Number Of Available Binding Sites Research Articles

Desorption of rare earth elements biosorbed on Euglena mutabilis suspensions and biofilms

AbstractIncreasing demand for rare earth elements (REEs) has stimulated research on novel recovery methods from sources like industrial effluents. Our recent study demonstrated that algal biofilms are good candidates for the adsorption of REEs and that the extracellular polymeric substances (EPS) within the biofilm were a key accumulator of REEs. In this work, we measured the desorption kinetics, extents, and selectivity of REEs from the algae Euglena mutabilis in suspensions and biofilms using aqueous solutions of HCl at pH between 1.5 and 4, and Na2EDTA at pH 5. We examined the potential for reusing suspended and biofilm biomass for repeated adsorption–desorption cycles. The desorption kinetics were similar for suspensions and biofilms, with equilibrium being reached within 20 min. The extent of desorption was higher in biofilms with 86% and 95% desorption, compared to suspensions which had 72% and 74% desorption using HCl and Na2EDTA, respectively. We postulate that the difference between suspensions and biofilms is attributed to the binding sites in the EPS matrix of the biofilm being more readily protonated than those at the algal cell wall surface. Heavy REEs were found to preferentially desorb at pH 4 over lighter REEs. After 3 repeated adsorption–desorption cycles, the adsorption capacity of biofilms increased by 280% and 80% using HCl and Na2EDTA, respectively, compared to an 80% decrease in biosorption capacity for the suspension. The increase in biofilm biosorption capacity was attributed to the simultaneous desorption of divalent cations alongside REEs from the EPS increasing the number of available binding sites.

Modelling ligand depletion for simultaneous affinity and binding site quantification on cells and tissue

The quantification of the number of targets in biological systems is an important parameter to assess the suitability of surface markers as targets for drugs, drug delivery and medical imaging. Likewise, quantifying the interaction with the target in terms of affinity and binding kinetics is essential during drug development. Commonly used approaches to quantify membrane antigens on live cells are based on manual saturation techniques that are labour-intensive, require careful calibration of the generated signal and do not quantify the binding rates. Here, we present how measuring interactions in real-time on live cells and tissue under ligand depletion conditions can be used to simultaneously quantify the kinetic binding parameters as well as the number of available binding sites in a biological system. Suitable assay design was explored with simulated data and feasibility of the method verified with experimental data for exemplary low molecular weight peptide and antibody radiotracers as well as fluorescent antibodies. In addition to revealing the number of accessible target sites and improving the accuracy of binding kinetics and affinities, the presented method does not require knowledge about the absolute signal generated per ligand molecule. This enables a simplified workflow for use with both radioligands and fluorescent binders.

Pediatrics: pharmacokinetics and dose calculation

The pediatrics has various pharmacodynamic, physiological and pharmacokinetic properties as well as growth rate in comparison with adults. Gastric pH is neutral at birth but falls to pH 1-3 within twenty four to forty eight hours after birth. At birth, the blood-brain barrier is still not fully mature and medicinal products perhaps obtain access to the central nervous system with sequence toxicity. Plasma protein attaching of compounds is dependent on the amount of present attaching proteins, the number of available binding sites, the affinity constant of the medicine for the protein(s), and the availability of pathophysiological conditions or endogenous compounds that perhaps change the medicine-protein attaching interaction. Total body water, explained as percentage of body weight, reduces with age, from comparatively 80% in newborns to 60% by 1 year of age. Reversibly, body fat accelerates with age, from one percent to two percent in a preterm neonate to ten percent to fifteen percent in a term neonate and 20 to 25% in a 1-year-old. First-pass metabolism of zidovudine was decrease in the first 14 days of life. At birth, renal blood flow is only 5 to 6% of cardiac output, 15 to 25% by one year of age and reaches adult values after two years of age. Children's dosage depends on factors such as their age and weight, their health status, their respiratory system, and the stage of development of their body systems for drugs metabolism (e.g., liver enzymes) and elimination (e.g., kidneys). Young’s rule can be applied quickly approach a situation in which the patients weight is unknown; this rule cannot be used for newborns and consideration must be made for growth variability in growth at any given age. A young’s rule for calculating the dose of medicine correct for a child by adding twelve to the child’s age, dividing the sum by the child’s age, then dividing the adult dose by the figure obtained, as it expressed beneath: (age in years / age (years) + 12)) × adult dose.

Expression of genes for selected plant aminoacyl-tRNA synthetases in the abiotic stress

Plants, as sessile organisms, have evolved intricate mechanisms to adapt to various environmental changes and challenges. Because various types of stress trigger significant decrease in global translation rates we examined the stress-related expression of aminoacyl-tRNA synthetases (aaRSs), enzymes that participate in the first step of protein biosynthesis. We have analyzed promoters of genes encoding cytosolic seryl-tRNA synthetase (SerRS), cytosolic aspartyl-tRNA synthetase (AspRS) and cytosolic cysteinyl-tRNA synthetase (CysRS) in Arabidopsis thaliana L., and examined SerRS, AspRS and CysRS gene expression in seedlings exposed to different abiotic stressors. Although global translation levels are repressed by stress, our results show that plant aaRSs expression is not decreased by osmotic, salt and heavy metal/cadmium stress. Moreover, during exposure to stress conditions we detected increased AspRS and CysRS transcript levels. SerRS gene expression did not change in stress conditions although participation of SerRS in stress response could be regulated at the protein level. Expression of the examined aaRS genes under stress correlated well with the length of their predicted promoters and the number of available binding sites for the stress related transcription factors. It thus appears that during stress it is important to keep steady state levels of aaRSs for translation of specific stress-related mRNAs and furthermore to rapidly continue with translation when stress conditions cease. Importantly, increased levels of plant aaRSs during stress may serve as a pool of aaRS proteins that can participate directly in stress responses through their noncanonical activities.

Bisphenol A Removal by Adsorption Using Waste Biomass: Isotherm and Kinetic Studies

BPA is categorized as a new emerging pollutant that mimics the structure and function of hormone estrogen for humans and animals. The removal of BPA had been concerned because human exposure toward BPA is widespread, and it is harmful to humans and the ecosystem. The aim of the study was to remove BPA by adsorption technique and understand the application of agricultural waste material as potential adsorbent. The increasing of the surface area of post-adsorption from 4.80 m2/g to 5.83 m2/g indicated that the sulphuric acid treatment responsible for the growth in the porosity of the banana bunches, increase the number of available binding sites and its ion-adsorption capacity. The highest removal of BPA by the banana bunch and coconut bunch was obtained in the following condition: the temperature at 25°, pH 3, dosage at 100 mg, and agitation at 150rpm. The adsorption process was well described with the Langmuir isotherm, while the best correlation with the kinetic study of BPA adsorption was the pseudo-second-order model. The negative values of ΔH° suggested that the adsorption of BPA onto banana bunch (-13.748 J mol−1) and coconut bunch (-5693.67 J mol−1) is exothermic in nature.

Complexation and conformation of lead ion with poly-γ-glutamic acid in soluble state.

Complexation of microbial polymer in soluble state could impact the solubility, mobility, and bioavailability of heavy metals in the environment. The complexation of a bacterial exopolymer, poly-γ-glutamic acid (γ-PGA), with Pb2+ was studied using the polarographic method and circular dichroism measurement in soluble state. The number of available binding sites was determined based on the Chau’s method and was found to be 0.04, 1.12, 3.56 and 4.51 mmol/(g dry weight of γ-PGA) at pH 3.4, 4.2, 5.0 and 6.2, respectively. Further, the number of binding sites was determined based on the Ruzic’s method and was found to be 3.60 and 4.41 mmol/(g dry weight of γ-PGA) for pH 5.0 and 6.2, respectively. The constant (expressed as log K) values were 5.8 and 6.0 at pH 5.0 and 6.2. Compared to biopolymers secreted by other microorganisms, such as extracellular polymeric substances extraction from activated sludge, γ-PGA was a more efficient Pb2+ carrier from pH 5.0 to 6.2. The secondary structure of γ-PGA varied significantly when Pb2+ added. Ca2+ or Mg2+ replace a portion of the adsorbed Pb2+. However, the portion of Pb2+ involved in changing the γ-PGA conformation was not easily replaced by Ca2+ and Mg2+.

Tetrasulfide‐Functionalized Mesoporous Silica on Nanowire Ring Resonators for Detection of Aqueous Lead, Pb(II)

AbstractSilicon‐on‐insulator (SOI) microring resonators coated with tetrasulfide‐functionalized mesoporous silica (MPS) (S4‐MPS) are reported as sensors to detect Pb(II) ions in aqueous solutions from 10 ppb to 1 ppm. The sensors are based on the interaction of the evanescent field of the guided light with the functionalized mesoporous silica films. Upon absorption of metal ions, the resonant wavelengths of the microresonators shift due to the increased refractive index of the film. The sensors are exposed to aqueous solutions of Pb(II) ions at different concentrations and time‐resolved absorption–desorption curves are obtained. The concentrations of Pb(II) ions are determined using the Lorentz–Lorenz model. It is found that two distinct binding sites with different affinities for Pb(II) are present in the S4‐MPS silica films, corresponding to tetrasulfide groups and, likely, silanol groups. The number of available binding sites corresponds to 1/25 of the total number of S4‐groups. Equilibrium constants for absorption of Pb(II) to both binding sites are obtained from the rate constants for the absorption and desorption processes. The initial absorption rates depend linearly on the concentration of Pb(II) and are used to determine the 3σ limit of detection as 58 ppbm in deionized water and 350 ppbm in tap water.

Bioinspired Hierarchically Structured Surfaces for Efficient Capture and Release of Circulating Tumor Cells.

The development of novel bioinspired surfaces with hierarchical micro- and nanoscale topographic structures for efficient capture and release of circulating tumor cells (CTCs) is reported. The capture of CTCs, facilitated by surface-immobilized epithelial cell adhesion molecule antibodies (anti-EpCAM), was shown to be significantly enhanced in novel three-dimensional hierarchically structured surfaces that were fabricated by replicating the natural micro- and nanostructures of rose petals. Under static conditions, these hierarchical capture substrates exhibited up to 6 times higher cell capture ability at concentrations of 100 cells mL-1 in contrast to flat anti-EpCAM-functionalized polydimethylsiloxane (PDMS) surfaces. As indicated by scanning electron microscopy (SEM) and immunofluorescent images, this enhancement can be in large part attributed to the topographical interaction between nanoscale cell surface components and nanostructures on the substrate. Similarly, the increased surface area affords a higher nominal coverage of anti-EpCAM, which increases the number of available binding sites for cell capture. By treating the substrates with the biocompatible reductant glutathione (GSH), up to 85% of the captured cells were released, which displayed over 98% cell viability after culturing on tissue culture polystyrene (TCP) for 24 h. Therefore, these bioinspired hierarchically structured and functionalized substrates can be successfully applied to capture CTCs, as well as release CTCs for subsequent analysis. These findings provide new prospects for designing cell-material interfaces for advanced cell-based biomedical studies in the future.

Self-Assembly Dynamics of Linear Virus-Like Particles: Theory and Experiment.

We experimentally and theoretically studied the self-assembly kinetics of linear virus-like particles (VLPs) consisting of double-stranded DNA and virus-like coat proteins. The polynucleotide acts as a self-assembly template for our proteins with engineered attractive protein-DNA and protein-protein interactions that imitate the physicochemical functionality of virus coat proteins. Inspired by our experimental observations, where we found that VLPs grow from one point onward, our model presumes a nucleation step before subsequent sequential cooperative binding from one of the ends of the polynucleotide. By numerically solving the pertinent reaction rate equations, we investigated the assembly dynamics as a function of the ratio between the number of available binding sites and proteins in the solution, i.e., the stoichiometry of the molecular building blocks. Depending on the stoichiometry, we found monotonic or nonmonotonic assembly kinetics. If the proteins in the solution vastly outnumber the binding sites on all of the polynucleotides, then the assembly kinetics were strictly monotonic and the assembled fraction increases steadily with time. However, if the concentration of proteins and binding sites is equal, then we found an overshoot in the concentration of fully covered polynucleotides. We compared our model with length distributions of two types of VLPs measured by atomic force microscopy imaging and found satisfactory agreement, suggesting that a relatively simple model may be useful in describing the assembly kinetics of chemically complex systems. We furthermore re-evaluated data by Hernandez-Garcia et al. (Nat. Nanotechnol. 2014, 9, 698-702) to include the effect of a finite protein concentration previously ignored. By fitting our model to the experimental data, we were able to pinpoint the sum of the protein-protein and protein-DNA interaction free energies, the binding rate of a protein to the DNA, and the nucleation free energy associated with switching a protein from the solution to the bound conformation. The values that we found for the VLPs are comparable to virus capsid binding energies of linear and spherical viruses.

Sequential switching of binding partners on PCNA during in vitro Okazaki fragment maturation.

The homotrimeric sliding clamp proliferating cell nuclear antigen (PCNA) mediates Okazaki fragment maturation through tight coordination of the activities of DNA polymerase δ (Pol δ), flap endonuclease 1 (FEN1) and DNA ligase I (Lig1). Little is known regarding the mechanism of partner switching on PCNA and the involvement of PCNA's three binding sites in coordinating such processes. To shed new light on PCNA-mediated Okazaki fragment maturation, we developed a novel approach for the generation of PCNA heterotrimers containing one or two mutant monomers that are unable to bind and stimulate partners. These heterotrimers maintain the native oligomeric structure of PCNA and exhibit high stability under various conditions. Unexpectedly, we found that PCNA heterotrimers containing only one functional binding site enable Okazaki fragment maturation by efficiently coordinating the activities of Pol δ, FEN1, and Lig1. The efficiency of switching between partners on PCNA was not significantly impaired by limiting the number of available binding sites on the PCNA ring. Our results provide the first direct evidence, to our knowledge, that simultaneous binding of multiple partners to PCNA is unnecessary, and if it occurs, does not provide significant functional advantages for PCNA-mediated Okazaki fragment maturation in vitro. In contrast to the "toolbelt" model, which was demonstrated for bacterial and archaeal sliding clamps, our results suggest a mechanism of sequential switching of partners on the eukaryotic PCNA trimer during DNA replication and repair.

Retention effect of human saliva on aroma release and respective contribution of salivary mucin and α-amylase

As great differences were observed in the amount of α-amylase in human saliva, there is a need to better understand the effect of this protein alone or in mixture with mucin on aroma compound partitioning. We report the respective role of mucin and α-amylase on the air/liquid partition coefficients of two series of 5 methyl-ketones and 5 ethyl-esters. We confirm that mucin affects the release of aroma compounds and, for the first time, we demonstrate the ability of α-amylase to decrease the release of aroma compounds. For both proteins, we report the involvement of hydrophobic effects. Interestingly, no cumulative effect was observed when both proteins were mixed together in solution. We hypothesize that protein–protein interactions occur between the two proteins and decrease the total number of available binding sites for aroma compounds. The effect of human saliva is also investigated and compared to that of artificial salivas. In the presence of human saliva the release of ketones is lower than in water and slightly higher than in the presence of artificial saliva composed of α-amylase and/or mucin. Esters are more affected by the presence of human saliva than ketones. This observation is due to the presence of an esterase activity in saliva, which activity increases with the hydrophobicity of esters. The difference observed in aroma release between artificial and human salivas could be explained by the presence of other salivary proteins in human saliva.

Reconstitution of a hierarchical +TIP interaction network controlling microtubule end tracking of dynein.

Growing microtubule end regions recruit a variety of proteins collectively termed +TIPs, which confer local functions to the microtubule cytoskeleton. +TIPs form dynamic interaction networks whose behaviour depends on a number of potentially competitive and hierarchical interaction modes. The rules that determine which of the various +TIPs are recruited to the limited number of available binding sites at microtubule ends remain poorly understood. Here we examined how the human dynein complex, the main minus-end-directed motor and an important +TIP (refs, , ), is targeted to growing microtubule ends in the presence of different +TIP competitors. Using a total internal reflection fluorescence microscopy-based reconstitution assay, we found that a hierarchical recruitment mode targets the large dynactin subunit p150Glued to growing microtubule ends via EB1 and CLIP-170 in the presence of competing SxIP-motif-containing peptides. We further show that the human dynein complex is targeted to growing microtubule ends through an interaction of the tail domain of dynein with p150Glued. Our results highlight how the connectivity and hierarchy within dynamic +TIP networks areorchestrated.

Cytotoxicity in the age of nano: The role of fourth period transition metal oxide nanoparticle physicochemical properties

A clear understanding of physicochemical factors governing nanoparticle toxicity is still in its infancy. We used a systematic approach to delineate physicochemical properties of nanoparticles that govern cytotoxicity. The cytotoxicity of fourth period metal oxide nanoparticles (NPs): TiO2, Cr2O3, Mn2O3, Fe2O3, NiO, CuO, and ZnO increases with the atomic number of the transition metal oxide. This trend was not cell-type specific, as observed in non-transformed human lung cells (BEAS-2B) and human bronchoalveolar carcinoma-derived cells (A549). Addition of NPs to the cell culture medium did not significantly alter pH. Physiochemical properties were assessed to discover the determinants of cytotoxicity: (1) point-of-zero charge (PZC) (i.e., isoelectric point) described the surface charge of NPs in cytosolic and lysosomal compartments; (2) relative number of available binding sites on the NP surface quantified by X-ray photoelectron spectroscopy was used to estimate the probability of biomolecular interactions on the particle surface; (3) band-gap energy measurements to predict electron abstraction from NPs which might lead to oxidative stress and subsequent cell death; and (4) ion dissolution. Our results indicate that cytotoxicity is a function of particle surface charge, the relative number of available surface binding sites, and metal ion dissolution from NPs. These findings provide a physicochemical basis for both risk assessment and the design of safer nanomaterials.

Understanding the in vivo uptake kinetics of a phosphatidylethanolamine-binding agent 99mTc-Duramycin

(99m)Tc-Duramycin is a peptide-based molecular probe that binds specifically to phosphatidylethanolamine (PE). The goal was to characterize the kinetics of molecular interactions between (99m)Tc-Duramycin and the target tissue. High level of accessible PE is induced in cardiac tissues by myocardial ischemia (30min) and reperfusion (120min) in Sprague-Dawley rats. Target binding and biodistribution of (99m)Tc-duramycin were captured using SPECT/CT. To quantify the binding kinetics, the presence of radioactivity in ischemic versus normal cardiac tissues was measured by gamma counting at 3, 10, 20, 60 and 180min after injection. A partially inactivated form of (99m)Tc-Duramycin was analyzed in the same fashion. A compartment model was developed to quantify the uptake kinetics of (99m)Tc-Duramycin in normal and ischemic myocardial tissue. (99m)Tc-duramycin binds avidly to the damaged tissue with a high target-to-background radio. Compartment modeling shows that accessibility of binding sites in myocardial tissue to (99m)Tc-Duramycin is not a limiting factor and the rate constant of target binding in the target tissue is at 2.2ml/nmol/min/g. The number of available binding sites for (99m)Tc-Duramycin in ischemic myocardium was estimated at 0.14nmol/g. Covalent modification of D15 resulted in a 9-fold reduction in binding affinity. (99m)Tc-Duramycin accumulates avidly in target tissues in a PE-dependent fashion. Model results reflect an efficient uptake mechanism, consistent with the low molecular weight of the radiopharmaceutical and the relatively high density of available binding sites. These data help better define the imaging utilities of (99m)Tc-Duramycin as a novel PE-binding agent.

Model for Adsorption of Ligands to Colloidal Quantum Dots with Concentration-Dependent Surface Structure

A study of the adsorption equilibrium of solution-phase CdS quantum dots (QDs) and acid-derivatized viologen ligands (N-[1-heptyl],N'-[3-carboxypropyl]-4,4'-bipyridinium dihexafluorophosphate, V(2+)) reveals that the structure of the surfaces of the QDs depends on their concentration. This adsorption equilibrium is monitored through quenching of the photoluminescence of the QDs by V(2+) upon photoinduced electron transfer. When modeled with a simple Langmuir isotherm, the equilibrium constant for QD-V(2+) adsorption, K(a), increases from 6.7 × 10(5) to 8.6 × 10(6) M(-1) upon decreasing the absolute concentration of the QDs from 1.4 × 10(-6) to 5.1 × 10(-8) M. The apparent increase in K(a) upon dilution results from an increase in the mean number of available adsorption sites per QD from 1.1 (for [QD] = 1.4 × 10(-6) M) to 37 (for [QD] = 5.1 × 10(-8) M) through desorption of native ligands from the surfaces of the QDs and through disaggregation of soluble QD clusters. A new model based on the Langmuir isotherm that treats both the number of adsorbed ligands per QD and the number of available binding sites per QD as binomially distributed quantities is described. This model yields a concentration-independent value for K(a) of 8.7 × 10(5) M(-1) for the QD-V(2+) system and provides a convenient means for quantitative analysis of QD-ligand adsorption in the presence of competing surface processes.

Adsorption of low-density lipoprotein, its oxidation, and subsequent binding of specific recombinant antibodies: An in situ ellipsometric study

Background Low-density lipoprotein (LDL) particles accumulate in the arterial wall and become oxidized during atherogenesis, leading to the formation of atherosclerotic plaques. The major protein of the LDL particle, apolipoprotein B-100 (apoB-100), becomes fragmented during oxidation and a target for the immune system. Methods In this study we used in situ ellipsometry to monitor the adsorption of LDL to solid silica surfaces and the effects of oxidation on the structure of the adsorbed LDL layer. We additionally investigated the binding kinetics of two recombinant human antibodies with different specificities recognizing epitopes of apoB-100 in surface-bound native and CuCl 2-oxidized LDL (oxLDL). The latter process was studied by adsorbing LDL and then adding the antibody and CuCl 2 while continuously monitoring adsorbed amount and the thickness of the film. The molar ratios between the antibodies and surface-bound LDL and oxLDL were calculated from these data. Results Our results indicate that oxidation of surface-bound LDL induces swelling of the layer, accompanied by a slight desorption. We further found that both antibodies were able to recognize LDL and oxLDL in its adsorbed orientation. Quantitative information was obtained on the number of available binding sites on surface-bound LDL and oxLDL for these two antibodies. General significance Using ellipsometry for real-time monitoring of adsorption, in situ oxidation of LDL and binding of specific recombinant antibodies to surface-bound LDL, will open up possibilities to map different conformations and orientations of LDL in the adsorbed state.

Quantitating Antibody Uptake In Vivo: Conditional Dependence on Antigen Expression Levels

Antibodies form an important class of cancer therapeutics, and there is intense interest in using them for imaging applications in diagnosis and monitoring of cancer treatment. Despite the expanding body of knowledge describing pharmacokinetic and pharmacodynamic interactions of antibodies in vivo, discrepancies remain over the effect of antigen expression level on tumoral uptake with some reports indicating a relationship between uptake and expression and others showing no correlation. Using a cell line with high epithelial cell adhesion molecule expression and moderate epidermal growth factor receptor expression, fluorescent antibodies with similar plasma clearance were imaged in vivo. A mathematical model and mouse xenograft experiments were used to describe the effect of antigen expression on uptake of these high-affinity antibodies. As predicted by the theoretical model, under subsaturating conditions, uptake of the antibodies in such tumors is similar because localization of both probes is limited by delivery from the vasculature. In a separate experiment, when the tumor is saturated, the uptake becomes dependent on the number of available binding sites. In addition, targeting of small micrometastases is shown to be higher than larger vascularized tumors. These results are consistent with the prediction that high affinity antibody uptake is dependent on antigen expression levels for saturating doses and delivery for subsaturating doses. It is imperative for any probe to understand whether quantitative uptake is a measure of biomarker expression or transport to the region of interest. The data provide support for a predictive theoretical model of antibody uptake, enabling it to be used as a starting point for the design of more efficacious therapies and timely quantitative imaging probes.

Inhibin-A Antagonizes TGFβ2 Signaling by Down-Regulating Cell Surface Expression of the TGFβ Coreceptor Betaglycan

Inhibin is an atypical member of the TGFbeta family of signaling ligands and is classically understood to function via competitive antagonism of activin ligand binding. Inhibin-null (Inha-/-) mice develop both gonadal and adrenocortical tumors, the latter of which depend upon gonadectomy for initiation. We have previously shown that gonadectomy initiates adrenal tumorigenesis in Inha-/- mice by elevating production of LH, which drives aberrant proliferation and differentiation of subcapsular adrenocortical progenitor cells. In this study, we demonstrate that LH signaling specifically up-regulates expression of TGFbeta2 in the subcapsular region of the adrenal cortex, which coincides with regions of aberrant Smad3 activation in Inha-/- adrenal glands. Consistent with a functional interaction between inhibin and TGFbeta2, we further demonstrate that recombinant inhibin-A antagonizes signaling by TGFbeta2 in cultured adrenocortical cells. The mechanism of this antagonism depends upon the mutual affinity of inhibin-A and TGFbeta2 for the signaling coreceptor betaglycan. Although inhibin-A cannot physically displace TGFbeta2 from its binding sites on betaglycan, binding of inhibin-A to the cell surface causes endocytic internalization of betaglycan, thereby reducing the number of available binding sites for TGFbeta2 on the cell surface. The mechanism by which inhibin-A induces betaglycan internalization is clathrin independent, making it distinct from the mechanism by which TGFbeta ligands themselves induce betaglycan internalization. These data indicate that inhibin can specifically antagonize TGFbeta2 signaling in cellular contexts where surface expression of betaglycan is limiting and provide a novel mechanism for activin-independent phenotypes in Inha-/- mice.

Simultaneous multiple element detection by particle beam/hollow cathode-optical emission spectroscopy as a tool for metallomic studies: Determinations of metal binding with apo-transferrin

Particle beam/hollow cathode-optical emission spectroscopy (PB/HC-OES) is presented as a tool for the determination of metal ion loading in transferrin (Tf). The elemental specificity of optical emission spectroscopy provides a means of assessing metal ion concentrations as well as the relative amounts of metal per unit protein concentration (up to 2 moles of Fe per mole of protein). The PB/HC-OES method allows for the simultaneous detection of metal content (Fe (I) 371.99, Ni (I) 341.41 nm, Zn (I) 213.86 nm, and Ag (I) 338.28 nm in this case), as well as elemental carbon and sulfur (C (I) 156.14 nm and S (I) 180.73 nm) that are reflective of the protein composition and concentration. Quantification for the metal species is based on calibration functions derived from aqueous solutions, with limits of detection for the entire suite being less than 1.0 μM. Determinations in this manner eliminate much of the ambiguity inherent in UV-VIS absorbance determinations of Tf metal binding. Validation of this method is obtained by analyzing loading response of Fe(3+) into Tf using the PB/HC-OES method and comparing the results with those of the standard UV-VIS absorbance method. Maximum Fe(3+) loading of Tf (based on the number of available binding sites) was determined to be 71.2 ± 4.7% by the PB/HC-OES method and 67.5 ± 2.5% for the UV-VIS absorbance method. Element emission ratios between the dopant metals and the carbon and sulfur protein constituents allow for concentration independent determinations of metal binding into Tf. Loading percentages were determined for Ni(2+), Zn(2+), and Ag(+) into Tf with maximum loading values of 19.5 ± 0.4%, 41.0 ± 4.4%, and 141.2 ± 4.3%, respectively. While of no apparent biological significance, Ag(+) presents an interesting case as a surrogate for Pt(2+), whose binding with Tf has shown to be quite different from the other metals. A different mode from the others is indeed observed, and is consistent with conjecture on the Pt(2+) mechanisms. Competitive binding studies not easily performed using absorbance spectroscopy are easily performed by simultaneous, multielement analysis, reflective of the metals and the protein content. In this work, there is clear competition between and Fe(3+) and Zn(2+) for binding in the C-terminus lobe of Tf, while Ni(2+) binds within the N-terminus lobe. Addition of Ag(+) to this mixture does not affect the other metals' distributions, but reflects binding at other protein sites.

Effects of aggressive encounters on plasma corticosteroid-binding globulin and its ligands in white-crowned sparrows

In birds, corticosteroid-binding globulin (CBG) binds corticosterone, progesterone and testosterone. The concentration of each ligand can alter the binding of the other ligands through competitive interactions. Thus, an increase in corticosterone or progesterone may displace testosterone bound to CBG, leading to an increase in bioactive free testosterone levels without affecting total testosterone levels in the circulation. Aggressive interactions increase plasma total testosterone levels in some birds but not in others. Here, we tested the hypothesis that aggressive encounters in the late breeding season would not increase total testosterone levels in plasma, but would alter CBG, total corticosterone or total progesterone levels in such a way as to modify the number of available binding sites and therefore occupancy by testosterone. A marked decrease in CBG occupancy by testosterone would indirectly suggest an increase in free testosterone levels in plasma. Wild male white-crowned sparrows were exposed to a simulated territorial intrusion (STI) or control for 30 min. Subjects were then caught and bled. We measured CBG using a ligand-binding assay and corticosterone, progesterone and testosterone using highly sensitive radioimmunoassays. STI significantly increased aggressive behaviors but did not affect plasma total testosterone levels. STI significantly increased plasma CBG and total corticosterone levels and decreased plasma total progesterone levels. We predict that CBG occupancy by corticosterone will increase slightly following an aggressive encounter. However, this small change is unlikely to increase free testosterone levels, because of the large number of seemingly unoccupied CBG binding sites in these subjects.