality also may affect the multimeric structure of the plasma factor VIII/vW factor protein. Images PMID:6601805

Analysis of human serum lipoprotein lipid composition using MALDI-TOF mass spectrometry.

PubMed

Hidaka, Hiroya; Hanyu, Noboru; Sugano, Mitsutoshi; Kawasaki, Kenji; Yamauchi, Kazuyoshi; Katsuyama, Tsutomu

2007-01-01

This study used matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify all lipid classes in human serum lipoproteins. After the major lipoproteins classes were isolated from serum by ultracentrifugation, the lipids were extracted and mixed with 2,5-dihydroxybenzoic acid (2,5-DHB) dissolved in Folch's solution (chloroform/methanol 2:1, v/v). MALDI-TOF MS analysis of the samples identified phospholipids (PLs), lysophospholipids (lysoPLs), sphingolipids (SLs), triglycerides (TGs), cholesteryl esters (CEs), and free cholesterol; it also showed the characteristics of individual fatty acid chains in serum lipids. MALDI-TOF MS allowed analysis of strongly hydrophobic and non-polar molecules such as CEs and TGs as well as hydrophilic molecules such as phospholipids. Direct analysis of fatty acids was not possible. The concentrations of lipids were not consistent with the ion peak intensities, since the extent of polarity affected the ionization characteristics of the molecules. However, lipid molecules with similar molecular structures but various fatty acid chains, such as phosphatidylcholine (PCs), were analyzed quantitatively by MALDI-TOF MS. Quantitative measurement of cholesterol was possible with the use of an internal standard. This study shows that MALDI-TOF MS can be used for direct investigation and quantitative analysis of the phospholipid composition of serum lipoproteins.

Effect of essential oil of traditional two Saudi mint types and its possible role in cardiovascular and throat health.

PubMed

Ahmed, Ahmed M; Ozbak, Hani A; Hemeg, Hassan A

2015-01-01

Essential Oil (EO) of two Mentha species (popularly used in Saudi Arabia), Mentha Longifolia (ML), and Mentha pulegium (MP) was isolated and subjected to inhibit LDL oxidation in 22 hypercholestrolemic samples collected from 22 subjects, and inhibited of 5 bacterial pathogen in vitro. LDL was isolated by ultracentrifugation and enhanced to oxidation with CuSO4 and EO was added to prevent this oxidation, free radical scavenging activity was measured (decrease 50% DPPH radicals). EO content of phenolic and flavonoid was estimated. Five bacterial strains infect human throat was tested against EO of ML and MP in diffusion agar method. EO of the two herbals possess highly significantresults, success to inhibit LDL oxidation (P<0.001 for both herbal than non addition against lipid peroxidase, P<0.001 ML better than MP P<0.001), decreased DPPH free radicals (P<0.001 for both than control, P<0.001 for ML than MP), and possess strong antioxidant activity for ML than MP (polyphenol and flavonoids contents was higher in EO of ML than MP, P<0.001). EO of ML possess strong antioxidant and antibacterial activity than MP, these attributed to its high flavonoid contents enable it to be a good for cardiovascular and throat health. The study supported the traditional uses of ML.

Purification and properties of glutamate binding protein from the periplasmic space of Escherichia coli K-12.

PubMed

Barash, H; Halpern, Y S

1975-03-28

Glutamate binding protein released from the periplasmic space of Escherichia coli K-12 by lysozyme-EDTA treatment was purified to homogeneity and its physical and chemical properties were studied. It is a basic protein with a pI of 9.1. Its molecular weight, determined in an analytical ultracentrifuge, and by gel filtration on Sephadex G-100 and dodecylsulphate acrylamide is 29 700, 27 800 and 32 000, respectively. The KD value for glutamate was 6.7 - 10- minus 6 M. L-Aspartate, reduced glutathione, G-glutamate-gamma-benzylester and L-glutamate-gamma-ethylester competitively inhibited glutamate binding with K-i; values of 7.8 - 10- minus 5, 1.1 - 10- minus 5, 1.0 - 10- minus 5 and 1.0 - 10- minus 5 M, respectively. Spheroplasts retained 40% of glutamate transport as compared to intact cells. The glutamate binding activity of a glutamate-utilizing strain (CS7), was 1.6 times as high as that of the glutamate non-utilizing parent strain (CS101). Similarly, the glutamate binding activity of a temperature conditional glutamate-utilizing mutant (CS2-TC) was 1.9 times higher when grown at the permissive temperature (42 degrees C) than when grown at the restrictive temperature (30 degrees C).

Mechanisms of Ricin Toxin Neutralization Revealed through Engineered Homodimeric and Heterodimeric Camelid Antibodies*

PubMed Central

Herrera, Cristina; Tremblay, Jacqueline M.; Shoemaker, Charles B.; Mantis, Nicholas J.

2015-01-01

Novel antibody constructs consisting of two or more different camelid heavy-chain only antibodies (VHHs) joined via peptide linkers have proven to have potent toxin-neutralizing activity in vivo against Shiga, botulinum, Clostridium difficile, anthrax, and ricin toxins. However, the mechanisms by which these so-called bispecific VHH heterodimers promote toxin neutralization remain poorly understood. In the current study we produced a new collection of ricin-specific VHH heterodimers, as well as VHH homodimers, and characterized them for their ability neutralize ricin in vitro and in vivo. We demonstrate that the VHH heterodimers, but not homodimers were able to completely protect mice against ricin challenge, even though the two classes of antibodies (heterodimers and homodimers) had virtually identical affinities for ricin holotoxin and similar IC50 values in a Vero cell cytotoxicity assay. The VHH heterodimers did differ from the homodimers in their ability to promote toxin aggregation in solution, as revealed through analytical ultracentrifugation. Moreover, the VHH heterodimers that were most effective at promoting ricin aggregation in solution were also the most effective at blocking ricin attachment to cell surfaces. Collectively, these data suggest that heterodimeric VHH-based neutralizing agents may function through the formation of antibody-toxin complexes that are impaired in their ability to access host cell receptors. PMID:26396190

Transmission of HBV DNA Mediated by Ceramide-Triggered Extracellular Vesicles.

PubMed

Sanada, Takahiro; Hirata, Yuichi; Naito, Yutaka; Yamamoto, Naoki; Kikkawa, Yoshiaki; Ishida, Yuji; Yamasaki, Chihiro; Tateno, Chise; Ochiya, Takahiro; Kohara, Michinori

2017-03-01

An extracellular vesicle (EV) is a nanovesicle that shuttles proteins, nucleic acids, and lipids, thereby influencing cell behavior. A recent crop of reports have shown that EVs are involved in infectious biology, influencing host immunity and playing a role in the viral life cycle. In the present work, we investigated the EV-mediated transmission of hepatitis B virus (HBV) infection. We investigated the EV-mediated transmission of HBV infection by using a HBV infectious culture system that uses primary human hepatocytes derived from humanized chimeric mice (PXB-cells). Purified EVs were isolated by ultracentrifugation. To analyze the EVs and virions, we used stimulated emission depletion microscopy. Purified EVs from HBV-infected PXB-cells were shown to contain HBV DNA and to be capable of transmitting HBV DNA to naive PXB-cells. These HBV-DNA-transmitting EVs were shown to be generated through a ceramide-triggered EV production pathway. Furthermore, we showed that these HBV-DNA-transmitting EVs were resistant to antibody neutralization; stimulated emission depletion microscopy showed that EVs lacked hepatitis B surface antigen, the target of neutralizing antibodies. These findings suggest that EVs harbor a DNA cargo capable of transmitting viral DNA into hepatocytes during HBV infection, representing an additional antibody-neutralization-resistant route of HBV infection.

Purification and immunogenicity study of human papillomavirus 58 virus-like particles expressed in Pichia pastoris.

PubMed

Jiang, Zijun; Tong, Guangjie; Cai, Beibei; Xu, Yihan; Lou, Jueren

2011-12-01

Two human papillomavirus (HPV) prophylactic vaccines are currently available in the market: Gardasil and Cervarix. These two vaccines work against tumor high-risk subtypes HPV 16 and HPV 18. However, they do not include other high-risk subtypes such as HPV 58. Epidemiological research in China shows that HPV 58 is a prevalent high-risk subtype, second only to HPV 16 and HPV 18. Thus, for cervical cancer prevention in China, developing a vaccine against HPV 58 is necessary. In this study, HPV 58 virus-like particles (VLPs) were expressed in the Pichia pastoris, and subsequently purified through pretreatment and a three-step purification process consisting of strong cation exchange chromatography, size-exclusion chromatography, and hydroxyapatite chromatography. The highly purified HPV 58 VLPs were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, electron microscopy, dynamic laser scattering, and ultracentrifugation. The purified VLPs were used to immunize mice to test their ability to induce humoral immunity. Enzyme-linked immunosorbent assays were performed on the sera of the immunized mice and significantly high anti-HPV 58 VLP antibody titers were observed. The immunogenicity study demonstrates that the purified HPV 58 VLPs are HPV vaccine candidates. Copyright © 2011 Elsevier Inc. All rights reserved.

Specific Inhibition of Herpes Simplex Virus DNA Polymerase by Helical Peptides Corresponding to the Subunit Interface

NASA Astrophysics Data System (ADS)

Digard, Paul; Williams, Kevin P.; Hensley, Preston; Brooks, Ian S.; Dahl, Charles E.; Coen, Donald M.

1995-02-01

The herpes simplex virus DNA polymerase consists of two subunits-a catalytic subunit and an accessory subunit, UL42, that increases processivity. Mutations affecting the extreme C terminus of the catalytic subunit specifically disrupt subunit interactions and ablate virus replication, suggesting that new antiviral drugs could be rationally designed to interfere with polymerase heterodimerization. To aid design, we performed circular dichroism (CD) spectroscopy and analytical ultracentrifugation studies, which revealed that a 36-residue peptide corresponding to the C terminus of the catalytic subunit folds into a monomeric structure with partial α-helical character. CD studies of shorter peptides were consistent with a model where two separate regions of α-helix interact to form a hairpin-like structure. The 36-residue peptide and a shorter peptide corresponding to the C-terminal 18 residues blocked UL42-dependent long-chain DNA synthesis at concentrations that had no effect on synthesis by the catalytic subunit alone or by calf thymus DNA polymerase δ and its processivity factor. These peptides, therefore, represent a class of specific inhibitors of herpes simplex virus DNA polymerase that act by blocking accessory-subunit-dependent synthesis. These peptides or their structures may form the basis for the synthesis of clinically effective drugs.

Surfactants at Single-Walled Carbon Nanotube-Water Interface: Physics of Surfactants, Counter-Ions, and Hydration Shell

NASA Astrophysics Data System (ADS)

Khare, Ketan S.; Phelan, Frederick R., Jr.

Specialized applications of single-walled carbon nanotubes (SWCNTs) require an efficient and reliable method to sort these materials into monodisperse fractions with respect to their defining metrics (chirality, length, etc.) while retaining their physical and chemical integrity. A popular method to achieve this goal is to use surfactants that individually disperse SWCNTs in water and then to separate the resulting colloidal mixture into fractions that are enriched in monodisperse SWCNTs. Recently, experiments at NIST have shown that subtle point mutations of chemical groups in bile salt surfactants have a large impact on the hydrodynamic properties of SWCNT-surfactant complexes during ultracentrifugation. These results provide strong motivation for understanding the rich physics underlying the assembly of surfactants around SWCNTs, the structure and dynamics of counter ions around the resulting complex, and propagation of these effects into the first hydration shell. Here, all-atom molecular dynamics simulations are used to investigate the thermodynamics of SWCNT-bile salt surfactant complexes in water with an emphasis on the buoyant characteristics of the SWCNT-surfactant complexes. Simulation results will be presented along with a comparison with experimental data. Official contribution of the National Institute of Standards and Technology; not subject to copyright in the United States.

Interactions between G-actin and myosin subfragment 1: immunochemical probing of the NH2-terminal segment on actin.

PubMed

DasGupta, G; White, J; Cheung, P; Reisler, E

1990-09-11

The role of the N-terminal segment of actin in myosin-induced polymerization of G-actin was studied by using peptide antibodies directed against the first seven N-terminal residues of alpha-skeletal actin. Light scattering, fluorescence, and analytical ultracentrifugation experiments showed that the Fab fragments of these antibodies inhibited the polymerization of G-actin by myosin subfragment 1 (S-1) by inhibiting the binding of these proteins to each other. Fluorescence measurements using actin labeled with pyrenyliodoacetamide revealed that Fab inhibited the initial step in the binding of S-1 to G-actin. It is deduced from these results and from other literature data that the initial contact between G-actin and S-1 involves residues 1-7 on actin and residues 633-642 on the S-1 heavy chain. This interaction appears to be of major importance for the binding of S-1 and G-actin. The presence of additional myosin contact sites on G-actin was indicated by concentration-dependent recovery of S-1 binding to G-actin without displacement of Fab. The reduced Fab inhibition of S-1 binding to polymerizing and polymerized actin is consistent with the tightening of acto-S-1 binding at these sites or the creation of new sites upon formation of F-actin.

Separation and characterization of metallothionein in the liver of sea turtles by high performance liquid chromatographylinductively coupled plasma-mass spectrometry

NASA Astrophysics Data System (ADS)

Shinsuke, T.; Yasumi, A.; Takashi, K.

2003-05-01

To investigate whether trace metals bind to metallothioneins (MTs) in the hepatocytosol of green turtles (Chelonia mydas) and hawksbill turtles (Eretmochelys imbricata), MT fraction was obtained by ultracentrifugation and gel filtration methods. MTs separated from hepatocytosol were further purified and characterized by high performance liquid chromatography/inductively coupled plasma-mass spectrometry. In addition, the involvement of MTs in the accumulation of trace metals in the liver of sea turtle was examine. Gel filtration analysis showed that significant amounts of Cu, Zn, Ag and Cd were bound to MT in the cytosol of sea turtles, suggesting that such trace metals were primarily detoxified by interaction with MTs in the liver. Elution profiles of these trace metals by anion-exchange chromatography were different between green turtles and hawksbill turtles. These results suggest the presence of multiple isoforms of MT in the liver of both sea turtles; however, constituents of isoforms were different between green and hawksbill turtles. In both species, we observed the elevation of the height of a specific peak in elution profile with an increase in Cu concentration in hepatocytosol. This result suggests the presence of a novel MT isoform related to copper accumulation in the liver of sea turtles.

Comparative Evaluation of U.S. Brand and Generic Intravenous Sodium Ferric Gluconate Complex in Sucrose Injection: Physicochemical Characterization

PubMed Central

Sun, Dajun; Rouse, Rodney; Patel, Vikram; Wu, Yong; Zheng, Jiwen; Karmakar, Alokita; Patri, Anil K.; Keire, David; Ma, Jia; Jiang, Wenlei

2018-01-01

The objective of this study was to evaluate physicochemical equivalence between brand (i.e., Ferrlecit) and generic sodium ferric gluconate (SFG) in sucrose injection by conducting a series of comparative in vitro characterizations using advanced analytical techniques. The elemental iron and carbon content, thermal properties, viscosity, particle size, zeta potential, sedimentation coefficient, and molecular weight were determined. There was no noticeable difference between brand and generic SFG in sucrose injection for the above physical parameters evaluated, except for the sedimentation coefficient determined by sedimentation velocity analytical ultracentrifugation (SV-AUC) and molecular weight by asymmetric field flow fractionation-multi-angle light scattering (AFFF-MALS). In addition, brand and generic SFG complex products showed comparable molecular weight distributions when determined by gel permeation chromatography (GPC). The observed minor differences between brand and generic SFG, such as sedimentation coefficient, do not impact their biological activities in separate studies of in vitro cellular uptake and rat biodistribution. Coupled with the ongoing clinical study comparing the labile iron level in healthy volunteers, the FDA-funded post-market studies intended to illustrate comprehensive surveillance efforts ensuring safety and efficacy profiles of generic SFG complex in sucrose injection, and also to shed new light on the approval standards on generic parenteral iron colloidal products. PMID:29303999

Current methods for the isolation of extracellular vesicles.

PubMed

Momen-Heravi, Fatemeh; Balaj, Leonora; Alian, Sara; Mantel, Pierre-Yves; Halleck, Allison E; Trachtenberg, Alexander J; Soria, Cesar E; Oquin, Shanice; Bonebreak, Christina M; Saracoglu, Elif; Skog, Johan; Kuo, Winston Patrick

2013-10-01

Extracellular vesicles (EVs), including microvesicles and exosomes, are nano- to micron-sized vesicles, which may deliver bioactive cargos that include lipids, growth factors and their receptors, proteases, signaling molecules, as well as mRNA and non-coding RNA, released from the cell of origin, to target cells. EVs are released by all cell types and likely induced by mechanisms involved in oncogenic transformation, environmental stimulation, cellular activation, oxidative stress, or death. Ongoing studies investigate the molecular mechanisms and mediators of EVs-based intercellular communication at physiological and oncogenic conditions with the hope of using this information as a possible source for explaining physiological processes in addition to using them as therapeutic targets and disease biomarkers in a variety of diseases. A major limitation in this evolving discipline is the hardship and the lack of standardization for already challenging techniques to isolate EVs. Technical advances have been accomplished in the field of isolation with improving knowledge and emerging novel technologies, including ultracentrifugation, microfluidics, magnetic beads and filtration-based isolation methods. In this review, we will discuss the latest advances in methods of isolation methods and production of clinical grade EVs as well as their advantages and disadvantages, and the justification for their support and the challenges that they encounter.

ISOLATION AND CHARACTERIZATION OF LAMELLAR BODIES AND TUBULAR MYELIN FROM RAT LUNG HOMOGENATES

PubMed Central

Gil, Joan; Reiss, Oscar K.

1973-01-01

Three surface-active fractions which differ in their morphology have been isolated from rat lung homogenates by ultracentrifugation in a discontinuous sucrose density gradient. In order of increasing density, the fractions consisted, as shown by electron microscopy, primarily of common myelin figures, lamellar bodies, and tubular myelin figures. The lipid of all three fractions contained approximately 94% polar lipids and 2% cholesterol. In the case of the common myelin figures and the lamellar bodies, the polar lipids consisted of 73% phosphatidylcholines, 9% phosphatidylserines and inositols, and 8% phosphatidylethanolamines. In the case of the tubular myelin figures, the respective percentages were 58, 19, and 5. Over 90% of the fatty acids of the lecithins of all three fractions were saturated. Electrophoresis of the proteins of the fractions in sodium dodecyl sulfate or Triton X-100 revealed that the lamellar bodies and the tubular myelin figures differed in the mobilities of their proteins. The common myelin figures, however, contained proteins from both of the other fractions. These data indicate that, whereas the lipids of the extracellular, alveolar surfactant(s) originate in the lamellar bodies, the proteins arise from another source. It is further postulated that the tubular myelin figures represent a liquid crystalline state of the alveolar surface-active lipoproteins. PMID:4726305

Cellular Uptake of the Clostridium perfringens Binary Iota-Toxin

PubMed Central

Blöcker, Dagmar; Behlke, Joachim; Aktories, Klaus; Barth, Holger

2001-01-01

The binary iota-toxin is produced by Clostridium perfringens type E strains and consists of two separate proteins, the binding component iota b (98 kDa) and an actin-ADP-ribosylating enzyme component iota a (47 kDa). Iota b binds to the cell surface receptor and mediates the translocation of iota a into the cytosol. Here we studied the cellular uptake of iota-toxin into Vero cells. Bafilomycin A1, but not brefeldin A or nocodazole, inhibited the cytotoxic effects of iota-toxin, indicating that toxin is translocated from an endosomal compartment into the cytoplasm. Acidification (pH ≤ 5.0) of the extracellular medium enabled iota a to directly enter the cytosol in the presence of iota b. Activation by chymotrypsin induced oligomerization of iota b in solution. An average mass of 530 ± 28 kDa for oligomers was determined by analytical ultracentrifugation, indicating heptamer formation. The entry of iota-toxin into polarized CaCo-2 cells was studied by measuring the decrease in transepithelial resistance after toxin treatment. Iota-toxin led to a significant decrease in resistance when it was applied to the basolateral surface of the cells but not following application to the apical surface, indicating a polarized localization of the iota-toxin receptor. PMID:11292715

Mitochondrial oxidative stress and dysfunction induced by isoniazid: study on isolated rat liver and brain mitochondria.

PubMed

Ahadpour, Morteza; Eskandari, Mohammad Reza; Mashayekhi, Vida; Haj Mohammad Ebrahim Tehrani, Kamaleddin; Jafarian, Iman; Naserzadeh, Parvaneh; Hosseini, Mir-Jamal

2016-01-01

Isoniazid (INH or isonicotinic hydrazide) is used for the treatment and prophylaxis of tuberculosis. Liver and brain are two important target organs in INH toxicity. However, the exact mechanisms behind the INH hepatotoxicity or neurotoxicity have not yet been completely understood. Considering the mitochondria as one of the possible molecular targets for INH toxicity, the aim of this study was to evaluate the mechanisms of INH mitochondrial toxicity on isolated mitochondria. Mitochondria were isolated by differential ultracentrifugation from male Sprague-Dawley rats and incubated with different concentrations of INH (25-2000 μM) for the investigation of mitochondrial parameters. The results indicated that INH could interact with mitochondrial respiratory chain and inhibit its activity. Our results showed an elevation in mitochondrial reactive oxygen species (ROS) formation, lipid peroxidation and mitochondrial membrane potential collapse after exposure of isolated liver mitochondria in INH. However, different results were obtained in brain mitochondria. Noteworthy, significant glutathione oxidation, adenosine triphosphate (ATP) depletion and lipid peroxidation were observed in higher concentration of INH, as compared to liver mitochondria. In conclusion, our results suggest that INH may initiate its toxicity in liver mitochondria through interaction with electron transfer chain, lipid peroxidation, mitochondrial membrane potential decline and cytochrome c expulsion which ultimately lead to cell death signaling.

The Amino Acid Specificity for Activation of Phenylalanine Hydroxylase Matches the Specificity for Stabilization of Regulatory Domain Dimers

PubMed Central

2016-01-01

Liver phenylalanine hydroxylase is allosterically activated by phenylalanine. The structural changes that accompany activation have not been identified, but recent studies of the effects of phenylalanine on the isolated regulatory domain of the enzyme support a model in which phenylalanine binding promotes regulatory domain dimerization. Such a model predicts that compounds that stabilize the regulatory domain dimer will also activate the enzyme. Nuclear magnetic resonance spectroscopy and analytical ultracentrifugation were used to determine the ability of different amino acids and phenylalanine analogues to stabilize the regulatory domain dimer. The abilities of these compounds to activate the enzyme were analyzed by measuring their effects on the fluorescence change that accompanies activation and on the activity directly. At concentrations of 10–50 mM, d-phenylalanine, l-methionine, l-norleucine, and (S)-2-amino-3-phenyl-1-propanol were able to activate the enzyme to the same extent as 1 mM l-phenylalanine. Lower levels of activation were seen with l-4-aminophenylalanine, l-leucine, l-isoleucine, and 3-phenylpropionate. The ability of these compounds to stabilize the regulatory domain dimer agreed with their ability to activate the enzyme. These results support a model in which allosteric activation of phenylalanine hydroxylase is linked to dimerization of regulatory domains. PMID:26252467

Non-ionic detergents facilitate non-specific binding of M13 bacteriophage to polystyrene surfaces.

PubMed

Hakami, Abdulrahim R; Ball, Jonathan K; Tarr, Alexander W

2015-09-01

Phage-displayed random peptide libraries are widely used for identifying peptide interactions with proteins and other substrates. Selection of peptide ligands involves iterative rounds of affinity enrichment. The binding properties of the selected phage clones are routinely tested using immunoassay after propagation to high titre in a bacterial host and precipitation using polyethylene glycol (PEG) and high salt concentration. These immunoassays can suffer from low sensitivity and high background signals. Polysorbate 20 (Tween(®) 20) is a non-ionic detergent commonly used in immunoassay washing buffers to reduce non-specific binding, and is also used as a blocking reagent. We have observed that Tween 20 enhances non-specific M13 library phage binding in a peptide-independent manner. Other non-ionic detergents were also found to promote significant, dose-dependent non-specific phage binding in ELISA. This effect was not observed for assays using phage concentrated by ultracentrifugation, suggesting that interactions occur between detergents and the PEG-precipitated phage, irrespective of the displayed peptide motif. This artefact may impact on successful affinity selection of peptides from phage-display libraries. We propose alternative methods for screening phage libraries for identifying binding interactions with target ligands. Copyright © 2015 Elsevier B.V. All rights reserved.

A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation.

PubMed

Nohara, Kazunari; Chen, Zheng; Yoo, Seung-Hee

2017-07-06

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a challenge for ChIP due to technical difficulty in isolation of high-quality nuclei with minimal contamination of myofibrils. Previous protocols have attempted to purify nuclei before cross-linking, which incurs the risk of altered DNA-protein interaction during the prolonged nuclei preparation process. In the current protocol, we first cross-linked the skeletal muscle tissue collected from mice, and the tissues were minced and sonicated. Since we found that ultracentrifugation was not able to separate nuclei from myofibrils using cross-linked muscle tissue, we devised a sequential filtration procedure to obtain high-quality nuclei devoid of significant myofibril contamination. We subsequently prepared chromatin by using an ultrasonicator, and ChIP assays with anti-BMAL1 antibody revealed robust circadian binding pattern of BMAL1 to target gene promoters. This filtration protocol constitutes an easily applicable method to isolate high-quality nuclei from cross-linked skeletal muscle tissue, allowing consistent sample processing for circadian and other time-sensitive studies. In combination with next-generation sequencing (NGS), our method can be deployed for various mechanistic and genomic studies focusing on skeletal muscle function.

Ultrathin Nanocrystalline Diamond Films with Silicon Vacancy Color Centers via Seeding by 2 nm Detonation Nanodiamonds.

PubMed

Stehlik, Stepan; Varga, Marian; Stenclova, Pavla; Ondic, Lukas; Ledinsky, Martin; Pangrac, Jiri; Vanek, Ondrej; Lipov, Jan; Kromka, Alexander; Rezek, Bohuslav

2017-11-08

Color centers in diamonds have shown excellent potential for applications in quantum information processing, photonics, and biology. Here we report chemical vapor deposition (CVD) growth of nanocrystalline diamond (NCD) films as thin as 5-6 nm with photoluminescence (PL) from silicon-vacancy (SiV) centers at 739 nm. Instead of conventional 4-6 nm detonation nanodiamonds (DNDs), we prepared and employed hydrogenated 2 nm DNDs (zeta potential = +36 mV) to form extremely dense (∼1.3 × 10 13 cm -2 ), thin (2 ± 1 nm), and smooth (RMS roughness < 0.8 nm) nucleation layers on an Si/SiO x substrate, which enabled the CVD growth of such ultrathin NCD films in two different and complementary microwave (MW) CVD systems: (i) focused MW plasma with an ellipsoidal cavity resonator and (ii) pulsed MW plasma with a linear antenna arrangement. Analytical ultracentrifuge, infrared and Raman spectroscopies, atomic force microscopy, and scanning electron microscopy are used for detailed characterization of the 2 nm H-DNDs and the nucleation layer as well as the ultrathin NCD films. We also demonstrate on/off switching of the SiV center PL in the NCD films thinner than 10 nm, which is achieved by changing their surface chemistry.

Application of Fourier-transform infrared (FT-IR) spectroscopy for simple and easy determination of chylomicron-triglyceride and very low density lipoprotein-triglyceride.

PubMed

Sato, Kenichi; Seimiya, Masanori; Kodera, Yoshio; Kitamura, Akihide; Nomura, Fumio

2010-02-01

Fourier-transform infrared (FT-IR) spectroscopy is a simple and reagent-free physicochemical analysis method, and is a potential alternative to more time-consuming and labor-intensive procedures. In this study, we aimed to use FT-IR spectroscopy to determine serum concentrations of chylomicron-triglyceride (TG) and very low density lipoprotein (VLDL)-TG. We analyzed a chylomicron fraction and VLDL fraction, which had been obtained by ultracentrifugation, to search for wavelengths to designate to each fraction. Then, partial least square (PLS) calibrations were developed using a training set of samples, for which TG concentrations had been determined by conventional procedures. Validation was conducted with another set of samples using the PLS model to predict serum TG concentrations on the basis of the samples' IR spectra. We analyzed a total of 150 samples. Serum concentrations of chylomicron-TG and VLDL-TG estimated by FT-IR spectroscopy agreed well with those obtained by the reference method (r=0.97 for both lipoprotein fractions). FT-IR spectrometric analysis required 15mul of serum and was completed within 1min. Serum chylomicron-TG and VLDL-TG concentrations can be determined with FT-IR spectroscopy. This rapid and simple test may have a great impact on the management of patients with dyslipidemia. Copyright 2009. Published by Elsevier B.V.

States of phage T3/T7 capsids: buoyant density centrifugation and cryo-EM.

PubMed

Serwer, Philip; Wright, Elena T; Demeler, Borries; Jiang, Wen

2018-04-01

Mature double-stranded DNA bacteriophages have capsids with symmetrical shells that typically resist disruption, as they must to survive in the wild. However, flexibility and associated dynamism assist function. We describe biochemistry-oriented procedures used to find previously obscure flexibility for capsids of the related phages, T3 and T7. The primary procedures are hydration-based buoyant density ultracentrifugation and purified particle-based cryo-electron microscopy (cryo-EM). We review the buoyant density centrifugation in detail. The mature, stable T3/T7 capsid is a shell flexibility-derived conversion product of an initially assembled procapsid (capsid I). During DNA packaging, capsid I expands and loses a scaffolding protein to form capsid II. The following are observations made with capsid II. (1) The in vivo DNA packaging of wild type T3 generates capsid II that has a slight (1.4%), cryo-EM-detected hyper-expansion relative to the mature phage capsid. (2) DNA packaging in some altered conditions generates more extensive hyper-expansion of capsid II, initially detected by hydration-based preparative buoyant density centrifugation in Nycodenz density gradients. (3) Capsid contraction sometimes occurs, e.g., during quantized leakage of DNA from mature T3 capsids without a tail.

The Influence of Age, Sex, Pregnancy, Starvation, and Other Factors on the Cytoplasmic Ribonucleoproteins of Rat Liver

PubMed Central

Petermann, Mary L.; Hamilton, Mary G.

1958-01-01

Rat liver was homogenized in 0.88 M sucrose. The DNA and total RNA were determined, and the homogenate was fractionated by differential centrifugation. The pellets obtained between 30 minutes at 20,000 g and 180 minutes at 105,000 g were analyzed for RNA and nitrogen. The ribonucleoproteins were determined in the analytical ultracentrifuge. The non-pellet RNA was calculated by difference. The results are reported as amounts per 6.7 x 10-9 mg. of DNA. In young, growing male rats the amounts of microsomal protein and ribonucleoprotein B (83S) increased with age. Non-pregnant adult females showed less non-pellet RNA and much more ribonucleoprotein C (63S) than did adult males. During pregnancy both of these cell constituents reverted to levels characteristic for male animals. Starvation for 5 days resulted in a reduction in the mass of liver tissue, the non-pellet RNA, the microsomal protein, and ribonucleoproteins B and C. During recovery from starvation the return of the liver to normal paralleled the rate at which body weight was restored. Treatment with cortisone, 25 mg. per rat per day for 5 days, caused an increase in microsomal protein and a decrease in ribonucleoprotein B. Treatment with 6-mercapto-purine, 50 mg. per kilo per day for 5 days, caused little change in liver composition in either males or females. PMID:13610943

Assembly of flammutoxin, a cytolytic protein from the edible mushroom Flammulina velutipes, into a pore-forming ring-shaped oligomer on the target cell.

PubMed

Tomita, T; Ishikawa, D; Noguchi, T; Katayama, E; Hashimoto, Y

1998-07-01

Flammutoxin has been previously isolated as a cardiotoxic and cytolytic polypeptide of 22 or 32 kDa from the fruiting bodies of the edible mushroom Flammulina velutipes. In the present study, we purified flammutoxin as a single haemolytic protein of 31 kDa and studied the mode of its cytolytic action. (1) Flammutoxin caused efflux of potassium ions from human erythrocytes and swelling of the cells before haemolysis. (2) Flammutoxin did not lyse human erythrocytes in the presence of non-electrolytes with hydrodynamic diameters of >5.0 nm, although it caused leakage of potassium ions and swelling of the cells under the same conditions. (3) Experiments including solubilization of cell-bound toxin with 2% (w/v) SDS at 20 degrees C and subsequent Western immunoblots showed that flammutoxin formed a band corresponding to 180 kDa under the conditions where it lysed erythrocytes. (4) Electron microscopy of flammutoxin-treated human erythrocytes revealed the presence of a ring-shaped structure with outer and inner diameters of 10 and 5 nm, respectively, on the cells. (5) A ring-shaped toxin oligomer of the same dimensions was solubilized from the toxin-treated human erythrocytes with 2% (w/v) SDS at 20 degrees C and isolated by a sucrose-gradient ultracentrifugation. These data indicated that flammutoxin assembles into a ring-shaped oligomer possessing a hydrophilic pore of 4-5 nm on target cells.

Amyloid-β Reduces Exosome Release from Astrocytes by Enhancing JNK Phosphorylation.

PubMed

Abdullah, Mohammad; Takase, Hiroshi; Nunome, Mari; Enomoto, Hiroyuki; Ito, Jin-Ichi; Gong, Jian-Sheng; Michikawa, Makoto

2016-07-02

Exosomes are small extracellular vesicles secreted by variety of cell types such as neurons, astrocytes, and oligodendrocytes. It is suggested that exosomes play essential role in the maintenance of the neuronal functions and also in the clearance of amyloid-β (Aβ) from the brain. Aβ is well known to cause neuronal cell death, whereas little is known about its effect on astrocytes. In this study, we examined the effect of Aβ on release of exosomes from astrocytes in culture. We analyzed release of exosomes and apoE, both of which are known to remove/clear Aβ from the brain, in the culture medium of astrocytes. We found that exosome and apoE-HDL were successfully separated by density gradient ultracentrifugation demonstrated by distribution of their specific markers, flotillin and HSP90, and cholesterol, and morphological analysis using electron microscopy. Exosome release was significantly reduced by Aβ1-42 treatment in cultured astrocytes accompanied by an increased JNK phosphorylation. Whereas, apoE-HDL release remained unchanged. A JNK inhibitor restored the decreased levels of exosome release induced by Aβ treatment to levels similar to those of control, suggesting that Aβ1-42 inhibits exosome release via stimulation of JNK signal pathway. Because exosomes are shown to remove Aβ in the brain, our findings suggest that increased Aβ levels in the brain may impair the exosome-mediated Aβ clearance pathway.

Exosome enrichment of human serum using multiple cycles of centrifugation.

PubMed

Kim, Jeongkwon; Tan, Zhijing; Lubman, David M

2015-09-01

In this work, we compared the use of repeated cycles of centrifugation at conventional speeds for enrichment of exosomes from human serum compared to the use of ultracentrifugation (UC). After removal of cells and cell debris, a speed of 110 000 × g or 40 000 × g was used for the UC or centrifugation enrichment process, respectively. The enriched exosomes were analyzed using the bicinchoninic acid assay, 1D gel separation, transmission electron microscopy, Western blotting, and high-resolution LC-MS/MS analysis. It was found that a five-cycle repetition of UC or centrifugation is necessary for successful removal of nonexosomal proteins in the enrichment of exosomes from human serum. More significantly, 5× centrifugation enrichment was found to provide similar or better performance than 5× UC enrichment in terms of enriched exosome protein amount, Western blot band intensity for detection of CD-63, and numbers of identified exosome-related proteins and cluster of differentiation (CD) proteins. A total of 478 proteins were identified in the LC-MS/MS analyses of exosome proteins obtained from 5× UCs and 5× centrifugations including many important CD membrane proteins. The presence of previously reported exosome-related proteins including key exosome protein markers demonstrates the utility of this method for analysis of proteins in human serum. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthesis, Characterization, and Evaluation of Pluronic-Based β-Cyclodextrin Polyrotaxanes for Mobilization of Accumulated Cholesterol from Niemann-Pick Type C Fibroblasts

PubMed Central

Collins, Christopher J.; McCauliff, Leslie A.; Hyun, Seok-Hee; Zhang, Zhaorui; Paul, Lake N.; Kulkarni, Aditya; Zick, Klaus; Wirth, Mary; Storch, Judith; Thompson, David H.

2015-01-01

Several lines of evidence suggest that β-cyclodextrin (β-CD) derivatives initiate the efflux of accumulated, unesterified cholesterol from the late endosomal/lysosomal compartment in Niemann Pick C (NPC) disease models. Unfortunately, repeated injections or continuous infusions of current β-CD therapies are required to sustain suppression of symptoms and prolong life. In an effort to make CD treatment a more viable option by boosting efficacy and improving pharmacokinetics, a library of Pluronic surfactant-based β-CD polyrotaxanes has been developed using biocompatible poly(ethylene glycol) (PEG)–polypropylene glycol (PPG)–PEG triblock copolymers. These compounds carry multiple copies of β-CD as shown by 1H NMR, 2D nuclear Overhouser effect spectroscopy, gel permeation chromatography/multiangle light scattering, analytical ultracentrifugation analysis, matrix assisted laser desorption/ionization mass spectrometry, and diffusion-ordered spectroscopy. Analyses of free β-cyclodextrin contamination in the compounds were made by reverse phase high pressure liquid chromatography and hydrophilic interaction liquid chromatography. Dethreading kinetics were studied by reverse phase high pressure liquid chromatography, UV/vis, and 1H NMR analysis. Filipin staining studies using npc2−/− fibroblasts show significant reversal of cholesterol accumulation after treatment with polyrotaxane compounds. The rate and efficacy of reversal is similar to that achieved by equivalent amounts of monomeric β-CD alone. PMID:23560535

NanoRelease: Pilot interlaboratory comparison of a ...

EPA Pesticide Factsheets

A major use of multi-walled carbon nanotubes (MWCNTs) is as functional fillers embedded in a solid matrix, such as plastics or coatings. Weathering and abrasion of the solid matrix during use can lead to environmental releases of the MWCNTs. Here we focus on a protocol to identify and quantify the primary release induced by weathering, and assess reproducibility, transferability, and sensitivity towards different materials and uses. We prepared 132 specimens of two polymer-MWCNT composites containing the same grade of MWCNTs used in earlier OECD hazard assessments but without UV stabilizer. We report on a pilot inter-laboratory comparison (ILC) with four labs (two US and two EU) aging by UV and rain, then shipping for analysis. Two labs (one US and one EU) conducted the release sampling and analysis by Transmission Electron Microscopy (TEM), Inductively Coupled Plasma- Mass Spectrometry (ICP-MS), Ultraviolet–Visible Spectroscopy (UV–Vis), Analytical Ultracentrifugation (AUC), and Asymmetric Flow Field Flow Fractionation (AF4). We compare results between aging labs, between analysis labs and between materials. Surprisingly, we found quantitative agreement between analysis labs for TEM, ICP-MS, UV–Vis; low variation between aging labs by all methods; and consistent rankings of release between TEM, ICP-MS, UV–Vis, AUC. Significant disagreement was related primarily to differences in aging, but even these cases remained within a factor of two. Published in t

Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates

PubMed Central

Tanase, Maya; Zolla, Valerio; Clement, Cristina C; Borghi, Francesco; Urbanska, Aleksandra M; Rodriguez-Navarro, Jose Antonio; Roda, Barbara; Zattoni, Andrea; Reschiglian, Pierluigi; Cuervo, Ana Maria; Santambrogio, Laura

2016-01-01

Herein we describe a protocol that uses hollow-fiber flow field-flow fractionation (FFF) coupled with multiangle light scattering (MALS) for hydrodynamic size-based separation and characterization of complex protein aggregates. The fractionation method, which requires 1.5 h to run, was successfully modified from the analysis of protein aggregates, as found in simple protein mixtures, to complex aggregates, as found in total cell lysates. In contrast to other related methods (filter assay, analytical ultracentrifugation, gel electrophoresis and size-exclusion chromatography), hollow-fiber flow FFF coupled with MALS allows a flow-based fractionation of highly purified protein aggregates and simultaneous measurement of their molecular weight, r.m.s. radius and molecular conformation (e.g., round, rod-shaped, compact or relaxed). The polyethersulfone hollow fibers used, which have a 0.8-mm inner diameter, allow separation of as little as 20 μg of total cell lysates. In addition, the ability to run the samples in different denaturing and nondenaturing buffer allows defining true aggregates from artifacts, which can form during sample preparation. The protocol was set up using Paraquat-induced carbonylation, a model that induces protein aggregation in cultured cells. This technique will advance the biochemical, proteomic and biophysical characterization of molecular-weight aggregates associated with protein mutations, as found in many CNS degenerative diseases, or chronic oxidative stress, as found in aging, and chronic metabolic and inflammatory conditions. PMID:25521790

Disruption of Membranes of Extracellular Vesicles Is Necessary for ELISA Determination of Urine AQP2: Proof of Disruption and Epitopes of AQP2 Antibodies

PubMed Central

Nameta, Masaaki; Saijo, Yoko; Ohmoto, Yasukazu; Katsuragi, Kiyonori; Yamamoto, Keiko; Yamamoto, Tadashi; Ishibashi, Kenichi; Sasaki, Sei

2016-01-01

Aquaporin-2 (AQP2) is present in urine extracellular vesicles (EVs) and is a useful biomarker for water balance disorders. We previously found that pre-treatment of urine with alkali/detergent or storage at −25 °C is required for enzyme-linked immunosorbent assay (ELISA) measurement. We speculated that disruptions of EVs membranes are necessary to allow for the direct contact of antibodies with their epitopes. Human urine EVs were prepared using an ultracentrifugation method. Urine EV samples were stored at different temperatures for a week. Electron microscopy showed abundant EVs with diameters of 20–100 nm, consistent with those of exosomes, in normal urine, whereas samples from alkali/detergent pre-treated urine showed fewer EVs with large swollen shapes and frequent membrane disruptions. The abundance and structures of EVs were maintained during storage at −80 °C, but were severely damaged at −25 °C. Binding and competitive inhibition assays showed that epitopes of monoclonal antibody and polyclonal antibody were the hydrophilic Loop D and C-terminus of AQP2, respectively, both of which are present on the inner surface of EVs. Thus, urine storage at −25 °C or pre-treatment with alkali/detergent disrupt EVs membranes and allow AQP2 antibodies to bind to their epitopes located inside EVs. PMID:27681727

Kinetic Analysis of Phospholipase C from Catharanthus roseus Transformed Roots Using Different Assays1

PubMed Central

Hernández-Sotomayor, S.M. Teresa; De Los Santos-Briones, César; Muñoz-Sánchez, J. Armando; Loyola-Vargas, Victor M.

1999-01-01

The properties of phospholipase C (PLC) partially purified from Catharanthus roseus transformed roots were analyzed using substrate lipids dispersed in phospholipid vesicles, phospholipid-detergent mixed micelles, and phospholipid monolayers spread at an air-water interface. Using [33P]phosphatidylinositol 4,5-bisphosphate (PIP2) of high specific radioactivity, PLC activity was monitored directly by measuring the loss of radioactivity from monolayers as a result of the release of inositol phosphate and its subsequent dissolution on quenching in the subphase. PLC activity was markedly affected by the surface pressure of the monolayer, with reduced activity at extremes of initial pressure. The optimum surface pressure for PIP2 hydrolysis was 20 mN/m. Depletion of PLC from solution by incubation with sucrose-loaded PIP2 vesicles followed by ultracentrifugation demonstrated stable attachment of PLC to the vesicles. A mixed micellar system was established to assay PLC activity using deoxycholate. Kinetic analyses were performed to determine whether PLC activity was dependent on both bulk PIP2 and PIP2 surface concentrations in the micelles. The interfacial Michaelis constant was calculated to be 0.0518 mol fraction, and the equilibrium dissociation constant of PLC for the lipid was 45.5 μm. These findings will add to our understanding of the mechanisms of regulation of plant PLC. PMID:10444091

A Supercomplex Spanning the Inner and Outer Membranes Mediates the Biogenesis of β-Barrel Outer Membrane Proteins in Bacteria.

PubMed

Wang, Yan; Wang, Rui; Jin, Feng; Liu, Yang; Yu, Jiayu; Fu, Xinmiao; Chang, Zengyi

2016-08-05

β-barrel outer membrane proteins (OMPs) are ubiquitously present in Gram-negative bacteria, mitochondria and chloroplasts, and function in a variety of biological processes. The mechanism by which the hydrophobic nascent β-barrel OMPs are transported through the hydrophilic periplasmic space in bacterial cells remains elusive. Here, mainly via unnatural amino acid-mediated in vivo photo-crosslinking studies, we revealed that the primary periplasmic chaperone SurA interacts with nascent β-barrel OMPs largely via its N-domain but with β-barrel assembly machine protein BamA mainly via its satellite P2 domain, and that the nascent β-barrel OMPs interact with SurA via their N- and C-terminal regions. Additionally, via dual in vivo photo-crosslinking, we demonstrated the formation of a ternary complex involving β-barrel OMP, SurA, and BamA in cells. More importantly, we found that a supercomplex spanning the inner and outer membranes and involving the BamA, BamB, SurA, PpiD, SecY, SecE, and SecA proteins appears to exist in living cells, as revealed by a combined analyses of sucrose-gradient ultra-centrifugation, Blue native PAGE and mass spectrometry. We propose that this supercomplex integrates the translocation, transportation, and membrane insertion events for β-barrel OMP biogenesis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The thermodynamic and hydrodynamic properties of macromolecules that influence the hydrodynamics of porous systems.

PubMed

Comper, W D

1994-06-21

The water flow across porous, semipermeable membranes associated with osmosis and filtration under a variety of conditions is analysed and compared to macromolecular diffusion across free-liquid boundaries, diffusion and sedimentation in the ultracentrifuge, and tracer diffusion of water. This study establishes that osmosis can be explained in terms of the irreversible thermodynamics of diffusion. For macromolecular osmotically active solutes in the semidilute concentration regime the water flows across semipermeable porous membranes are interpreted in terms of a rate-limiting solute-solvent exchange layer that exists on the solution side of the membrane adjacent to the membrane pore; both osmosis and filtration will be governed by these exchange layers. These exchange layers also yield unique properties of their constituent molecules in systems where there is osmotic equilibration between solutions of different solutes. This study also establishes the need to consider the internal osmotic pressure of membranes in the pressure balance associated with the flow across the membrane. The complex situation of partially permeable membranes is analysed for the simple case where there are no mechanical gradients and there is only one osmotically active solution that creates a rate-limiting exchange layer. This treatment predicts that the flow will be governed primarily by the osmotic pressure difference associated with the partitioning of the solute at the membrane-solution interface.

Morphologic and proteomic characterization of exosomes released by cultured extravillous trophoblast cells

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

Atay, Safinur; Gercel-Taylor, Cicek; Kesimer, Mehmet

Exosomes represent an important intercellular communication vehicle, mediating events essential for the decidual microenvironment. While we have demonstrated exosome induction of pro-inflammatory cytokines, to date, no extensive characterization of trophoblast-derived exosomes has been provided. Our objective was to provide a morphologic and proteomic characterization of these exosomes. Exosomes were isolated from the conditioned media of Swan71 human trophoblast cells by ultrafiltration and ultracentrifugation. These were analyzed for density (sucrose density gradient centrifugation), morphology (electron microscopy), size (dynamic light scattering) and protein composition (Ion Trap mass spectrometry and western immunoblotting). Based on density gradient centrifugation, microvesicles from Sw71 cells exhibit amore » density between 1.134 and 1.173 g/ml. Electron microscopy demonstrated that microvesicles from Sw71 cells exhibit the characteristic cup-shaped morphology of exosomes. Dynamic light scattering showed a bell-shaped curve, indicating a homogeneous population with a mean size of 165 nm {+-} 0.5 nm. Ion Trap mass spectrometry demonstrated the presence of exosome marker proteins (including CD81, Alix, cytoskeleton related proteins, and Rab family). The MS results were confirmed by western immunoblotting. Based on morphology, density, size and protein composition, we defined the release of exosomes from extravillous trophoblast cells and provide their first extensive characterization. This characterization is essential in furthering our understanding of 'normal' early pregnancy.« less

The use of analytical sedimentation velocity to extract thermodynamic linkage.

PubMed

Cole, James L; Correia, John J; Stafford, Walter F

2011-11-01

For 25 years, the Gibbs Conference on Biothermodynamics has focused on the use of thermodynamics to extract information about the mechanism and regulation of biological processes. This includes the determination of equilibrium constants for macromolecular interactions by high precision physical measurements. These approaches further reveal thermodynamic linkages to ligand binding events. Analytical ultracentrifugation has been a fundamental technique in the determination of macromolecular reaction stoichiometry and energetics for 85 years. This approach is highly amenable to the extraction of thermodynamic couplings to small molecule binding in the overall reaction pathway. In the 1980s this approach was extended to the use of sedimentation velocity techniques, primarily by the analysis of tubulin-drug interactions by Na and Timasheff. This transport method necessarily incorporates the complexity of both hydrodynamic and thermodynamic nonideality. The advent of modern computational methods in the last 20 years has subsequently made the analysis of sedimentation velocity data for interacting systems more robust and rigorous. Here we review three examples where sedimentation velocity has been useful at extracting thermodynamic information about reaction stoichiometry and energetics. Approaches to extract linkage to small molecule binding and the influence of hydrodynamic nonideality are emphasized. These methods are shown to also apply to the collection of fluorescence data with the new Aviv FDS. Copyright © 2011 Elsevier B.V. All rights reserved.

Analysis of PKR-RNA interactions by sedimentation velocity

PubMed Central

Wong, C. Jason; Launer-Felty, Katherine; Cole, James L.

2010-01-01

PKR is an interferon-induced kinase that plays a pivotal role in the innate immunity pathway for defense against viral infection. PKR is activated to undergo autophosphorylation upon binding to RNAs that contain duplex regions. Some highly structured viral RNAs do not activate and function as PKR inhibitors. In order to define the mechanisms of activation and inhibition of PKR by RNA it is necessary to characterize the stoichiometries, affinities and free energy couplings governing the assembly of the relevant complexes. We have found sedimentation velocity analytical ultracentrifugation to be particularly useful in the study of PKR-RNA interactions. Here, we describe protocols for designing and analyzing sedimentation velocity experiments that are generally applicable to studies of protein-nucleic interactions. Initially, velocity data obtained at multiple protein:RNA ratios are analyzed using the dc/dt method to define the association model and to test whether the system is kinetically limited. The sedimentation velocity data obtained at multiple loading concentrations are then globally fit to this model to determine the relevant association constants. The frictional ratios of the complexes are calculated using the fitted sedimentation coefficients to determine whether the hydrodynamic properties are physically reasonable. We demonstrate the utility of this approach using examples from our studies of PKR interactions with simple dsRNAs, the HIV TAR RNA and the VAI RNA from Adenovirus. PMID:21195224

Visualization and in vivo tracking of the exosomes of murine melanoma B16-BL6 cells in mice after intravenous injection.

PubMed

Takahashi, Yuki; Nishikawa, Makiya; Shinotsuka, Haruka; Matsui, Yuriko; Ohara, Saori; Imai, Takafumi; Takakura, Yoshinobu

2013-05-20

The development of exosomes as delivery vehicles requires understanding how and where exogenously administered exosomes are distributed in vivo. In the present study, we designed a fusion protein consisting of Gaussia luciferase and a truncated lactadherin, gLuc-lactadherin, and constructed a plasmid expressing the fusion protein. B16-BL6 murine melanoma cells were transfected with the plasmid, and exosomes released from the cells were collected by ultracentrifugation. Strong luciferase activity was detected in the fraction containing exosomes, indicating their efficient labeling with gLuc-lactadherin. Then, the labeled B16-BL6 exosomes were intravenously injected into mice, and their tissue distribution was evaluated. Pharmacokinetic analysis of the exosome blood concentration-time profile revealed that B16-BL6 exosomes disappeared very quickly from the blood circulation with a half-life of approximately 2min. Little luciferase activity was detected in the serum at 4h after exosome injection, suggesting rapid clearance of B16-BL6 exosomes in vivo. Moreover, sequential in vivo imaging revealed that the B16-BL6 exosome-derived signals distributed first to the liver and then to the lungs. These results indicate that gLuc-lactadherin labeling is useful for tracing exosomes in vivo and that B16-BL6 exosomes are rapidly cleared from the blood circulation after systemic administration. Copyright © 2013 Elsevier B.V. All rights reserved.

Influence of electronic type purity on the lithiation of single-walled carbon nanotubes.

PubMed

Jaber-Ansari, Laila; Iddir, Hakim; Curtiss, Larry A; Hersam, Mark C

2014-03-25

Single-walled carbon nanotubes (SWCNTs) have emerged as one of the leading additives for high-capacity nanocomposite lithium ion battery electrodes due to their ability to improve electrode conductivity, current collection efficiency, and charge/discharge rate for high power applications. However, since as-grown SWCNTs possess a distribution of physical and electronic structures, it is of high interest to determine which subpopulations of SWCNTs possess the highest lithiation capacity and to develop processing methods that can enhance the lithiation capacity of underperforming SWCNT species. Toward this end, SWCNT electronic type purity is controlled via density gradient ultracentrifugation, enabling a systematic study of the lithiation of SWCNTs as a function of metal versus semiconducting content. Experimentally, vacuum-filtered freestanding films of metallic SWCNTs are found to accommodate lithium with an order of magnitude higher capacity than their semiconducting counterparts, which is consistent with ab initio molecular dynamics and density functional theory calculations in the limit of isolated SWCNTs. In contrast, SWCNT film densification leads to the enhancement of the lithiation capacity of semiconducting SWCNTs to levels comparable to metallic SWCNTs, which is corroborated by theoretical calculations that show increased lithiation of semiconducting SWCNTs in the limit of small SWCNT-SWCNT spacing. Overall, these results will inform ongoing efforts to utilize SWCNTs as conductive additives in nanocomposite lithium ion battery electrodes.

Nucleoplasmin-like domain of FKBP39 from Drosophila melanogaster forms a tetramer with partly disordered tentacle-like C-terminal segments

PubMed Central

Kozłowska, Małgorzata; Tarczewska, Aneta; Jakób, Michał; Bystranowska, Dominika; Taube, Michał; Kozak, Maciej; Czarnocki-Cieciura, Mariusz; Dziembowski, Andrzej; Orłowski, Marek; Tkocz, Katarzyna; Ożyhar, Andrzej

2017-01-01

Nucleoplasmins are a nuclear chaperone family defined by the presence of a highly conserved N-terminal core domain. X-ray crystallographic studies of isolated nucleoplasmin core domains revealed a β-propeller structure consisting of a set of five monomers that together form a stable pentamer. Recent studies on isolated N-terminal domains from Drosophila 39-kDa FK506-binding protein (FKBP39) and from other chromatin-associated proteins showed analogous, nucleoplasmin-like (NPL) pentameric structures. Here, we report that the NPL domain of the full-length FKBP39 does not form pentameric complexes. Multi-angle light scattering (MALS) and sedimentation equilibrium ultracentrifugation (SE AUC) analyses of the molecular mass of the full-length protein indicated that FKBP39 forms homotetrameric complexes. Molecular models reconstructed from small-angle X-ray scattering (SAXS) revealed that the NPL domain forms a stable, tetrameric core and that FK506-binding domains are linked to it by intrinsically disordered, flexible chains that form tentacle-like segments. Analyses of full-length FKBP39 and its isolated NPL domain suggested that the distal regions of the polypeptide chain influence and determine the quaternary conformation of the nucleoplasmin-like protein. These results provide new insights regarding the conserved structure of nucleoplasmin core domains and provide a potential explanation for the importance of the tetrameric structural organization of full-length nucleoplasmins. PMID:28074868

Low pH overrides the need of calcium ions for the shape-function relationship of calmodulin: resolving prevailing debates.

PubMed

Pandey, Kalpana; Dhoke, Reema R; Rathore, Yogendra Singh; Nath, Samir K; Verma, Neha; Bawa, Simranjot; Ashish

2014-05-15

Calmodulin (CaM) regulates numerous cellular functions by sensing Ca(2+) levels inside cells. Although its structure as a function of the Ca(2+)-bound state remains hotly debated, no report is available on how pH independently or in interaction with Ca(2+) ions regulates shape and function of CaM. From SAXS data analysis of CaM at different levels of Ca(2+)-ion concentration and buffer pH, we found that (1) CaM molecules possess a Gaussian-chain-like shape in solution even in the presence of Ca(2+) ion or at low pH, (2) the global shape of apo CaM is very similar to its NMR structure rather than the crystal structures, (3) about 16 Ca(2+) ions or more are required per CaM molecule in solution to achieve the four-Ca(2+)-bound crystal structure, (4) low pH alone can impart shape changes in CaM similar to Ca(2+) ions, and (5) at different [Ca(2+)]/[CaM] ratio or pH values, the predominant shape of CaM is essentially a weighted average of its apo and fully activated shape. Results were further substantiated by analysis of sedimentation coefficient values from analytical ultracentrifugation and peptide binding assays using two peptides, each known to preferentially bind the apo or the Ca(2+)-activated state.

Evaporative concentration of skimmed milk: effect on casein micelle hydration, composition, and size.

PubMed

Liu, Dylan Z; Dunstan, David E; Martin, Gregory J O

2012-10-01

Understanding the effect of evaporative concentration on casein micelle composition is of high importance for milk processing. Alterations to the hydration, composition and size of casein micelles were investigated in skimmed milk evaporated to concentrations of 12-45% total solids content. The size of casein micelles was determined by dynamic light scattering, and the water content and composition determined by analysis of supernatants and pellets obtained by ultracentrifugation. The mass balance and hydration results showed that during the evaporation process, while micelles were dehydrated, water was removed preferentially from the serum. The amount of soluble casein and calcium in the serum decreased as a function of increasing solids content, indicating a shift of these components to the micelles. The formation of a small proportion of micelle aggregates at high concentrations appeared dependent on the time kept at these concentrations. Upon redilution with water, casein micelles were immediately rehydrated and aggregates were broken up in a matter of minutes. Soluble calcium and pH returned to their original state over a number of hours; however, only a small percentage of original soluble casein returned to the serum over the 5h period investigated. These results showed that casein micelles are significantly affected by evaporative concentration and that the alterations are not completely and rapidly reversible. Copyright © 2012. Published by Elsevier Ltd.

Tissue distribution and lipophorin transport of hydrocarbons and sex pheromones in the house fly, Musca domestica

PubMed Central

Schal, Coby; Sevala, Veeresh; de L.Capurro, Margareth; Snyder, Theodore E.; Blomquist, Gary J.; Bagnères, Anne–Geneviève

2001-01-01

We investigated the relationship between epicuticular and internal hydrocarbons in the adult house fly, Musca domestica and the distribution of hydrocarbons, including the female sex pheromone component, (Z)-9-tricosene, in tissues. Internal hydrocarbons increased dramatically in relation to sexual maturation and were found in the hemolymph, ovaries, digestive tract, and fat body. (Z)-9-Tricosene comprised a relatively large fraction of the hydrocarbons in the female carcass and hemolymph, and less so in other tissues, while other hydrocarbons were represented in greater amounts in the ovaries than in other tissues. It therefore appears that certain hydrocarbons were selectively provisioned to certain tissues such as the ovaries, from which pheromone was relatively excluded. Both KBr gradient ultracentrifugation and specific immunoprecipitation indicated that > 90% of hemolymph hydrocarbons were associated with a high-density lipophorin (density = 1.09 g ml−1), composed of two apoproteins under denaturing conditions, apolipophorin I (∼240 kD) and apolipophorin II (∼85 kD). Our results support a predicted model (Chino, 1985) that lipophorin is involved in the transport of sex pheromone in M. domestica. In addition to delivering hydrocarbons and sex pheromones to the cuticular surface, we suggest that lipophorin may play an important role in an active mechanism that selectively deposits certain subsets of hydrocarbons at specific tissues. PMID:15455072

CO binding improves the structural, functional, physical and anti-oxidation properties of the PEGylated hemoglobin.

PubMed

Wang, Qingqing; Hu, Tao; Sun, Lijing; Ji, Shaoyang; Zhao, Dawei; Liu, Jiaxin; Ma, Guanghui; Su, Zhiguo

2015-02-01

PEGylated hemoglobin (Hb) is a promising oxygen therapeutic agent for clinical application. However, it suffered from structural perturbation, functional instability and methemoglobin (metHb) formation. To improve the structural, functional, physical and anti-oxidation properties of the PEGylated Hb. PEGylation of Hb with CO binding (HbCO) was conducted using maleimide and acylation chemistry, respectively. Physical and chemical parameters were measured for Hb samples. The circular dichroism spectra, dynamic light scattering and analytical ultracentrifugation were used to investigate the structure and conformation of PEGylated HbCO. CO binding can inhibit the autoxidation of the PEGylated Hb, structurally stabilize its tetramer and improve its thermal and pH stability. Importantly, the circular dichroism spectra showed that CO binding can decrease the structural perturbation of Hb induced by PEGylation. The PEGylated HbCO with CO release showed slightly higher oxygen-delivery capacity than the PEGylated Hb. The PEGylated HbCO did not show metHb formation after 30-day storage at 4°C. CO binding structurally stabilized the PEGylated Hb, abolished its metHb formation, and significantly increased its physical stability. In particular, it also avoided the perturbation of PEG chains on the heme microenvironment. The functional property of the PEGylated HbCO can be maintained during its long-term storage, which is of great significance for field transfusion.

A plasma membrane sucrose-binding protein that mediates sucrose uptake shares structural and sequence similarity with seed storage proteins but remains functionally distinct.

PubMed

Overvoorde, P J; Chao, W S; Grimes, H D

1997-06-20

Photoaffinity labeling of a soybean cotyledon membrane fraction identified a sucrose-binding protein (SBP). Subsequent studies have shown that the SBP is a unique plasma membrane protein that mediates the linear uptake of sucrose in the presence of up to 30 mM external sucrose when ectopically expressed in yeast. Analysis of the SBP-deduced amino acid sequence indicates it lacks sequence similarity with other known transport proteins. Data presented here, however, indicate that the SBP shares significant sequence and structural homology with the vicilin-like seed storage proteins that organize into homotrimers. These similarities include a repeated sequence that forms the basis of the reiterated domain structure characteristic of the vicilin-like protein family. In addition, analytical ultracentrifugation and nonreducing SDS-polyacrylamide gel electrophoresis demonstrate that the SBP appears to be organized into oligomeric complexes with a Mr indicative of the existence of SBP homotrimers and homodimers. The structural similarity shared by the SBP and vicilin-like proteins provides a novel framework to explore the mechanistic basis of SBP-mediated sucrose uptake. Expression of the maize Glb protein (a vicilin-like protein closely related to the SBP) in yeast demonstrates that a closely related vicilin-like protein is unable to mediate sucrose uptake. Thus, despite sequence and structural similarities shared by the SBP and the vicilin-like protein family, the SBP is functionally divergent from other members of this group.

Association of canalicular membrane enzymes with bile acid micelles and lipid aggregates in human and rat bile.

PubMed

Accatino, L; Pizarro, M; Solís, N; Koenig, C S

1995-01-18

This study was undertaken to gain insights into the characteristics of the polymolecular association between canalicular membrane enzymes, bile acids, cholesterol and phospholipids in bile and into the celular mechanisms whereby the enzymes are secreted into bile. With this purpose, we studied the distribution of bile acids, cholesterol, phospholipids, proteins and representative canalicular membrane enzymes (alkaline phosphatase, 5'-nucleotidase and gamma-glutamyl transpeptidase), which can be considered specific marker constituents, in bile fractions enriched in phospholipid-cholesterol lamellar structures (multilamellar and unilamellar vesicles) and bile acid-mixed micelles. These fractions were isolated by ultracentrifugation from human hepatic bile, normal rat bile and bile of rats treated with diosgenin, a steroid that induces a marked increase in biliary cholesterol secretion, and were characterized by density, lipid composition and transmission electron microscopy. These studies demonstrate that alkaline phosphatase, 5'-nucleotidase and gamma-glutamyl transpeptidase are secreted into both human and rat bile where they are preferentially associated with bile acid-mixed micelles, suggesting a role for bile acids in both release of these enzymes and lipids from the canalicular membrane and solubilization in bile. In addition, heterogeneous association of these enzymes with nonmicellar, lamellar structures in human and rat bile is consistent with the hypothesis that processes independent of the detergent effects of bile acids might also result in the release of specific intrinsic membrane proteins into bile.

Purification and properties of Renilla reniformis luciferase

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

Matthews, J.C.; Hori, K.; Cormier, M.J.

1977-01-11

Luciferase from the anthozoan coelenterate Renilla reniformis (Renilla luciferin:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.5.) catalyzes the bioluminescent oxidation of Renilla luciferin producing light (lambda/sub B/ 480 nm, Q/sub B/ 5.5 percent), oxyluciferin, and CO/sub 2/. Using a combination of ion-exchange, molecular-sieve, sulfhydryl-exchange, and affinity chromatography, luciferase has been purified, approximately 12,000-fold with 24 percent recovery, to homogeneity as judged by analysis with disc and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and ultracentrifugation. Renilla luciferase is active as a nearly spherical single polypeptide chain monomer of 3.5 x 10/sup 4/ daltons having a specific activity of 1.8 x 10/sup 15/ h..nu..s/supmore » -1/ mg/sup -1/ and a turnover number of 111 ..mu..mol min/sup -/1 ..mu..mol/sup -1/ of enzyme. This enzyme has a high content of aromatic and hydrophobic amino acids such that it has an epsilon/sub 280 nm/ 0.1 percent/ of 2.1 and an average hydrophobicity of 1200 cal residue/sup -1/. The high average hydrophobicity of luciferase, which places it among the more hydrophobic proteins reported, is believed to account, at least in part, for its tendency to self-associate forming inactive dimers and higher molecular weight species.« less

pH-dependent structural change of the extracellular sensor domain of the DraK histidine kinase from Streptomyces coelicolor.

PubMed

Yeo, Kwon Joo; Kim, Eun Hye; Hwang, Eunha; Han, Young-Hyun; Eo, Yumi; Kim, Hyun Jung; Kwon, Ohsuk; Hong, Young-Soo; Cheong, Chaejoon; Cheong, Hae-Kap

2013-02-15

Recently, the DraR/DraK (Sco3063/Sco3062) two-component system (TCS) of Streptomycescoelicolor has been reported to be involved in the differential regulation of antibiotic biosynthesis. However, it has not been shown that under which conditions and how the DraR/DraK TCS is activated to initiate the signal transduction process. Therefore, to understand the sensing mechanism, structural study of the sensory domain of DraK is highly required. Here, we report the biochemical and biophysical properties of the extracellular sensory domain (ESD) of DraK. We observed a reversible pH-dependent conformational change of the ESD in a pH range of 2.5-10. Size-exclusion chromatography and AUC (analytical ultracentrifugation) data indicated that the ESD is predominantly monomeric in solution and exists in equilibrium between monomer and dimer states in acidic condition. Using NMR (nuclear magnetic resonance) and CD (circular dichroism) spectroscopy, our findings suggest that the structure of the ESD at low pH is more structured than that at high pH. In particular, the glutamate at position 83 is an important residue for the pH-dependent conformational change. These results suggest that this pH-dependent conformational change of ESD may be involved in signal transduction process of DraR/DraK TCS. Copyright © 2013 Elsevier Inc. All rights reserved.

Protective Effect of Ozone against Hemiscorpius lepturus Envenomation in Mice.

PubMed

Naserzadeh, Parvaneh; Shahi, Farshad; Shahbazzadeh, Delavar; Ghanei, Mostafa; Ashtari, Khadijeh; Panahi, Yoones; Hosseini, Mir-Jamal; Izadi, Morteza

2017-08-01

Scorpion (Hemiscorpius lepturus) stings are a public health concern in Iran, particularly in south and southwestern regions of Iran. The gold standard for the treatment of a scorpion sting is anti-venom therapy. However, immunotherapy can have serious side effects, such as anaphylactic shock (which can sometimes even lead to death). The aim of the current study was to demonstrate the protective effect of ozone against toxicity induced by Hemiscorpius lepturus (H. lepturus) venom in mice. Eight hours after the injection of ozone to the experimental design groups, the male mice were decapitated and mitochondria were isolated from five different tissues (liver, kidney, heart, brain, and spinal cord) using differential ultracentrifugation. Then, assessment of mitochondrial parameters including mitochondrial reactive oxidative species (ROS) production, mitochondrial membrane potential (MMP), ATP level, and the release of cytochrome c from the mitochondria was performed. Our results showed that H. lepturus venom-induced oxidative stress is related to ROS production and MMP collapse, which is correlated with cytochrome c release and ATP depletion, indicating the predisposition to the cell death signaling. In general, ozone therapy in moderate dose can be considered as clinically effective for the treatment of H. lepturus sting as a protective and antioxidant agent. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Production and purification of non replicative canine adenovirus type 2 derived vectors.

PubMed

Szelechowski, Marion; Bergeron, Corinne; Gonzalez-Dunia, Daniel; Klonjkowski, Bernard

2013-12-03

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 10(10) infectious particles per ml and can be directly administrated in vivo.

The efficiency of concentration methods used to detect enteric viruses in anaerobically digested sludge

PubMed Central

Prado, Tatiana; Guilayn, Wilma de Carvalho Pereira Bonet; Gaspar, Ana Maria Coimbra; Miagostovich, Marize Pereira

2013-01-01

The presence of enteric viruses in biosolids can be underestimated due to the inefficient methods (mainly molecular methods) used to recover the viruses from these matrices. Therefore, the goal of this study was to evaluate the different methods used to recover adenoviruses (AdV), rotavirus species A (RVA), norovirus genogroup II (NoV GII) and the hepatitis A virus (HAV) from biosolid samples at a large urban wastewater treatment plant in Brazil after they had been treated by mesophilic anaerobic digestion. Quantitative polymerase chain reaction (PCR) was used for spiking experiments to compare the detection limits of feasible methods, such as beef extract elution and ultracentrifugation. Tests were performed to detect the inhibition levels and the bacteriophage PP7 was used as an internal control. The results showed that the inhibitors affected the efficiency of the PCR reaction and that beef extract elution is a suitable method for detecting enteric viruses, mainly AdV from biosolid samples. All of the viral groups were detected in the biosolid samples: AdV (90%), RVA, NoV GII (45%) and HAV (18%), indicating the viruses' resistance to the anaerobic treatment process. This is the first study in Brazil to detect the presence of RVA, AdV, NoV GII and HAV in anaerobically digested sludge, highlighting the importance of adequate waste management. PMID:23440119

Mechanisms of Ricin Toxin Neutralization Revealed through Engineered Homodimeric and Heterodimeric Camelid Antibodies.

PubMed

Herrera, Cristina; Tremblay, Jacqueline M; Shoemaker, Charles B; Mantis, Nicholas J

2015-11-13

Novel antibody constructs consisting of two or more different camelid heavy-chain only antibodies (VHHs) joined via peptide linkers have proven to have potent toxin-neutralizing activity in vivo against Shiga, botulinum, Clostridium difficile, anthrax, and ricin toxins. However, the mechanisms by which these so-called bispecific VHH heterodimers promote toxin neutralization remain poorly understood. In the current study we produced a new collection of ricin-specific VHH heterodimers, as well as VHH homodimers, and characterized them for their ability neutralize ricin in vitro and in vivo. We demonstrate that the VHH heterodimers, but not homodimers were able to completely protect mice against ricin challenge, even though the two classes of antibodies (heterodimers and homodimers) had virtually identical affinities for ricin holotoxin and similar IC50 values in a Vero cell cytotoxicity assay. The VHH heterodimers did differ from the homodimers in their ability to promote toxin aggregation in solution, as revealed through analytical ultracentrifugation. Moreover, the VHH heterodimers that were most effective at promoting ricin aggregation in solution were also the most effective at blocking ricin attachment to cell surfaces. Collectively, these data suggest that heterodimeric VHH-based neutralizing agents may function through the formation of antibody-toxin complexes that are impaired in their ability to access host cell receptors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Precise determination of protein extinction coefficients under native and denaturing conditions using SV-AUC.

PubMed

Hoffmann, Andreas; Grassl, Kerstin; Gommert, Janine; Schlesak, Christian; Bepperling, Alexander

2018-04-17

The accurate determination of protein concentration is an important though non-trivial task during the development of a biopharmaceutical. The fundamental prerequisite for this is the availability of an accurate extinction coefficient. Common approaches for the determination of an extinction coefficient for a given protein are either based on the theoretical prediction utilizing the amino acid sequence or the photometric determination combined with a measurement of absolute protein concentration. Here, we report on an improved SV-AUC based method utilizing an analytical ultracentrifuge equipped with absorbance and Rayleigh interference optics. Global fitting of datasets helped to overcome some of the obstacles encountered with the traditional method employing synthetic boundary cells. Careful calculation of dn/dc values taking glycosylation and solvent composition into account allowed the determination of the extinction coefficients of monoclonal antibodies and an Fc-fusion protein under native as well as under denaturing conditions. An intra-assay precision of 0.9% and an accuracy of 1.8% compared to the theoretical value was achieved for monoclonal antibodies. Due to the large number of data points of a single dataset, no meaningful difference between the ProteomeLab XL-I and the new Optima AUC platform could be observed. Thus, the AUC-based approach offers a precise, convenient and versatile alternative to conventional methods like total amino acid analysis (AAA).

[Effect of Coriolus versicolor polysaccharide B on membrane glycosaminoglycans and cellular glutathione changes in RAW264.7 macrophages exposed to angiotensin II].

PubMed

Lou, Ning; Ma, Gang; Wang, Dao-feng; Zhu, Zhi-wei; Su, Quan-guan; Fang, Yi

2007-12-01

To investigate the effect of Coriolus versicolor polysaccharide B (CVP-B) on increased membrane glycosaminoglycans (GAG) expression and intracellular glutathione (GSH) of RAW264.7 macrophages exposed to angiotensin II (Ang II). The plasma membrane of RAW264.7 macrophages exposed to Ang II treatment was isolated by ultracentrifugation, and the membrane GAG expression was analyzed using 1, 9-dimethylmethylene blue (DMMB) spectrophotometric assay for sulfated GAG. The intracellular reduced GSH was determined using fluorophotometry. The GAG content in the macrophage membranes increased by up to 54% following cell exposure to 1.0 micromol/L Ang II, whereas in presence of 1.0 micromol;/L Ang II, CVP-B at 1, 10, and 50 microg/ml decreased the GAG content by 13%, 43% (P<0.01), and 52% (P<0.01), respectively. The macrophage GSH activity decreased by 69% following incubation with 1.0 micromol;/L Ang II for 24 h, and CVP-B treatment at 1, 10, and 50 microg/ml in presence of 1.0 micromol;/L Ang II resulted in significant increment of GSH activity by 31%(P<0.05), 104% (P<0.01), and 168% (P<0.01), respectively. These data provide the first evidence that CVP-B inhibits elevated GAG expression in RAW264.7 macrophage membrane induced by Ang II.

Urinary excretion of glycosaminoglycans in the various forms of gargoylism

PubMed Central

Manley, G.; Williams, U.

1969-01-01

The urinary excretion of glycosaminoglycans in 28 cases of gargoylism, embracing the Hurler, Hunter, Sanfilippo, Morquio, and Scheie syndromes (McKusick, 1966), has been examined using the cetylpyridinium chloride (CPC) turbidity test, the uronic acid/creatinine ratio, and the electrophoretic pattern of urine concentrates, as routine procedures. Ion-exchange column chromatographic techniques were also employed for the fractionation of glycosaminoglycans and aminosugars. Molecular weights were investigated by gel filtration and ultracentrifugation. The CPC turbidity test was positive in every case. The uronic acid/creatinine ratio provided a sensitive index of increased glycosaminoglycan excretion. Cases of the Hurler syndrome showed the highest, and cases of the Morquio and Scheie syndromes the lowest, ratios. A correlation was observed between the uronic acid/creatinine ratio and the clinical severity of the disease. Cellulose acetate electrophoresis differentiated clearly between the two major forms of gargoylism, the Hurler and Sanfilippo syndromes, but differentiation between the Hurler, Hunter, and Scheie syndromes was more difficult on electrophoretic data alone. Results obtained with cases diagnosed as the Morquio syndrome were disappointing. The existence of formes frustes of the Sanfilippo syndrome among the mentally subnormal is predicted. Errors caused by bacterial contamination of urine samples are emphasized. The atypical behaviour of urinary glycosaminoglycans in analytical procedures is discussed. Molecular weight studies suggested heterogeneity. The nature of the basic defect in gargoylism is discussed. Images PMID:4239429

Immunoglobulin E (IgE) and IgE-containing cells in human gastrointestinal fluids and tissues.

PubMed Central

Brown, W R; Borthistle, B K; Chen, S T

1975-01-01

Human gastric, small intestinal, colonic and rectal mucosae were examined for IgE-containing cells by single- and double-antibody immunofluorescence techniques, and IgE in intesinal fluids was measured by a double-antibody radioimmunoassay. IgE-containing cells were identified in all tissue specimens and comprised about 2% of all immunoglobulin-containing cells. Although less numerous than cells containing IgA, IgM or IgG, they were remarkably numerous in relation to the concentration of IgE in serum (about 0-001% of total immunoglobulin). IgE immunocytes were significantly more numerous in stomach and proximal small bowel than in colon and rectum, and were very numerous at bases of gastric and duodenal peptic ulcers. Measurable IgE was found in seventy-eight of eighty-five (92%) intestinal fluids. Sucrose gradient ultracentrifugation analysis of four of the fluids revealed that the immunologically reactive IgE was largely in fractions corresponding to molecules of lower molecular weight than that of albumin, which suggests that IgE in gut contents is degraded by proteolytic enzymes. The presence of IgE-forming cells in gastrointestinal tissues, and IgE or a fragment of IgE in intestinal fluids, suggests that IgE antibodies are available for participation in local reaginic-type reactions in the gut. Images FIG. 1 PMID:813925

Structure and hydrodynamics of a DNA G-quadruplex with a cytosine bulge.

PubMed

Meier, Markus; Moya-Torres, Aniel; Krahn, Natalie J; McDougall, Matthew D; Orriss, George L; McRae, Ewan K S; Booy, Evan P; McEleney, Kevin; Patel, Trushar R; McKenna, Sean A; Stetefeld, Jörg

2018-06-01

The identification of four-stranded G-quadruplexes (G4s) has highlighted the fact that DNA has additional spatial organisations at its disposal other than double-stranded helices. Recently, it became clear that the formation of G4s is not limited to the traditional G3+NL1G3+NL2G3+NL3G3+ sequence motif. Instead, the G3 triplets can be interrupted by deoxythymidylate (DNA) or uridylate (RNA) where the base forms a bulge that loops out from the G-quadruplex core. Here, we report the first high-resolution X-ray structure of a unique unimolecular DNA G4 with a cytosine bulge. The G4 forms a dimer that is stacked via its 5'-tetrads. Analytical ultracentrifugation, static light scattering and small angle X-ray scattering confirmed that the G4 adapts a predominantly dimeric structure in solution. We provide a comprehensive comparison of previously published G4 structures containing bulges and report a special γ torsion angle range preferentially populated by the G4 core guanylates adjacent to bulges. Since the penalty for introducing bulges appears to be negligible, it should be possible to functionalize G4s by introducing artificial or modified nucleotides at such positions. The presence of the bulge alters the surface of the DNA, providing an opportunity to develop drugs that can specifically target individual G4s.

Neuronal nuclei isolation from human postmortem brain tissue.

PubMed

Matevossian, Anouch; Akbarian, Schahram

2008-10-01

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan. Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays.

Production and characterization of a recombinant chimeric antigen consisting botulinum neurotoxin serotypes A, B and E binding subdomains.

PubMed

Ebrahimi, Firouz; Rasaee, Mohammad Javad; Mousavi, Seyed Latif; Babaeipour, Valiollah

2010-02-01

Botulinum neurotoxins (BoNTs) are potent toxicant proteins composed of a heavy chain (100 kDa) and a light chain (50 kDa) of seven (A-G) serotypes that is responsible for botulism syndrome. In this study, polypeptides from C-terminal heavy chain of BoNTs serotypes A, B and E to the length of 54, 45 and 48 amino acid respectively were selected, linked together using a hydrophobic linker and expressed in E. coli. The expression efficiency of the chimeric protein was found to be 51%. The chimeric protein was produced in the form of inclusion body (IB) both at two studied temperatures, 30 degrees C and 37 degrees C. This IB was extracted by ultracentrifugation and followed for chimeric protein solubilization and purification using of ultrafiltration and preparative electrophoresis. The purified chimeric protein was characterized using blotting and ELISA. To evaluate the protection ability of this chimeric antigen against their active toxins, it was injected to mice and the antibody titer as well as the extent of protectivity were determined. Mice given three injections (10 microg/mice) of the antigen were protected against an intra-peritoneal administration of 10 LD(50 )of serotypes A and E, but 100 LD(50) of serotype B. We conclude that a significant correlation exists between the antigenic characteristics and protection capability of the chimeric protein prepared in this study.

Biotic and abiotic interactions in aquatic microcosms determine fate and toxicity of Ag nanoparticles. Part 1. Aggregation and dissolution.

PubMed

Unrine, Jason M; Colman, Benjamin P; Bone, Audrey J; Gondikas, Andreas P; Matson, Cole W

2012-07-03

To better understand their fate and toxicity in aquatic environments, we compared the aggregation and dissolution behavior of gum arabic (GA) and polyvinylpyrrolidone (PVP) coated Ag nanoparticles (NPs) in aquatic microcosms. There were four microcosm types: surface water; water and sediment; water and aquatic plants; or water, sediment, and aquatic plants. Dissolution and aggregation behavior of AgNPs were examined using ultracentrifugation, ultrafiltration, and asymmetrical flow field flow fractionation coupled to ultraviolet-visible spectroscopy, dynamic and static laser light scattering, and inductively coupled plasma mass spectrometry. Plants released dissolved organic matter (DOM) into the water column either through active or passive processes in response to Ag exposure. This organic matter fraction readily bound Ag ions. The plant-derived DOM had the effect of stabilizing PVP-AgNPs as primary particles, but caused GA-AgNPs to be removed from the water column, likely by dissolution and binding of released Ag ions on sediment and plant surfaces. The destabilization of the GA-AgNPs also corresponded with X-ray absorption near edge spectroscopy results which suggest that 22-28% of the particulate Ag was associated with thiols and 5-14% was present as oxides. The results highlight the potential complexities of nanomaterial behavior in response to biotic and abiotic modifications in ecosystems, and may help to explain differences in toxicity of Ag observed in realistic exposure media compared to simplified laboratory exposures.

Analysis of Active Methylotrophic Communities: When DNA-SIP Meets High-Throughput Technologies.

PubMed

Taubert, Martin; Grob, Carolina; Howat, Alexandra M; Burns, Oliver J; Chen, Yin; Neufeld, Josh D; Murrell, J Colin

2016-01-01

Methylotrophs are microorganisms ubiquitous in the environment that can metabolize one-carbon (C1) compounds as carbon and/or energy sources. The activity of these prokaryotes impacts biogeochemical cycles within their respective habitats and can determine whether these habitats act as sources or sinks of C1 compounds. Due to the high importance of C1 compounds, not only in biogeochemical cycles, but also for climatic processes, it is vital to understand the contributions of these microorganisms to carbon cycling in different environments. One of the most challenging questions when investigating methylotrophs, but also in environmental microbiology in general, is which species contribute to the environmental processes of interest, or "who does what, where and when?" Metabolic labeling with C1 compounds substituted with (13)C, a technique called stable isotope probing, is a key method to trace carbon fluxes within methylotrophic communities. The incorporation of (13)C into the biomass of active methylotrophs leads to an increase in the molecular mass of their biomolecules. For DNA-based stable isotope probing (DNA-SIP), labeled and unlabeled DNA is separated by isopycnic ultracentrifugation. The ability to specifically analyze DNA of active methylotrophs from a complex background community by high-throughput sequencing techniques, i.e. targeted metagenomics, is the hallmark strength of DNA-SIP for elucidating ecosystem functioning, and a protocol is detailed in this chapter.

Lag periods during the self-assembly of {Mo(72)Fe(30)} macroions: connection to the virus capsid formation process.

PubMed

Zhang, Jie; Li, Dong; Liu, Guang; Glover, Kerney Jebrell; Liu, Tianbo

2009-10-28

The kinetic properties of the self-assembly of hydrophilic Keplerate-type polyoxometalate (POM) {Mo(72)Fe(30)} macroanions into single-layer, vesicle-like blackberry structures in solutions were monitored by the static and dynamic laser light scattering techniques. In the presence of additional electrolytes, an obvious lag period at the initial stage of self-assembly was observed, followed by a fast increase of the scattered intensity. The whole kinetic curve is sigmoidal with a lag phase. A two-step nucleation-growth mechanism is proposed to explain this lag phase: the {Mo(72)Fe(30)} macroanions slowly associate into oligomers (mostly dimers), which are the thermodynamically unfavorable intermediates, at the initial stage; once the oligomers reach a critical concentration, the blackberry formation process is accelerated. Analytical ultracentrifugation (AUC) was used to confirm the oligomeric state in {Mo(72)Fe(30)} solution during the lag period. The length of the lag period is dependent on temperature, ionic strength, and the valent states of the additional salts, as well as the solvent content. The kinetics (including the lag period) of the blackberry formation of the {Mo(72)Fe(30)} macroanions show similarities to the self-assembly of virus capsid proteins (which are also soluble macroions) into spherical capsid shells, suggesting possible connections between the self-assembly behaviors of inorganic species and biological macromolecules.

The Micellar Sink

PubMed Central

Scharschmidt, Bruce F.; Schmid, Rudi

1978-01-01

Although the importance of mixed micelles in the solubilization and biliary excretion of lipids is established, little is known about a possible role of mixed micelles in the excretion of other biliary solutes. Ultrafiltration and ultracentrifugation techniques were used to investigate the interaction between substances that are excreted in bile and biliary mixed micelles. Substances (urea, erythritol, sucrose) excreted in bile at concentrations equal to, or less than, that in plasma did not show an association with mixed micelles, whereas substances (indocyanine green, iopanoic acid, rose bengal, unconjugated and conjugated sulfobromophthalein, and conjugated bilirubin) excreted in bile at high concentration relative to plasma did. The percentage of these latter substances in bile associated with micelles varied from 26 to 93% and was relatively independent of concentration. In addition to their association with mixed micelles, these test solutes formed self-aggregates that were stabilized primarily by ionic bonds, and only a small percentage (range = 0-5%) of these solutes were present in bile in the form of monomer or complexes small enough to pass a 5,000-mol wt membrane. These findings offer a possible explanation for the increase in sulfobromophthalein, bilirubin, and indocyanine green maximal biliary excretory rate produced by bile salt infusion, and suggest that the concentrative transport into bile of endogenous compounds and xenobiotics may result from their incorporation into mixed micelles and other macromolecular complexes. PMID:748371

RNA-Based Stable Isotope Probing Suggests Allobaculum spp. as Particularly Active Glucose Assimilators in a Complex Murine Microbiota Cultured In Vitro

PubMed Central

Herrmann, Elena; Young, Wayne; Rosendale, Douglas; Reichert-Grimm, Verena; Conrad, Ralf

2017-01-01

RNA-based stable isotope probing (RNA-SIP) and metabolic profiling were used to detect actively glucose-consuming bacteria in a complex microbial community obtained from a murine model system. A faeces-derived microbiota was incubated under anaerobic conditions for 0, 2, and 4 h with 40 mM [U13C]glucose. Isopycnic density gradient ultracentrifugation and fractionation of isolated RNA into labeled and unlabeled fractions followed by 16S rRNA sequencing showed a quick adaptation of the bacterial community in response to the added sugar, which was dominated by unclassified Lachnospiraceae species. Inspection of distinct fractions of isotope-labeled RNA revealed Allobaculum spp. as particularly active glucose utilizers in the system, as the corresponding RNA showed significantly higher proportions among the labeled RNA. With time, the labeled sugar was used by a wider spectrum of faecal bacteria. Metabolic profiling indicated rapid fermentation of [U13C]glucose, with lactate, acetate, and propionate being the principal 13C-labeled fermentation products, and suggested that “cross-feeding” occurred in the system. RNA-SIP combined with metabolic profiling of 13C-labeled products allowed insights into the microbial assimilation of a general model substrate, demonstrating the appropriateness of this technology to study assimilation processes of nutritionally more relevant substrates, for example, prebiotic carbohydrates, in the gut microbiota of mice as a model system. PMID:28299315

Arp2/3 Complex from Acanthamoeba Binds Profilin and Cross-links Actin Filaments

PubMed Central

Mullins, R. Dyche; Kelleher, Joseph F.; Xu, James; Pollard, Thomas D.

1998-01-01

The Arp2/3 complex was first purified from Acanthamoeba castellanii by profilin affinity chromatography. The mechanism of interaction with profilin was unknown but was hypothesized to be mediated by either Arp2 or Arp3. Here we show that the Arp2 subunit of the complex can be chemically cross-linked to the actin-binding site of profilin. By analytical ultracentrifugation, rhodamine-labeled profilin binds Arp2/3 complex with a Kd of 7 μM, an affinity intermediate between the low affinity of profilin for barbed ends of actin filaments and its high affinity for actin monomers. These data suggest the barbed end of Arp2 is exposed, but Arp2 and Arp3 are not packed together in the complex exactly like two actin monomers in a filament. Arp2/3 complex also cross-links actin filaments into small bundles and isotropic networks, which are mechanically stiffer than solutions of actin filaments alone. Arp2/3 complex is concentrated at the leading edge of motile Acanthamoeba, and its localization is distinct from that of α-actinin, another filament cross-linking protein. Based on localization and actin filament nucleation and cross-linking activities, we propose a role for Arp2/3 in determining the structure of the actin filament network at the leading edge of motile cells. PMID:9529382

Role of surfactants in carbon nanotubes density gradient separation.

PubMed

Carvalho, Elton J F; dos Santos, Maria Cristina

2010-02-23

Several strategies aimed at sorting single-walled carbon nanotubes (SWNT) by diameter and/or electronic structure have been developed in recent years. A nondestructive sorting method was recently proposed in which nanotube bundles are dispersed in water-surfactant solutions and submitted to ultracentrifugation in a density gradient. By this method, SWNTs of different diameters are distributed according to their densities along the centrifuge tube. A mixture of two anionic amphiphiles, namely sodium dodecylsulfate (SDS) and sodium cholate (SC), presented the best performance in discriminating nanotubes by diameter. We present molecular dynamics studies of the water-surfactant-SWNT system. The simulations revealed one aspect of the discriminating power of surfactants: they can actually be attracted toward the interior of the nanotube cage. The binding energies of SDS and SC on the outer nanotube surface are very similar and depend weakly on diameter. The binding inside the tubes, on the contrary, is strongly diameter dependent: SDS fits best inside tubes with diameters ranging from 8 to 9 A, while SC is best accommodated in larger tubes, with diameters in the range 10.5-12 A. The dynamics at room temperature showed that, as the amphiphile moves to the hollow cage, water molecules are dragged together, thereby promoting the nanotube filling. The resulting densities of filled SWNT are in agreement with measured densities.

Quantitative proteome analysis of plasma microparticles for the characterization of HCV-induced hepatic cirrhosis and hepatocellular carcinoma.

PubMed

Taleb, Raghda Saad Zaghloul; Moez, Pacint; Younan, Doreen; Eisenacher, Martin; Tenbusch, Matthias; Sitek, Barbara; Bracht, Thilo

2017-12-01

Hepatocellular carcinoma (HCC) is the most common primary malignant liver tumor and a leading cause of cancer-related deaths worldwide. Cirrhosis induced by hepatitis-C virus (HCV) infection is the most critical risk factor for HCC. However, the mechanism of HCV-induced carcinogenesis is not fully understood. Plasma microparticles (PMP) contribute to numerous physiological and pathological processes and contain proteins whose composition correlates to the respective pathophysiological conditions. We analyzed PMP from 22 HCV-induced cirrhosis patients, 16 HCV-positive HCC patients with underlying cirrhosis and 18 healthy controls. PMP were isolated using ultracentrifugation and analyzed via label-free LC-MS/MS. We identified 840 protein groups and quantified 507 proteins. 159 proteins were found differentially abundant between the three experimental groups. PMP in both disease entities displayed remarkable differences in the proteome composition compared to healthy controls. Conversely, the proteome difference between both diseases was minimal. GO analysis revealed that PMP isolated from both diseases were significantly enriched in proteins involved in complement activation, while endopeptidase activity was downregulated exclusively in HCC patients. This study reports for the first time a quantitative proteome analysis for PMP from patients with HCV-induced cirrhosis and HCC. Data are available via ProteomeXchange with identifier PXD005777. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Protective Effects of Scutellarin on Human Cardiac Microvascular Endothelial Cells against Hypoxia-Reoxygenation Injury and Its Possible Target-Related Proteins

PubMed Central

Shi, Meina; Liu, Yingting; Feng, Lixing; Cui, Yingbo; Chen, Yajuan; Wang, Peng; Wu, Wenjuan; Chen, Chen; Liu, Xuan; Yang, Weimin

2015-01-01

Scutellarin (SCU) is one of the main components of traditional Chinese medicine plant Erigeron breviscapus (Vant.) Hand.-Mazz. In this paper, we studied the protective effects of SCU on human cardiac microvascular endothelial cells (HCMECs) against hypoxia-reoxygenation (HR) injury and its possible target-related proteins. Results of MTT assay showed that pretreatment of SCU at doses of 1, 5, and 10 μM for 2 h could significantly inhibit the decrease in cell viability of HCMECs induced by HR injury. Subcellular fractions of cells treated with vehicle control, 1 μM SCU, HR injury, or 1 μM SCU + HR injury were separated by ultracentrifugation. The protein expression profiles of cytoplasm and membrane/nuclei fractions were checked using protein two-dimensional electrophoresis (2-DE). Proteins differentially expressed between control and SCU-treated group, control and HR group, or HR and SCU + HR group were identified using mass spectrometry (MS/MS). Possible interaction network of these target-related proteins was predicted using bioinformatic analysis. The influence of SCU on the expression levels of these proteins was confirmed using Western blotting assay. The results indicated that proteins such as p27BBP protein (EIF6), heat shock 60 kDa protein 1 (HSPD1), and chaperonin containing TCP1 subunit 6A isoform (CCT6A) might play important roles in the effects of SCU. PMID:26557144

Protective Effects of Scutellarin on Human Cardiac Microvascular Endothelial Cells against Hypoxia-Reoxygenation Injury and Its Possible Target-Related Proteins.

PubMed

Shi, Meina; Liu, Yingting; Feng, Lixing; Cui, Yingbo; Chen, Yajuan; Wang, Peng; Wu, Wenjuan; Chen, Chen; Liu, Xuan; Yang, Weimin

2015-01-01

Scutellarin (SCU) is one of the main components of traditional Chinese medicine plant Erigeron breviscapus (Vant.) Hand.-Mazz. In this paper, we studied the protective effects of SCU on human cardiac microvascular endothelial cells (HCMECs) against hypoxia-reoxygenation (HR) injury and its possible target-related proteins. Results of MTT assay showed that pretreatment of SCU at doses of 1, 5, and 10 μM for 2 h could significantly inhibit the decrease in cell viability of HCMECs induced by HR injury. Subcellular fractions of cells treated with vehicle control, 1 μM SCU, HR injury, or 1 μM SCU + HR injury were separated by ultracentrifugation. The protein expression profiles of cytoplasm and membrane/nuclei fractions were checked using protein two-dimensional electrophoresis (2-DE). Proteins differentially expressed between control and SCU-treated group, control and HR group, or HR and SCU + HR group were identified using mass spectrometry (MS/MS). Possible interaction network of these target-related proteins was predicted using bioinformatic analysis. The influence of SCU on the expression levels of these proteins was confirmed using Western blotting assay. The results indicated that proteins such as p27BBP protein (EIF6), heat shock 60 kDa protein 1 (HSPD1), and chaperonin containing TCP1 subunit 6A isoform (CCT6A) might play important roles in the effects of SCU.

Influenza Vaccine Manufacturing: Effect of Inactivation, Splitting and Site of Manufacturing. Comparison of Influenza Vaccine Production Processes

PubMed Central

Kon, Theone C.; Onu, Adrian; Berbecila, Laurentiu; Lupulescu, Emilia; Ghiorgisor, Alina; Kersten, Gideon F.; Cui, Yi-Qing; Amorij, Jean-Pierre; Van der Pol, Leo

2016-01-01

The aim of this study was to evaluate the impact of different inactivation and splitting procedures on influenza vaccine product composition, stability and recovery to support transfer of process technology. Four split and two whole inactivated virus (WIV) influenza vaccine bulks were produced and compared with respect to release criteria, stability of the bulk and haemagglutinin recovery. One clarified harvest of influenza H3N2 A/Uruguay virus prepared on 25.000 fertilized eggs was divided equally over six downstream processes. The main unit operation for purification was sucrose gradient zonal ultracentrifugation. The inactivation of the virus was performed with either formaldehyde in phosphate buffer or with beta-propiolactone in citrate buffer. For splitting of the viral products in presence of Tween®, either Triton™ X-100 or di-ethyl-ether was used. Removal of ether was established by centrifugation and evaporation, whereas removal of Triton-X100 was performed by hydrophobic interaction chromatography. All products were sterile filtered and subjected to a 5 months real time stability study. In all processes, major product losses were measured after sterile filtration; with larger losses for split virus than for WIV. The beta-propiolactone inactivation on average resulted in higher recoveries compared to processes using formaldehyde inactivation. Especially ether split formaldehyde product showed low recovery and least stability over a period of five months. PMID:26959983

Isolation of Exosome-Like Nanoparticles and Analysis of MicroRNAs Derived from Coconut Water Based on Small RNA High-Throughput Sequencing.

PubMed

Zhao, Zhehao; Yu, Siran; Li, Min; Gui, Xin; Li, Ping

2018-03-21

In this study, the presence of microRNAs in coconut water was identified by real-time polymerase chain reaction (PCR) based on the results of high-throughput small RNA sequencing. In addition, the differences in microRNA content between immature and mature coconut water were compared. A total of 47 known microRNAs belonging to 25 families and 14 new microRNAs were identified in coconut endosperm. Through analysis using a target gene prediction software, potential microRNA target genes were identified in the human genome. Real-time PCR showed that the level of most microRNAs was higher in mature coconut water than in immature coconut water. Then, exosome-like nanoparticles were isolated from coconut water. After ultracentrifugation, some particle structures were seen in coconut water samples using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate fluorescence staining. Subsequent scanning electron microscopy observation and dynamic light scattering analysis also revealed some exosome-like nanoparticles in coconut water, and the mean diameters of the particles detected by the two methods were 13.16 and 59.72 nm, respectively. In conclusion, there are extracellular microRNAs in coconut water, and their levels are higher in mature coconut water than in immature coconut water. Some exosome-like nanoparticles were isolated from coconut water, and the diameter of these particles was smaller than that of animal-derived exosomes.

Crystal structure of the inactive state of the receiver domain of Spo0A from Paenisporosarcina sp. TG-14, a psychrophilic bacterium isolated from an Antarctic glacier.

PubMed

Lee, Chang Woo; Park, Sun-Ha; Lee, Sung Gu; Shin, Seung Chul; Han, Se Jong; Kim, Han-Woo; Park, Hyun Ho; Kim, Sunghwan; Kim, Hak Jun; Park, Hyun; Park, HaJeung; Lee, Jun Hyuck

2017-06-01

The two-component phosphorelay system is the most prevalent mechanism for sensing and transducing environmental signals in bacteria. Spore formation, which relies on the two-component phosphorelay system, enables the long-term survival of the glacial bacterium Paenisporosarcina sp. TG-14 in the extreme cold environment. Spo0A is a key response regulator of the phosphorelay system in the early stage of spore formation. The protein is composed of a regulatory N-terminal phospho-receiver domain and a DNA-binding C-terminal activator domain. We solved the three-dimensional structure of the unphosphorylated (inactive) form of the receiver domain of Spo0A (PaSpo0A-R) from Paenisporosarcina sp. TG-14. A structural comparison with phosphorylated (active form) Spo0A from Bacillus stearothermophilus (BsSpo0A) showed minor notable differences. A molecular dynamics study of a model of the active form and the crystal structures revealed significant differences in the α4 helix and the preceding loop region where phosphorylation occurs. Although an oligomerization study of PaSpo0A-R by analytical ultracentrifugation (AUC) has shown that the protein is in a monomeric state in solution, both crosslinking and crystal-packing analyses indicate the possibility of weak dimer formation by a previously undocumented mechanism. Collectively, these observations provide insight into the mechanism of phosphorylation-dependent activation unique to Spo0A.

Enhanced Methodologies for Detecting Phenotypic Resistance in Mycobacteria.

PubMed

Hammond, Robert J H; Baron, Vincent O; Lipworth, Sam; Gillespie, Stephen H

2018-01-01

Lipid droplets found in algae and other microscopic organisms have become of interest to many researchers partially because they carry the capacity to produce bio-oil for the mass market. They are of importance in biology and clinical practice because their presence can be a phenotypic marker of an altered metabolism, including reversible resistance to antibiotics, prompting intense research.A useful stain for detecting lipid bodies in the lab is Nile red. It is a dye that exhibits solvatochromism; its absorption band varies in spectral position, shape and intensity with the nature of its solvent environment, it will fluoresce intensely red in polar environment and blue shift with the changing polarity of its solvent. This makes it ideal for the detection of lipid bodies within Mycobacterium spp. This is because mycobacterial lipid bodies' primary constituents are nonpolar lipids such as triacylglycerols but bacterial cell membranes are primarily polar lipid species. In this chapter we describe an optimal method for using Nile red to distinguish lipid containing (Lipid rich or LR cells) from those without lipid bodies (Lipid Poor or LP). As part of the process we have optimized a method for separating LP and LR cells that does not require the use of an ultracentrifuge or complex separation media. We believe that these methods will facilitate further research in these enigmatic, transient and important cell states.

Persistent replication of a hepatitis C virus genotype 1b-based chimeric clone carrying E1, E2 and p6 regions from GB virus B in a New World monkey.

PubMed

Suzuki, Saori; Mori, Ken-Ichi; Higashino, Atsunori; Iwasaki, Yuki; Yasutomi, Yasuhiro; Maki, Noboru; Akari, Hirofumi

2016-01-01

The development of effective hepatitis C virus (HCV) vaccines is essential for the prevention of further HCV dissemination, especially in developing countries. Therefore the aim of this study is to establish a feasible and immunocompetent surrogate animal model of HCV infection that will help in evaluation of the protective efficacy of newly developing HCV vaccine candidates. To circumvent the narrow host range of HCV, an HCV genotype 1b-based chimeric clone carrying E1, E2 and p6 regions from GB virus B (GBV-B), which is closely related to HCV, was generated. The chimera between HCV and GBV-B, named HCV/G, replicated more efficiently as compared with the HCV clone in primary marmoset hepatocytes. Furthermore, it was found that the chimera persistently replicated in a tamarin for more than 2 years after intrahepatic inoculation of the chimeric RNA. Although relatively low (<200 copies/mL), the viral RNA loads in plasma were detectable intermittently during the observation period. Of note, the chimeric RNA was found in the pellet fraction obtained by ultracentrifugation of the plasma at 73 weeks, indicating production of the chimeric virus. Our results will help establish a novel non-human primate model for HCV infection on the basis of the HCV/G chimera in the major framework of the HCV genome. © 2015 The Societies and John Wiley & Sons Australia, Ltd.

Toxicological Profiling of Highly Purified Single-Walled Carbon Nanotubes with Different Lengths in the Rodent Lung and Escherichia Coli.

PubMed

Wang, Xiang; Lee, Jae-Hyeok; Li, Ruibin; Liao, Yu-Pei; Kang, Joohoon; Chang, Chong Hyun; Guiney, Linda M; Mirshafiee, Vahid; Li, Linjiang; Lu, Jianqin; Xia, Tian; Hersam, Mark C; Nel, André E

2018-06-01

Carbon nanotubes (CNTs) exhibit a number of physicochemical properties that contribute to adverse biological outcomes. However, it is difficult to define the independent contribution of individual properties without purified materials. A library of highly purified single-walled carbon nanotubes (SWCNTs) of different lengths is prepared from the same base material by density gradient ultracentrifugation, designated as short (318 nm), medium (789 nm), and long (1215 nm) SWCNTs. In vitro screening shows length-dependent interleukin-1β (IL-1β) production, in order of long > medium > short. However, there are no differences in transforming growth factor-β1 production in BEAS-2B cells. Oropharyngeal aspiration shows that all the SWCNTs induce profibrogenic effects in mouse lung at 21 d postexposure, but there are no differences between tube lengths. In contrast, these SWCNTs demonstrate length-dependent antibacterial effects on Escherichia coli, with the long SWCNT exerting stronger effects than the medium or short tubes. These effects are reduced by Pluronic F108 coating or supplementing with glucose. The data show length-dependent effects on proinflammatory response in macrophage cell line and antibacterial effects, but not on collagen deposition in the lung. These data demonstrate that over the length scale tested, the biological response to highly purified SWCNTs is dependent on the complexity of the nano/bio interface. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Functional Genomics Analysis of Singapore Grouper Iridovirus: Complete Sequence Determination and Proteomic Analysis

PubMed Central

Song, Wen Jun; Qin, Qi Wei; Qiu, Jin; Huang, Can Hua; Wang, Fan; Hew, Choy Leong

2004-01-01

Here we report the complete genome sequence of Singapore grouper iridovirus (SGIV). Sequencing of the random shotgun and restriction endonuclease genomic libraries showed that the entire SGIV genome consists of 140,131 nucleotide bp. One hundred sixty-two open reading frames (ORFs) from the sense and antisense DNA strands, coding for lengths varying from 41 to 1,268 amino acids, were identified. Computer-assisted analyses of the deduced amino acid sequences revealed that 77 of the ORFs exhibited homologies to known virus genes, 23 of which matched functional iridovirus proteins. Forty-two putative conserved domains or signatures were detected in the National Center for Biotechnology Information CD-Search database and PROSITE database. An assortment of enzyme activities involved in DNA replication, transcription, nucleotide metabolism, cell signaling, etc., were identified. Viruses were cultured on a cell line derived from the embryonated egg of the grouper Epinephelus tauvina, isolated, and purified by sucrose gradient ultracentrifugation. The protein extract from the purified virions was analyzed by polyacrylamide gel electrophoresis followed by in-gel digestion of protein bands. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and database searching led to identification of 26 proteins. Twenty of these represented novel or previously unidentified genes, which were further confirmed by reverse transcription-PCR (RT-PCR) and DNA sequencing of their respective RT-PCR products. PMID:15507645

Different populations of Wnt-containing vesicles are individually released from polarized epithelial cells

PubMed Central

Chen, Qiuhong; Takada, Ritsuko; Noda, Chiyo; Kobayashi, Satoru; Takada, Shinji

2016-01-01

Accumulating evidence suggests that exosomes are heterogeneous in molecular composition and physical properties. Here we examined whether epithelial cells secrete a heterogeneous population of exosomes, and if that is the case, whether epithelial cell polarity affects release of different populations of exosomes, especially that of those carrying Wnt. Sucrose-density ultracentrifugation and molecular marker analysis revealed that different populations of exosomes or exosome-like vesicles were released from MDCK cells depending on the cell polarity. Wnt3a associated with these vesicles were detectable in culture media collected from both apical and basolateral sides of the cells. Basolaterally secreted Wnt3a were co-fractionated with a typical exosomal protein TSG101 in fractions having typical exosome densities. In contrast, most of apically secreted Wnt3a, as well as Wnt11, were co-fractionated with CD63 and Hsp70, which are also common to the most exosomes, but recovered in higher density fractions. Wnt3a exhibiting similar floatation behavior to the apically secreted ones were also detectable in the culture media of Wnt3a-expressing L and HEK293 cells. The lipidation of Wnt3a was required for its basolateral secretion in exosomes but was dispensable for the apical one. Thus, epithelial cells release Wnt via distinct populations of vesicles differing in secretion polarity and lipidation dependency. PMID:27765945

First production of fluorescent anti-ribonucleoproteins conjugate for diagnostic of rabies in Brazil.

PubMed

Medeiros Caporale, Graciane Maria; Rodrigues da Silva, Andréa de Cássia; Peixoto, Zélia Maria Pinheiro; Chaves, Luciana Botelho; Carrieri, Maria Luiza; Vassão, Ruth Camargo

2009-01-01

The laboratory tests recommended by the World Health Organization for detection of rabies virus and evaluation of specific antibodies are performed with fluorescent antibodies against the virus, the ribonucleoproteins (RNPs), or by monoclonal antibodies. In this study, we purified the rabies virus RNPs for the production of a conjugate presenting sensibility and specificity compatible with commercial reagents. The method employed for the purification of RNPs was ultracentrifugation in cesium chloride gradient, the obtained product being used for immunizing rabbits, from which the hyperimmune sera were collected. The serum used for conjugate production was the one presenting the highest titer (1/2,560) when tested by indirect immunofluorescence. The antibodies were purified by anion exchange chromatography (QAE-Sephadex A-50),conjugated to fluorescein isothiocyanate and separated by gel filtration (Sephadex G-50). The resulting conjugate presented titers of 1/400 and 1/500 when assayed by direct immunofluorescence (DIF) and simplified fluorescence inhibition microtest, respectively. Sensibility and specificity tests were performed by DIF in 100 central nervous system samples of different animal species, presenting 100% matches when compared with the commercial reagent used as standard, independent of the conservation state of the samples. The quality reached by our conjugate will enable the standardization of this reagent for use by the laboratories performing diagnosis of rabies in Brazil, contributing to the intensification of the epidemiological vigilance and research on this disease. Copyright 2009 Wiley-Liss, Inc.

Diverse oligomeric states of CEACAM IgV domains

DOE PAGES

Bonsor, Daniel A.; Günther, Sebastian; Beadenkopf, Robert; ...

2015-10-19

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) comprise a large family of cell surface adhesion molecules that bind to themselves and other family members to carry out numerous cellular functions, including proliferation, signaling, differentiation, tumor suppression, and survival. They also play diverse and significant roles in immunity and infection. The formation of CEACAM oligomers is caused predominantly by interactions between their N-terminal IgV domains. Although X-ray crystal structures of CEACAM IgV domain homodimers have been described, how CEACAMs form heterodimers or remain monomers is poorly understood. To address this key aspect of CEACAM function, we determined in this paper the crystalmore » structures of IgV domains that form a homodimeric CEACAM6 complex, monomeric CEACAM8, and a heterodimeric CEACAM6–CEACAM8 complex. To confirm and quantify these interactions in solution, we used analytical ultracentrifugation to measure the dimerization constants of CEACAM homodimers and isothermal titration calorimetry to determine the thermodynamic parameters and binding affinities of CEACAM heterodimers. We found the CEACAM6–CEACAM8 heterodimeric state to be substantially favored energetically relative to the CEACAM6 homodimer. Finally, our data provide a molecular basis for the adoption of the diverse oligomeric states known to exist for CEACAMs and suggest ways in which CEACAM6 and CEACAM8 regulate the biological functions of one another, as well as of additional CEACAMs with which they interact, both in cis and in trans.« less

Investigation of the effect of mutations of rat albumin on the binding affinity to the alpha(4)beta(1) integrin antagonist, 4-[1-[3-chloro-4-[N'-(2-methylphenyl)ureido]phenylacetyl]-(4S)-fluoro-(2S)-pyrrolidine-2-yl]methoxybenzoic acid (D01-4582), using recombinant rat albumins.

PubMed

Ito, Takashi; Takahashi, Masayuki; Okazaki, Osamu; Sugiyama, Yuichi

2010-08-02

The authors reported previously rat strain differences in plasma protein binding to alpha(4)beta(1) antagonist D01-4582, resulting in a great strain difference in its pharmacokinetics (19-fold differences in the AUC). The previous study suggested that amino acid changes of V238L and/or T293I in albumin reduced the binding affinity. In order to elucidate the relative significance of these mutations, an expression system was developed to obtain recombinant rat albumins (rRSA) using Pichia pastoris, followed by a binding analysis of four rRSAs by the ultracentrifugation method. The equilibrium dissociation constant (K(d)) of wild-type rRSA was 210 nM, while K(d) of rRSA that carried both V238L and T293I mutations was 974 nM. K(d) of artificial rRSA that carried only V238L was 426 nM, and K(d) of artificial rRSA that carried only T293I was 191 nM. These results suggested that V238L would be more important in the alteration of K(d). However, since none of the single mutations were sufficient to explain the reduction of affinity, the possibility was also suggested that T293I interacted cooperatively to reduce the binding affinity of rat albumin to D01-4582. Further investigation is required to elucidate the mechanism of the possible cooperative interaction.

[The incidence of the pituitary autoantibodies in Addison disease].

PubMed

Gut, Paweł; Kosowicz, Jerzy; Ziemnicka, Katarzyna; Baczyk, Maciej; Sowiński, Jerzy

2008-01-01

Addison disease (primary insufficience of adrenal cortex) characterized by clinical signs and symptoms associated with deficiency of adrenal hormones. The most frequent etiopathogenesis of Addison disease is related with autoimmunization. In sera of Addison patients are detectable autoantibodies against another endocrine glands. The aim of the study was evaluation of pituitary autoantibodies in Addison disease patients using immunoblotting methods. Studies were performed in 19 Addison disease patients, 16 women (age range: 28-63 yrs, median: 43.5 +/- 8.9) and 3 men (age range: 18-45 yrs, median: 30.6 +/- 9.8). All patients presented signs and symptoms typical of primary insufficiency of adrenal cortex. Sera of control subjects were obtained from 10 healthy blood donors, 7 women, 3 men (age range 21-45 yrs, median: 30.6 +/- 7.1). Incidence of pituitary autoantibodies was assessed by polyacrylamide electrophoresis gel and western-blotting. Pituitary microsomes were obtained from human pituitary tissues by ultracentrifugation and solubilisation in 1% desoxycholic acid. In 14 sera from 19 we detected autoantibodies against pituitary microsomal antigen 67 kDa, 12 sera were recting with 60 kDa and 10 sera with 55 kDa. It is important to note that 10 sera were reacting with 67 and 55 kDa, and 9 sera with 55, 60 and 67 kDa. In sera of Addison disease patients autoantibodies against pituitary microsomal antigens can be frequently detected. The most frequent are antibodies against 55, 60 and 67 kDa antigens.

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

Thuy, Nguyen Thanh, E-mail: ngtthuy02@yahoo.com; Huy, Tran Quang, E-mail: huytq@nihe.org.vn; Nga, Phan Thi

We describe the ultrastructure of the NamDinh virus (NDiV), a new member of the order Nidovirales grown in the C6/36 mosquito cell line. Uninfected and NDiV-infected cells were investigated by electron microscopy 24–48 h after infection. The results show that the viral nucleocapsid-like particles form clusters concentrated in the vacuoles, the endoplasmic reticulum, and are scattered in the cytoplasm. Mature virions of NDiV were released as budding particles on the cell surface where viral components appear to lie beneath and along the plasma membrane. Free homogeneous virus particles were obtained by ultracentrifugation on sucrose gradients of culture fluids. The sizemore » of the round-shaped particles with a complete internal structure was 80 nm in diameter. This is the first study to provide information on the morphogenesis and ultrastructure of the first insect nidovirus NDiV, a missing evolutionary link in the emergence of the viruses with the largest RNA genomes. - Highlights: • NamDinh virus (NDiV), a new member of the order Nidovirales was tested in cultured cell line. • The morphogenesis and ultrastructure of NDiV were investigated by electron microscopy. • The viral nucleocapsid-like particles clustered and scattered in the cytoplasm. • NDiVs were released as budding particles on the cell surface. • The size of the viral particles with a complete internal structure was 80 nm in diameter.« less

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

Bonsor, Daniel A.; Günther, Sebastian; Beadenkopf, Robert

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) comprise a large family of cell surface adhesion molecules that bind to themselves and other family members to carry out numerous cellular functions, including proliferation, signaling, differentiation, tumor suppression, and survival. They also play diverse and significant roles in immunity and infection. The formation of CEACAM oligomers is caused predominantly by interactions between their N-terminal IgV domains. Although X-ray crystal structures of CEACAM IgV domain homodimers have been described, how CEACAMs form heterodimers or remain monomers is poorly understood. To address this key aspect of CEACAM function, we determined in this paper the crystalmore » structures of IgV domains that form a homodimeric CEACAM6 complex, monomeric CEACAM8, and a heterodimeric CEACAM6–CEACAM8 complex. To confirm and quantify these interactions in solution, we used analytical ultracentrifugation to measure the dimerization constants of CEACAM homodimers and isothermal titration calorimetry to determine the thermodynamic parameters and binding affinities of CEACAM heterodimers. We found the CEACAM6–CEACAM8 heterodimeric state to be substantially favored energetically relative to the CEACAM6 homodimer. Finally, our data provide a molecular basis for the adoption of the diverse oligomeric states known to exist for CEACAMs and suggest ways in which CEACAM6 and CEACAM8 regulate the biological functions of one another, as well as of additional CEACAMs with which they interact, both in cis and in trans.« less

Structural, Bioinformatic, and In Vivo Analyses of Two Treponema pallidum Lipoproteins Reveal a Unique TRAP Transporter

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

Deka, Ranjit K.; Brautigam, Chad A.; Goldberg, Martin

2012-05-25

Treponema pallidum, the bacterial agent of syphilis, is predicted to encode one tripartite ATP-independent periplasmic transporter (TRAP-T). TRAP-Ts typically employ a periplasmic substrate-binding protein (SBP) to deliver the cognate ligand to the transmembrane symporter. Herein, we demonstrate that the genes encoding the putative TRAP-T components from T. pallidum, tp0957 (the SBP), and tp0958 (the symporter), are in an operon with an uncharacterized third gene, tp0956. We determined the crystal structure of recombinant Tp0956; the protein is trimeric and perforated by a pore. Part of Tp0956 forms an assembly similar to those of 'tetratricopeptide repeat' (TPR) motifs. The crystal structure ofmore » recombinant Tp0957 was also determined; like the SBPs of other TRAP-Ts, there are two lobes separated by a cleft. In these other SBPs, the cleft binds a negatively charged ligand. However, the cleft of Tp0957 has a strikingly hydrophobic chemical composition, indicating that its ligand may be substantially different and likely hydrophobic. Analytical ultracentrifugation of the recombinant versions of Tp0956 and Tp0957 established that these proteins associate avidly. This unprecedented interaction was confirmed for the native molecules using in vivo cross-linking experiments. Finally, bioinformatic analyses suggested that this transporter exemplifies a new subfamily of TPATs (TPR-protein-associated TRAP-Ts) that require the action of a TPR-containing accessory protein for the periplasmic transport of a potentially hydrophobic ligand(s).« less

Formation and biochemical characterization of tube/pelle death domain complexes: critical regulators of postreceptor signaling by the Drosophila toll receptor.

PubMed

Schiffmann, D A; White, J H; Cooper, A; Nutley, M A; Harding, S E; Jumel, K; Solari, R; Ray, K P; Gay, N J

1999-09-07

In Drosophila, the Toll receptor signaling pathway is required for embryonic dorso-ventral patterning and at later developmental stages for innate immune responses. It is thought that dimerization of the receptor by binding of the ligand spätzle causes the formation of a postreceptor activation complex at the cytoplasmic surface of the membrane. Two components of this complex are the adaptor tube and protein kinase pelle. These proteins both have "death domains", protein interaction motifs found in a number of signaling pathways, particularly those involved in apoptotic cell death. It is thought that pelle is bound by tube during formation of the activation complexes, and that this interaction is mediated by the death domains. In this paper, we show using the yeast two-hybrid system that the wild-type tube and pelle death domains bind together. Mutant tube proteins which do not support signaling in the embryo are also unable to bind pelle in the 2-hybrid assay. We have purified proteins corresponding to the death domains of tube and pelle and show that these form corresponding heterodimeric complexes in vitro. Partial proteolysis reveals a smaller core consisting of the minimal death domain sequences. We have studied the tube/pelle interaction with the techniques of surface plasmon resonance, analytical ultracentrifugation and isothermal titration calorimetry. These measurements produce a value of K(d) for the complex of about 0.5 microM.

Structure of the USP15 N-terminal domains: a β-hairpin mediates close association between the DUSP and UBL domains.

PubMed

Harper, Stephen; Besong, Tabot M D; Emsley, Jonas; Scott, David J; Dreveny, Ingrid

2011-09-20

Ubiquitin specific protease 15 (USP15) functions in COP9 signalosome mediated regulation of protein degradation and cellular signaling through catalyzing the ubiquitin deconjugation reaction of a discrete number of substrates. It influences the stability of adenomatous polyposis coli, IκBα, caspase-3, and the human papillomavirus type 16 E6. USP15 forms a subfamily with USP4 and USP11 related through a shared presence of N-terminal "domain present in ubiquitin specific proteases" (DUSP) and "ubiquitin-like" (UBL) domains (DU subfamily). Here we report the 1.5 Å resolution crystal structure of the human USP15 N-terminal domains revealing a 80 Å elongated arrangement with the DU domains aligned in tandem. This architecture is generated through formation of a defined interface that is dominated by an intervening β-hairpin structure (DU finger) that engages in an intricate hydrogen-bonding network between the domains. The UBL domain is closely related to ubiquitin among β-grasp folds but is characterized by the presence of longer loop regions and different surface characteristics, indicating that this domain is unlikely to act as ubiquitin mimic. Comparison with the related murine USP4 DUSP-UBL crystal structure reveals that the main DU interdomain contacts are conserved. Analytical ultracentrifugation, small-angle X-ray scattering, and gel filtration experiments revealed that USP15 DU is monomeric in solution. Our data provide a framework to advance study of the structure and function of the DU subfamily. © 2011 American Chemical Society

Methodological assessment of the reduction of the content of impurities in nimodipine emulsion via the use of 21 amino acid protection

PubMed Central

Xie, Yiqiao; Zhuang, Zhiquan; Zhang, Shu; Xia, Zihua; Chen, De; Fan, Kaiyan; Ren, Jialin; Lin, CuiCui; Chen, Yanzhong; Yang, Fan

2017-01-01

Purpose The present study examined the factors affecting the content of impurities of nimodipine (NMP) emulsion and the associated methods of compound protection. Methods Destructive testing of NMP emulsion and its active pharmaceutical ingredient (API) were conducted, and ultracentrifugation was used to study the content of impurities in two phases. The impurity of NMP was measured under different potential of hydrogen (pH) conditions, antioxidants and pH-adjusting agents. Results Following destruction, the degradation of NMP notably occurred in the basic environment. The consumption of the pH-adjusting agent NaOH was proportional to the production of impurities since the inorganic base and/or acid promoted the degradation of NMP. The organic antioxidants, notably amino acids with an appropriate length of intermediate chain and electron-donating side group, exhibited improved antioxidant effects compared with inorganic antioxidants. The minimal amount of impurities was produced following addition of 0.04% lysine and 0.06% leucine in the aqueous phase and adjustment of the pH to a range of 7.5–8.0 in the presence of acetic acid solution. Conclusion NMP was more prone to degradation in an oxidative environment, in an aqueous phase and/or in the presence of inorganic pH-adjusting agents and antioxidants. The appropriate antioxidant and pH-adjusting agent should be selected according to the chemical structure, while destructive testing of the drug is considered to play the optimal protective effect. PMID:28490879

Methods for the extraction and RNA profiling of exosomes

PubMed Central

Zeringer, Emily; Li, Mu; Barta, Tim; Schageman, Jeoffrey; Pedersen, Ketil Winther; Neurauter, Axl; Magdaleno, Susan; Setterquist, Robert; Vlassov, Alexander V

2013-01-01

AIM: To develop protocols for isolation of exosomes and characterization of their RNA content. METHODS: Exosomes were extracted from HeLa cell culture media and human blood serum using the Total exosome isolation (from cell culture media) reagent, and Total exosome isolation (from serum) reagent respectively. Identity and purity of the exosomes was confirmed by Nanosight® analysis, electron microscopy, and Western blots for CD63 marker. Exosomal RNA cargo was recovered with the Total exosome RNA and protein isolation kit. Finally, RNA was profiled using Bioanalyzer and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) methodology. RESULTS: Here we describe a novel approach for robust and scalable isolation of exosomes from cell culture media and serum, with subsequent isolation and analysis of RNA residing within these vesicles. The isolation procedure is completed in a fraction of the time, compared to the current standard protocols utilizing ultracentrifugation, and allows to recover fully intact exosomes in higher yields. Exosomes were found to contain a very diverse RNA cargo, primarily short sequences 20-200 nt (such as miRNA and fragments of mRNA), however longer RNA species were detected as well, including full-length 18S and 28S rRNA. CONCLUSION: We have successfully developed a set of reagents and a workflow allowing fast and efficient extraction of exosomes, followed by isolation of RNA and its analysis by qRT-PCR and other techniques. PMID:25237619

[Metabolic kinetics of MN9202 in Beagle dog liver microsomes].

PubMed

Yang, Zhi-fu; Zhou, Si-yuan; Mei, Qi-bing; Yang, Tie-hong; Liu, Zhen-guo

2005-11-01

To study the metabolic kinetics of MN9202 in Beagle dog liver microsome. Beagle dog liver microsomes were prepared by using ultracentrifuge method. After incubating 0.4 micromol x L(-1) MN9202 with 1 g x L(-1) microsomes for 30 min at 37 degrees C, the reaction was terminated by adding 0.5 mL alkalization. The RP-HPLC was used to determine the drug in the incubation mixture. The Michaelis-Menten parameters Km, and Vmax in Beagle dog liver microsomes were initially estimated by analyzing Lineweave-Brurk plot. Various selective CYP inhibitors were used to investigate their inhibitory effect on the metabolism of MN9202. The Km, Vmax and CLint of MN9202 were (22.6 +/- 8.0) micromol x L(-1), (0.54 +/- 0.17) micromol x g(-1) x min(-1) and (0.0242 +/- 0.0009) L x g(-1) x min(-1), respectively. The metabolism of MN9202 was significantly inhibited by ketoconazole (Ket) and troleandomycin (Tro) in Beagle dog liver microsomes. Tranylcypromine (Tra) could inhibit the metabolism of drug as well. While other inhibitors showed little inhibitory effect on the metabolism of MN9202. It was shown that CYP3A and CYP2C19 were involved in MN9202 metabolism. The inhibitors of human CYP3A and CYP2C19 may have potential interaction with MN9202, and this can reduce the metabolism rate and increase the toxicity of MN9202.

The mechanism of GTP hydrolysis by dynamin II: a transient kinetic study.

PubMed

Binns, D D; Helms, M K; Barylko, B; Davis, C T; Jameson, D M; Albanesi, J P; Eccleston, J F

2000-06-20

Dynamin II is a 98 kDa protein (870 amino acids) required for the late stages of clathrin-mediated endocytosis. The GTPase activity of dynamin is required for its function in the budding stages of receptor-mediated endocytosis and synaptic vesicle recycling. This activity is stimulated when dynamin self-associates on multivalent binding surfaces, such as microtubules and anionic liposomes. We first investigated the oligomeric state of dynamin II by analytical ultracentrifuge sedimentation equilibrium measurements at high ionic strength and found that it was best described by a monomer-tetramer equilibrium. We then studied the intrinsic dynamin GTPase mechanism by using a combination of fluorescence stopped-flow and HPLC methods using the fluorescent analogue of GTP, mantdGTP (2'-deoxy-3'-O-(N-methylanthraniloyl) guanosine-5'-triphosphate), under the same ionic strength conditions. The results are interpreted as showing that mantdGTP binds to dynamin in a two-step mechanism. The dissociation constant of mantdGTP binding to dynamin, calculated from the ratio of the off-rate to the on-rate (k(off)/k(on)), was 0.5 microM. Cleavage of mantdGTP then occurs to mantdGDP and P(i) followed by the rapid release of mantdGDP and P(i). No evidence of reversibility of hydrolysis was observed. The cleavage step itself is the rate-limiting step in the mechanism. This mechanism more closely resembles that of the Ras family of proteins involved in cell signaling than the myosin ATPase involved in cellular motility.

Characterization of the stability and folding of H2A.Z chromatin particles: implications for transcriptional activation.

PubMed

Abbott, D W; Ivanova, V S; Wang, X; Bonner, W M; Ausió, J

2001-11-09

H2A.Z and H2A.1 nucleosome core particles and oligonucleosome arrays were obtained using recombinant versions of these histones and a native histone H2B/H3/H4 complement reconstituted onto appropriate DNA templates. Analysis of the reconstituted nucleosome core particles using native polyacrylamide gel electrophoresis and DNase I footprinting showed that H2A.Z nucleosome core particles were almost structurally indistinguishable from its H2A.1 or native chicken erythrocyte counterparts. While this result is in good agreement with the recently published crystallographic structure of the H2A.Z nucleosome core particle (Suto, R. K., Clarkson, M J., Tremethick, D. J., and Luger, K. (2000) Nat. Struct. Biol. 7, 1121-1124), the ionic strength dependence of the sedimentation coefficient of these particles exhibits a substantial destabilization, which is most likely the result of the histone H2A.Z-H2B dimer binding less tightly to the nucleosome. Analytical ultracentrifuge analysis of the H2A.Z 208-12, a DNA template consisting of 12 tandem repeats of a 208-base pair sequence derived from the sea urchin Lytechinus variegatus 5 S rRNA gene, reconstituted oligonucleosome complexes in the absence of histone H1 shows that their NaCl-dependent folding ability is significantly reduced. These results support the notion that the histone H2A.Z variant may play a chromatin-destabilizing role, which may be important for transcriptional activation.

Biodegradable nanoparticles loaded with insulin-phospholipid complex for oral delivery: preparation, in vitro characterization and in vivo evaluation.

PubMed

Cui, Fude; Shi, Kai; Zhang, Liqiang; Tao, Anjin; Kawashima, Yoshiaki

2006-08-28

Biodegradable nanoparticles loaded with insulin-phospholipid complex were prepared by a novel reverse micelle-solvent evaporation method, in which soybean phosphatidylcholine (SPC) was employed to improve the liposolubility of insulin, and biodegradable polymers as carrier materials to control drug release. Solubilization study, IR and X-ray diffraction analysis were employed to prove the complex formation. The effects of key parameters such as polymer/SPC weight ratio, organic phase and polymer type on the properties of the nanoparticles were investigated. Spherical particles of 200 nm mean diameter and a narrow size distribution were obtained under optimal conditions. The drug entrapment efficiency was up to 90%. The in vitro drug release was characterized by an initial burst and subsequent delayed release in both pH 6.8 and pH 1.2 dissolution mediums. The specific modality of drug release, i.e., free or SPC-combined, was investigated in the aid of ultracentrifugation and ultrafiltration methods. The influence of polymer type on the drug release was also discussed. The pharmacological effects of the nanoparticles made of PLGA 50/50 (Av.Mw 9500) were further evaluated to confirm their potential suitability for oral delivery. Intragastric administration of the 20 IU/kg nanoparticles reduced fasting plasma glucose levels to 57.4% within the first 8 h of administration and this continued for 12 h. PK/PD analysis indicated that 7.7% of oral bioavailability relative to subcutaneous injection was obtained.

A new method for the extraction and purification of K99 pili from enterotoxigenic Escherichia coli and their characterization.

PubMed Central

Altmann, K; Pyliotis, N A; Mukkur, T K

1982-01-01

It was found that K99 pili from enterotoxigenic Escherichia coli (of bovine origin) could be extracted by treatment with 3M-KSCN solution. The K99 pili were purified by preparative isoelectric focusing to apparent homogeneity as judged by the presence of a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; the molecular weight of this component was calculated to be 12 600 +/- 300. This indicated that the K99 pili were composed of a single subunit. On analytical ultracentrifugation, a single boundary with an s20,w of 12.2 S at a concentration of 0.42 mg/ml was observed. The average length of purified pili at zero concentration was approx. 160 nm and the diameter was 7.4 +/- 0.6 nm. Amino acid analysis of the purified K99 pili revealed that sulphur-containing amino acids, cysteine and methionine, were absent. Aromatic amino acids, phenylalanine and tyrosine, previously reported to be absent [Isaacson (1977) Infect. Immun. 15. 272-279], constituted 7.14% of the total amino acid residues present. On immunoelectrophoresis, purified K99 pili migrated towards the cathode and caused mannose-resistant haemagglutination of horse, but not of sheep or guinea-pig, red blood cells. Pili from enterotoxigenic E. coli of porcine and human origin and from another bacterial species, namely Fusiformis nodosus, could also be extracted by the treatment of respective micro-organisms with 3 M-KSCN. Images PLATE 1 Fig. 3. Fig. 5. PMID:6124240

STUDIES ON PNEUMONIA VIRUS OF MICE (PVM) IN CELL CULTURE

PubMed Central

Harter, Donald H.; Choppin, Purnell W.

1967-01-01

Pneumonia virus of mice (PVM) has been serially propagated in a line of baby hamster kidney (BHK21) cells. A maximum titer of 6.3 x 106 TCID50 per ml was obtained, and there was little variation in yield on serial passage. PVM grown in BHK21 cells was antigenically similar to virus obtained from the mouse lung, but was somewhat less virulent for the mouse after 10 serial passages in these cells. Virus produced by BHK21 cells agglutinated mouse erythrocytes without prior heating or other treatment. Sedimentation of PVM in the ultracentrifuge or precipitation by ammonium sulfate resulted in a loss in infectivity but an increase in hemagglutinating activity, presumably due to disruption of the virus particle. In a potassium tartrate density gradient, the major portion of infective virus sedimented at a density of approximately 1.15, and noninfective hemagglutinin, at a density of approximately 1.13. Stock virus preparations appear to contain a large amount of noninfective hemagglutinin. The replication of PVM was not inhibited by 5-fluoro-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, or 5-iodo-2'-deoxyuridine. Infected cells contained eosinophilic cytoplasmic inclusions which showed the acridine orange staining characteristic of single-stranded RNA. Foci of viral antigen were observed in the cytoplasm of infected cells by fluorescent antibody staining. The results suggest that PVM is an RNA virus that replicates in the cytoplasm. PMID:4165740

An Overview of Magnetic Bearing Technology for Gas Turbine Engines

NASA Technical Reports Server (NTRS)

Clark, Daniel J.; Jansen, Mark J.; Montague, Gerald T.

2004-01-01

The idea of the magnetic bearing and its use in exotic applications has been conceptualized for many years, over a century, in fact. Patented, passive systems using permanent magnets date back over 150 years. More recently, scientists of the 1930s began investigating active systems using electromagnets for high-speed ultracentrifuges. However, passive magnetic bearings are physically unstable and active systems only provide proper stiffness and damping through sophisticated controllers and algorithms. This is precisely why, until the last decade, magnetic bearings did not become a practical alternative to rolling element bearings. Today, magnetic bearing technology has become viable because of advances in micro-processing controllers that allow for confident and robust active control. Further advances in the following areas: rotor and stator materials and designs which maximize flux, minimize energy losses, and minimize stress limitations; wire materials and coatings for high temperature operation; high-speed micro processing for advanced controller designs and extremely robust capabilities; back-up bearing technology for providing a viable touchdown surface; and precision sensor technology; have put magnetic bearings on the forefront of advanced, lubrication free support systems. This paper will discuss a specific joint program for the advancement of gas turbine engines and how it implies the vitality of magnetic bearings, a brief comparison between magnetic bearings and other bearing technologies in both their advantages and limitations, and an examination of foreseeable solutions to historically perceived limitations to magnetic bearing.

The John Charnley Award: an accurate and sensitive method to separate, display, and characterize wear debris: part 1: polyethylene particles.

PubMed

Billi, Fabrizio; Benya, Paul; Kavanaugh, Aaron; Adams, John; Ebramzadeh, Edward; McKellop, Harry

2012-02-01

Numerous studies indicate highly crosslinked polyethylenes reduce the wear debris volume generated by hip arthroplasty acetabular liners. This, in turns, requires new methods to isolate and characterize them. We describe a method for extracting polyethylene wear particles from bovine serum typically used in wear tests and for characterizing their size, distribution, and morphology. Serum proteins were completely digested using an optimized enzymatic digestion method that prevented the loss of the smallest particles and minimized their clumping. Density-gradient ultracentrifugation was designed to remove contaminants and recover the particles without filtration, depositing them directly onto a silicon wafer. This provided uniform distribution of the particles and high contrast against the background, facilitating accurate, automated, morphometric image analysis. The accuracy and precision of the new protocol were assessed by recovering and characterizing particles from wear tests of three types of polyethylene acetabular cups (no crosslinking and 5 Mrads and 7.5 Mrads of gamma irradiation crosslinking). The new method demonstrated important differences in the particle size distributions and morphologic parameters among the three types of polyethylene that could not be detected using prior isolation methods. The new protocol overcomes a number of limitations, such as loss of nanometer-sized particles and artifactual clumping, among others. The analysis of polyethylene wear particles produced in joint simulator wear tests of prosthetic joints is a key tool to identify the wear mechanisms that produce the particles and predict and evaluate their effects on periprosthetic tissues.

A PI 4. 6 peroxidase that specifically crosslinks extensin precursors

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

Upham, B.L; Alizadeh, H.; Ryan, K.J.

1991-05-01

The primary cell wall is a microcomposite of cellulose, pectin, hemicellulose and protein. The warp-weft model of the primary cell wall hypothesize that extensin monomers are intermolecularly crosslinked orthogonal to the cellulose microfibril thus mechanically coupling the major load-bearing polymer: cellulose. Media of tomato cell cultures contains heat labile, peroxide dependent crosslinking activity, as determined by the rate of decrease in monomer concentration analyzed via Superose-6. Isoelectric focusing of tomato cell culture media indicated crosslinking was predominantly in the acidic peroxidase fraction (pI4.6). This peroxidase was partially purified by ultracentrifugation, DEAE-Trisacryl and HPLC-DEAE chromatography techniques resulting in a 90 foldmore » purification and 45% yield. A second acidic peroxidase eluted from the HPLC-DEAE column had 25% of the crosslinking activity of the pI 4.6 peroxidase. Purified basic peroxidase had only 0.7% of the activity of the pI 4.6 peroxidase. The specific activity of the pI 4.6 peroxidase was 5,473 mg extensin crosslinked/min/mg peroxidase. The pI 4.6 peroxidase crosslinked the following extensins: tomato I and II, carrot, Ginkgo II and did not crosslink Ginkgo I, Douglas Fir, Maize, Asparagus I and II, and sugarbeet extensins as well as bovine serum albumin. Comparison of motifs common to extensins that are crosslinked by the pI 4.6 peroxidase may help identify the crosslink domain(s) of extension.« less

The Clp Chaperones and Proteases of the Human Malaria Parasite Plasmodium falciparum

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

Bakkouri, Majida El; Pow, Andre; Mulichak, Anne

The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, weremore » also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.« less

Membrane Vesicles Nucleate Mineralo-organic Nanoparticles and Induce Carbonate Apatite Precipitation in Human Body Fluids*

PubMed Central

Wu, Cheng-Yeu; Martel, Jan; Cheng, Wei-Yun; He, Chao-Chih; Ojcius, David M.; Young, John D.

2013-01-01

Recent studies indicate that membrane vesicles (MVs) secreted by various cells are associated with human diseases, including arthritis, atherosclerosis, cancer, and chronic kidney disease. The possibility that MVs may induce the formation of mineralo-organic nanoparticles (NPs) and ectopic calcification has not been investigated so far. Here, we isolated MVs ranging in size between 20 and 400 nm from human serum and FBS using ultracentrifugation and sucrose gradient centrifugation. The MV preparations consisted of phospholipid-bound vesicles containing the serum proteins albumin, fetuin-A, and apolipoprotein A1; the mineralization-associated enzyme alkaline phosphatase; and the exosome proteins TNFR1 and CD63. Notably, we observed that MVs induced mineral precipitation following inoculation and incubation in cell culture medium. The mineral precipitates consisted of round, mineralo-organic NPs containing carbonate hydroxyapatite, similar to previous descriptions of the so-called nanobacteria. Annexin V-immunogold staining revealed that the calcium-binding lipid phosphatidylserine (PS) was exposed on the external surface of serum MVs. Treatment of MVs with an anti-PS antibody significantly decreased their mineral seeding activity, suggesting that PS may provide nucleating sites for calcium phosphate deposition on the vesicles. These results indicate that MVs may represent nucleating agents that induce the formation of mineral NPs in body fluids. Given that mineralo-organic NPs represent precursors of calcification in vivo, our results suggest that MVs may initiate ectopic calcification in the human body. PMID:23990473

Membrane vesicles nucleate mineralo-organic nanoparticles and induce carbonate apatite precipitation in human body fluids.

PubMed

Wu, Cheng-Yeu; Martel, Jan; Cheng, Wei-Yun; He, Chao-Chih; Ojcius, David M; Young, John D

2013-10-18

Recent studies indicate that membrane vesicles (MVs) secreted by various cells are associated with human diseases, including arthritis, atherosclerosis, cancer, and chronic kidney disease. The possibility that MVs may induce the formation of mineralo-organic nanoparticles (NPs) and ectopic calcification has not been investigated so far. Here, we isolated MVs ranging in size between 20 and 400 nm from human serum and FBS using ultracentrifugation and sucrose gradient centrifugation. The MV preparations consisted of phospholipid-bound vesicles containing the serum proteins albumin, fetuin-A, and apolipoprotein A1; the mineralization-associated enzyme alkaline phosphatase; and the exosome proteins TNFR1 and CD63. Notably, we observed that MVs induced mineral precipitation following inoculation and incubation in cell culture medium. The mineral precipitates consisted of round, mineralo-organic NPs containing carbonate hydroxyapatite, similar to previous descriptions of the so-called nanobacteria. Annexin V-immunogold staining revealed that the calcium-binding lipid phosphatidylserine (PS) was exposed on the external surface of serum MVs. Treatment of MVs with an anti-PS antibody significantly decreased their mineral seeding activity, suggesting that PS may provide nucleating sites for calcium phosphate deposition on the vesicles. These results indicate that MVs may represent nucleating agents that induce the formation of mineral NPs in body fluids. Given that mineralo-organic NPs represent precursors of calcification in vivo, our results suggest that MVs may initiate ectopic calcification in the human body.

Hepatitis E virus capsid protein assembles in 4M urea in the presence of salts.

PubMed

Yang, Chunyan; Pan, Huirong; Wei, Minxi; Zhang, Xiao; Wang, Nan; Gu, Ying; Du, Hailian; Zhang, Jun; Li, Shaowei; Xia, Ningshao

2013-03-01

The hepatitis E virus (HEV) capsid protein has been demonstrated to be able to assemble into particles in vitro. However, this process and the mechanism of protein-protein interactions during particle assembly remain unclear. In this study, we investigated the assembly mechanism of HEV structural protein subunits, the capsid protein p239 (aa368-606), using analytical ultracentrifugation. It was the first to observe that the p239 can form particles in 4M urea as a result of supplementation with salt, including ammonium sulfate [(NH₄)₂SO₄], sodium sulfate (Na₂SO₄), sodium chloride (NaCl), and ammonium chloride (NH₄Cl). Interestingly, it is the ionic strength that determines the efficiency of promoting particle assembly. The assembly rate was affected by temperature and salt concentration. When (NH₄)₂SO₄ was used, assembling intermediates of p239 with sedimentation coefficient values of approximately 5 S, which were mostly dodecamers, were identified for the first time. A highly conserved 28-aa region (aa368-395) of p239 was found to be critical for particle assembly, and the hydrophobic residues Leu³⁷², Leu³⁷⁵, and Leu³⁹⁵ of p239 was found to be critical for particle assembly, which was revealed by site-directed mutagenesis. This study provides new insights into the assembly mechanism of native HEV, and contributes a valuable basis for further investigations of protein assembly by hydrophobic interactions under denaturing conditions. Copyright © 2012 The Protein Society.

Diverse oligomeric states of CEACAM IgV domains

PubMed Central

Bonsor, Daniel A.; Günther, Sebastian; Beadenkopf, Robert; Beckett, Dorothy; Sundberg, Eric J.

2015-01-01

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) comprise a large family of cell surface adhesion molecules that bind to themselves and other family members to carry out numerous cellular functions, including proliferation, signaling, differentiation, tumor suppression, and survival. They also play diverse and significant roles in immunity and infection. The formation of CEACAM oligomers is caused predominantly by interactions between their N-terminal IgV domains. Although X-ray crystal structures of CEACAM IgV domain homodimers have been described, how CEACAMs form heterodimers or remain monomers is poorly understood. To address this key aspect of CEACAM function, we determined the crystal structures of IgV domains that form a homodimeric CEACAM6 complex, monomeric CEACAM8, and a heterodimeric CEACAM6–CEACAM8 complex. To confirm and quantify these interactions in solution, we used analytical ultracentrifugation to measure the dimerization constants of CEACAM homodimers and isothermal titration calorimetry to determine the thermodynamic parameters and binding affinities of CEACAM heterodimers. We found the CEACAM6–CEACAM8 heterodimeric state to be substantially favored energetically relative to the CEACAM6 homodimer. Our data provide a molecular basis for the adoption of the diverse oligomeric states known to exist for CEACAMs and suggest ways in which CEACAM6 and CEACAM8 regulate the biological functions of one another, as well as of additional CEACAMs with which they interact, both in cis and in trans. PMID:26483485

Diverse oligomeric states of CEACAM IgV domains.

PubMed

Bonsor, Daniel A; Günther, Sebastian; Beadenkopf, Robert; Beckett, Dorothy; Sundberg, Eric J

2015-11-03

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) comprise a large family of cell surface adhesion molecules that bind to themselves and other family members to carry out numerous cellular functions, including proliferation, signaling, differentiation, tumor suppression, and survival. They also play diverse and significant roles in immunity and infection. The formation of CEACAM oligomers is caused predominantly by interactions between their N-terminal IgV domains. Although X-ray crystal structures of CEACAM IgV domain homodimers have been described, how CEACAMs form heterodimers or remain monomers is poorly understood. To address this key aspect of CEACAM function, we determined the crystal structures of IgV domains that form a homodimeric CEACAM6 complex, monomeric CEACAM8, and a heterodimeric CEACAM6-CEACAM8 complex. To confirm and quantify these interactions in solution, we used analytical ultracentrifugation to measure the dimerization constants of CEACAM homodimers and isothermal titration calorimetry to determine the thermodynamic parameters and binding affinities of CEACAM heterodimers. We found the CEACAM6-CEACAM8 heterodimeric state to be substantially favored energetically relative to the CEACAM6 homodimer. Our data provide a molecular basis for the adoption of the diverse oligomeric states known to exist for CEACAMs and suggest ways in which CEACAM6 and CEACAM8 regulate the biological functions of one another, as well as of additional CEACAMs with which they interact, both in cis and in trans.

Influence of aggregate size on the binding and activation of the first component of human complement by soluble IgG aggregates.

PubMed Central

Doekes, G; Vanes, L A; Daha, M R

1982-01-01

The interaction between small aggregates of human IgG and the first component of human complement was studied. Stabilized soluble IgG aggregates of restricted size were prepared by heat aggregation of human IgG, followed by sucrose-density ultracentrifugation. Human C1 was isolated in its precursor form by euglobulin precipitation, followed by gel filtration and immunoadsorption. A C1 preparation was obtained of which more than 90% was still in its unactivated form. Soluble aggregates containing 20, 10 or 5 molecules IgG, and monomeric IgG were tested for their ability to bind and to activate C1. The binding of C1 was determined by C1 consumption, whereas the activation of C1 was measured as the increased ability of the C1 preparation to consume purified human C4 after the incubation with the aggregates. The three aggregates tested and monomeric IgG were all able to bind and to activate C1, but the efficiency of both processes markedly increased with increasing aggregate-size. Furthermore, it was found that all four preparations activated an appreciable amount of C1 at concentrations that did not result in any detectable C1 fixation. These results confirm earlier suggestion that C1 can be activated during a short, transient binding to small aggregates or immune complexes that have a low avidity for C1, after which the activated form, C1, is released into the medium. PMID:7068172

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

PubMed

Di Silvio, Desirè; Rigby, Neil; Bajka, Balazs; Mackie, Alan; Baldelli Bombelli, Francesca

2016-06-01

Nanoparticles (NPs) in biological fluids immediately interact with proteins forming a biomolecular corona (PC) that imparts their biological identity. While several studies on the formation of the PC in human plasma have been reported, the PC of orally administrated NPs has been less investigated, mostly in the presence of a food matrix. In fact, food matrixes when digested are subject of several dynamic changes that will certainly affect the PC formed on the NPs. The lack of studies on this topic is clearly related to the difficulty in isolating representative PC NPs from such a complex environment. In this work magnetite NPs were added to in vitro simulated digestion simultaneously with bread and PC NPs were isolated after gastric and duodenal phases by sucrose gradient ultracentrifugation (UC). The PC NPs were characterized in terms of size and protein composition. Translocation studies were then performed on Caco-2 monolayers in a serum free environment and cell morphology was characterized by confocal microscopy. PC NPs isolated from gastric and duodenal phases were different in size, surface charge and protein corona composition. NP cellular uptake was enhanced by the digestive PC inducing morphology changes in the cell monolayer. Overall, in this work we were able to isolate PC NPs from digested fluids in the presence of a food matrix and study their biological response on Caco-2 cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

The chondroitin sulfate/dermatan sulfate 4-O-endosulfatase from marine bacterium Vibrio sp FC509 is a dimeric species: Biophysical characterization of an endosulfatase.

PubMed

Neira, José L; Medina-Carmona, Encarnación; Hernández-Cifre, José G; Montoliu-Gaya, Laia; Cámara-Artigás, Ana; Seffouh, Ilham; Gonnet, Florence; Daniel, Régis; Villegas, Sandra; de la Torre, José García; Pey, Angel L; Li, Fuchuan

2016-12-01

Sulfatases catalyze hydrolysis of sulfate groups. They have a key role in regulating the sulfation states that determine the function of several scaffold molecules. Currently, there are no studies of the conformational stability of endosulfatases. In this work, we describe the structural features and conformational stability of a 4-O-endosulfatase (EndoV) from a marine bacterium, which removes specifically the 4-O-sulfate from chondroitin sulfate/dermatan sulfate. For that purpose, we have used several biophysical techniques, namely, fluorescence, circular dichroism (CD), FTIR spectroscopy, analytical ultracentrifugation (AUC), differential scanning calorimetry (DSC), mass spectrometry (MS), dynamic light scattering (DLS) and size exclusion chromatography (SEC). The protein was a dimer with an elongated shape. EndoV acquired a native-like structure in a narrow pH range (7.0-9.0); it is within this range where the protein shows the maximum of enzymatic activity. The dimerization did not involve the presence of disulphide-bridges as suggested by AUC, SEC and DLS experiments in the presence of β-mercaptoethanol (β-ME). EndoV secondary structure is formed by a mixture of α and β-sheet topology, as judged by deconvolution of CD and FTIR spectra. Thermal and chemical denaturations showed irreversibility and the former indicates that protein did not unfold completely during heating. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Formation of nano/micro-dispersions with improved dissolution properties upon dispersion of ritonavir melt extrudate in aqueous media.

PubMed

Tho, Ingunn; Liepold, Bernd; Rosenberg, Joerg; Maegerlein, Markus; Brandl, Martin; Fricker, Gert

2010-04-16

The objective of the study was to characterise the aqueous dispersions of ritonavir melt extrudates. More specifically to look into the particular system formed when melt extrudate of a poorly soluble drug dissolved in a hydrophilic polymer matrix containing a surfactant is dispersed in an aqueous medium. Melt extrudates with and without ritonavir were studied. The drug containing extrudate was confirmed to be molecular dispersions of drug in a polymer/surfactant matrix. Particulate dispersions were formed in water from both drug and placebo extrudates. The dispersions were investigated with respect to mean particle size and particle size distribution (photon correlation spectroscopy and optical particle counting), surface charge (zeta potential), particle composition (ultracentrifugation), tendency to form aggregates and precipitate (turbidity), in vitro dissolution rate and drug release. It was concluded that dispersion of melt extrudates in aqueous medium give rise to nano/micro-dispersions. The stability of the nano/micro-dispersion is sensitive to anions and may be subjected to association/aggregation/flocculation as time proceeds after preparation of dispersion. Melt extrudate showed improved dissolution rate and drug release properties compared to crystalline raw material. From studies of single components and physical mixtures of the formulation composition it can be concluded that the drug delivery system itself, namely solid dispersion prepared by melt extrusion technology, plays a key role for the formation of the observed particles. 2010 Elsevier B.V. All rights reserved.

Biophysical properties of intrinsically disordered p130Cas substrate domain--implication in mechanosensing.

PubMed

Hotta, Kinya; Ranganathan, Soumya; Liu, Ruchuan; Wu, Fei; Machiyama, Hiroaki; Gao, Rong; Hirata, Hiroaki; Soni, Neelesh; Ohe, Takashi; Hogue, Christopher W V; Madhusudhan, M S; Sawada, Yasuhiro

2014-04-01

Mechanical stretch-induced tyrosine phosphorylation in the proline-rich 306-residue substrate domain (CasSD) of p130Cas (or BCAR1) has eluded an experimentally validated structural understanding. Cellular p130Cas tyrosine phosphorylation is shown to function in areas without internal actomyosin contractility, sensing force at the leading edge of cell migration. Circular dichroism shows CasSD is intrinsically disordered with dominant polyproline type II conformations. Strongly conserved in placental mammals, the proline-rich sequence exhibits a pseudo-repeat unit with variation hotspots 2-9 residues before substrate tyrosine residues. Atomic-force microscopy pulling experiments show CasSD requires minimal extension force and exhibits infrequent, random regions of weak stability. Proteolysis, light scattering and ultracentrifugation results show that a monomeric intrinsically disordered form persists for CasSD in solution with an expanded hydrodynamic radius. All-atom 3D conformer sampling with the TraDES package yields ensembles in agreement with experiment when coil-biased sampling is used, matching the experimental radius of gyration. Increasing β-sampling propensities increases the number of prolate conformers. Combining the results, we conclude that CasSD has no stable compact structure and is unlikely to efficiently autoinhibit phosphorylation. Taking into consideration the structural propensity of CasSD and the fact that it is known to bind to LIM domains, we propose a model of how CasSD and LIM domain family of transcription factor proteins may function together to regulate phosphorylation of CasSD and effect machanosensing.

Low-density-lipoprotein (LDL)-bound flavonoids increase the resistance of LDL to oxidation and glycation under pathophysiological concentrations of glucose in vitro.

PubMed

Wu, Chi-Hao; Lin, Jer-An; Hsieh, Wen-Ching; Yen, Gow-Chin

2009-06-10

The higher susceptibility of low-density lipoprotein (LDL) to oxidation and glycation in diabetes has been shown to be related to poor glycemic control. The aim of this study was to determine whether LDL-bound flavonoids attenuate high-glucose (HG)-mediated LDL oxidation and glycation. For this purpose, human plasma was preincubated with individual flavonoids for 3 h, followed by sequential ultracentrifugation and extensive dialysis to remove unbound flavonoid samples. Enriched LDL was subsequently isolated and challenged for its resistance to oxidation and glycation. Results showed that glucose (5-30 mM) dose-dependently accelerates copper (Cu(2+))-mediated LDL oxidative modification. The enrichment of flavonoids such as luteolin, naringenin, and kaempferol significantly increased the resistance of LDL to oxidation and prevented endogenous alpha-tocopherol consumption caused by HG/Cu(2+) (p < 0.05). The long-term glycation of LDL, which was measured by advanced glycation endproducts (AGEs)-related fluorescence and boronate affinity chromatography, was found to be inhibited by LDL-bound flavonoids in the following order: rutin > luteolin > quercetin > kaempferol > naringenin > catechin approximately EC > naringin. Moreover, a solid-phase extraction system with HPLC-diode array detection provided evidence that flavonoids were bound to LDL particles to a certain extent concurrently facilitating the lipoprotein antioxidant and antiglycation activities. In conclusion, this study supports the hypothesis that HG promoted oxidative and glycative modifications of LDL. This is the first study to show that the introduction of flavonoids into LDL particles protects the lipoprotein against glycotoxin-mediated adverse effects.