Density Of Binding Sites Research Articles

Genetic Deletion of Sterol Carrier Protein 2 Selectively Enhances Amygdalar Endocannabinoid Signaling

Sterol carrier protein 2 (SCP-2) is an intracellular lipid transport protein that transports cholesterol, fatty acids and the endocannabinoids (eCBs). Cellular accumulation of the eCBs contributes to inactivation of endocannabinoid signaling (ECS); and we have shown that SCP-2 can facilitate this process. These data lead to the hypothesis that reduction of SCP-2 enhances ECS through increased ligand availability. SCP-2 is expressed in the amygdala, where CB1 cannabinoid receptor (CB1R) signaling regulates extinction of fear. SCP-2 KO mice indeed exhibit enhanced extinction of fear, and this enhancement is reversed by CB1R blockade. However, concentrations of the eCBs anandamide and 2-arachidonoylglycerol (2-AG) were not greater in SCP-2 KO amygdala; in fact, 2-AG concentrations were significantly less than wild type. On the other hand, amygdalar CB1R mRNA expression and binding site density were both significantly increased in the SCP-2 KO mice compared to wild type, suggesting that the hypercannabinergic phenotype is the result of increased CB1R rather than eCB ligand. CB1R expression and binding site density were not increased in hippocampus, prefrontal cortex and cerebellum. These findings suggest that reduced SCP-2 function results in enhanced CB1R expression in the amygdala through an as-yet unknown mechanism. These data have relevance for discovery of processes by which CB1R function can be selectively enhanced in the CNS. Supported by R01 DA09155 and the Advancing a Healthier Wisconsin Endowment at the Medical College of Wisconsin.

Sensitivity Enhancement of Electrolyte–Insulator–Semiconductor Sensors Using Mesotextured and Nanotextured Dielectric Surfaces

We report the fabrication of an electrolyte–insulator–semiconductor (EIS) device with an improved sensitivity brought about by texturization of the functionalized dielectric at the dielectric/electrolyte interface by electrostatic attachment of nanoparticle and mesoparticle. Improvement in sensitivities up to 42% over the nontextured (control) devices was obtained with SiO2 particles on SiO2 dielectric. The sensitivity was a function of the size of the particles with higher sensitivities being obtained with smaller particle sizes. The possible contributions of the chemical modification of the dielectric surface by the aminosilanes resulting in the modification of the surface charge, and of the physical modifications by particle texturization leading likely to enhancement of the surface densities of the active binding sites, were highlighted. A novel design for the EIS reservoir for possible improved circuit integration and manufacturability, involving a two-step reservoir in photoresist was demonstrated. This device was capable of characterizing sample volumes as small as $20~\mu $ L. A $2 \times 1$ and a $2 \times 2$ array was also demonstrated, which could be utilized for multianalyte detection.

Mechanisms Contributing to Lack of Antidepressant Efficacy in Juveniles and Adolescents

Depression is a major health problem where most patients are not effectively treated. This problem is compounded in children where only two antidepressants are clinically approved. Both are selective serotonin (5‐HT) reuptake inhibitors (SSRIs), which are often less efficacious in young populations compared to adults. We found the antidepressant‐like effects of SSRIs in juvenile mice (21 days post‐partum) was reduced relative to adult mice; however, there was no difference in expression of hippocampal 5‐HT transporter (SERT), the target protein of SSRIs, to account for the reduced SSRI efficacy. Increased extracellular 5‐HT after SSRI treatment is thought to trigger downstream events for therapeutic response. Our data suggests transporters capable of 5‐HT uptake other than SERT, such as organic cation transporters (OCTs) and the plasma membrane monoamine transporter (PMAT), may be preventing extracellular 5‐HT from climbing to therapeutically relevant levels after SSRI treatment. The binding site density of [3H]decynium‐22 (D22), an OCTs/PMAT blocker, was greater in juvenile mice than adults. Western blots revealed the greater [3H]D22 binding was likely driven by greater PMAT protein expression in juvenile mice. In our preliminary studies, D22 (0.01mg/kg) produced antidepressant‐like effects in juvenile but not adult mice. Using in vivo chronoamperometry, a technique which measures region specific 5‐HT clearance in brain, studies are underway to determine whether the antidepressant‐like effects of D22 in juvenile and adolescent mice are related to its ability to inhibit 5‐HT clearance.Support: NIH MH093320 / MH064489

Fluorescence Quenching Determination of Uranium (VI) Binding Properties by Two Functional Proteins: Acetylcholinesterase (AChE) and Vitellogenin (Vtg).

The interactions between uranium and two functional proteins (AChE and Vtg) were investigated using fluorescence quenching measurements. The combined use of a microplate spectrofluorometer and logarithmic additions of uranium into protein solutions allowed us to define the fluorescence quenching over a wide range of [U]/[Pi] ratios (from 1 to 3235) at physiologically relevant conditions of pH. Results showed that fluorescence from the two functional proteins was quenched by UO2 (2+). Stoichiometry reactions, fluorescence quenching mechanisms and complexing properties of proteins, i.e. binding constants and binding sites densities, were determined using classic fluorescence quenching methods and curve-fitting software (PROSECE). It was demonstrated that in our test conditions, the protein complexation by uranium could be simulated by two specific sites (L1 and L2). The obtained complexation constant values are log K1 = 5.7 (±1.0), log K2 = 4.9 (±1.1); L1 = 83 (±2), L2 = 2220 (±150) for U(VI) - Vtg and log K1 = 8.1 (±0.9), log K2 = 6.6 (±0.5), L1 = 115 (±16), L2 = 530 (±23) for U(VI)-AChE (Li is expressed in mol/mol of protein).

Electropolymerized molecularly imprinted polypyrrole film for sensing of clofibric acid.

Piezoelectric quartz crystals and analogous gold substrates were electrochemically coated with molecularly imprinted polypyrrole films for pulsed amperometric detection (PAD) of clofibric acid, a metabolite of clofibrate. Cyclic voltammetry data obtained during polymerization and deposited weight estimations revealed a decrease of the polymerization rate with increasing clofibric acid concentration. XPS measurements indicated that clofibric acid could be removed after imprinting with an aqueous ethanol solution, which was further optimized by using PAD. Zeta potential and contact angle measurements revealed differences between molecularly imprinted (MIP) and non-imprinted polymer (NIP) layers. Binding experiments with clofibric acid and other substances showed a pronounced selectivity of the MIP for clofibric acid vs. carbamazepine, but the response of MIP and NIP to 2,4-dichlorophenoxyacetic acid was higher than that for clofibric acid. A smooth surface, revealed by AFM measurements, with roughness of 6–8 nm for imprinted and non-imprinted layers, might be a reason for an excessively low density of specific binding sites for clofibric acid. Furthermore, the decreased polymerization rate in the presence of clofibric acid might not result in well-defined polymer structures, which could be the reason for the lower sensitivity.

Characterisation of bioaccumulation dynamics of three differently coated silver nanoparticles and aqueous silver in a simple freshwater food chain

Environmental context Nanoparticles may be passed from primary producers to predators higher up the food chain, but little is currently known about this transfer. We studied the accumulation dynamics of silver nanoparticles by algae, and then from algae to zooplankton. Using the biodynamic approach, we reconstructed the accumulation process to show that diet is the primary route of uptake for silver nanoparticles. Abstract This study investigated the bioaccumulation dynamics of silver nanoparticles (Ag NPs) with different coatings (polyvinyl pyrrolidone, polyethylene glycol and citrate), in comparison with aqueous Ag (added as AgNO3), in a simplified freshwater food chain comprising the green alga Chlorella vulgaris and the crustacean Daphnia magna. Algal uptake rate constants (ku) and membrane transport characteristics (binding site density, transporter affinity and strength of binding) were determined after exposing algae to a range of either aqueous Ag or Ag NP concentrations. In general, higher ku values were related to higher toxicity in the algae. Transmission electron microscopy images were used to investigate the internalisation of Ag NPs in algal cells following exposure to low concentrations for 72h (mimicking inhibition tests) or high concentrations for 4h (mimicking preparation for daphnia dietary exposure). Ag NPs were only visualised in algal cells exposed to high Ag NP concentrations. To establish D. magna biodynamic model constants, organisms were fed Ag-contaminated algae and depurated for 96h. Assimilation efficiencies ranged from 10 to 25% and the elimination of accumulated Ag followed a two-compartmental model, indicating lower loss rate constants for polyvinyl pyrrolidone-, and polyethylene glycol-coated Ag NPs. Biodynamic model results revealed that in most cases, food is the dominant pathway of Ag uptake in D. magna. Despite the predicted low steady-state body burdens in D. magna, dietary uptake of Ag was possible from aqueous and particulate forms of Ag.

Tuning neuron adhesion and neurite guiding using functionalized AuNPs and backfill chemistry

Gold nanoparticles are used to investigate the dependence of neuron adhesion on the density of cell binding sites and particle backfill. Neurons viability and neurite development depend differently on cell attractive and cell repellant surface cues.

Modelling metal accumulation using humic acid as a surrogate for plant roots

Metal accumulation in roots was modelled with WHAM VII using humic acid (HA) as a surrogate for root surface. Metal accumulation was simulated as a function of computed metal binding to HA, with a correction term (EHA) to account for the differences in binding site density between HA and root surface. The approach was able to model metal accumulation in roots to within one order of magnitude for 95% of the data points. Total concentrations of Mn in roots of Vigna unguiculata, total concentrations of Ni, Zn, Cu and Cd in roots of Pisum sativum, as well as internalized concentrations of Cd, Ni, Pb and Zn in roots of Lolium perenne, were significantly correlated to the computed metal binding to HA. The method was less successful at modelling metal accumulation at low concentrations and in soil experiments. Measured concentrations of Cu internalized in L. perenne roots were not related to Cu binding to HA modelled and deviated from the predictions by over one order of magnitude. The results indicate that metal uptake by roots may under certain conditions be influenced by conditional physiological processes that cannot simulated by geochemical equilibrium. Processes occurring in chronic exposure of plants grown in soil to metals at low concentrations complicate the relationship between computed metal binding to HA and measured metal accumulation in roots.

Cardioprotection by ranolazine in perfused rat heart.

: We used the isolated working rat model to evaluate the effect of therapeutic concentrations (5-10 μM) of ranolazine on contractile performance, oxygen consumption, irreversible ischemic injury, and sarcoplasmic reticulum (SR) function. Ischemic injury was induced by 30 minutes of global ischemia followed by 120 minutes of Langendorff reperfusion and evaluated on the basis of triphenyltetrazolium chloride staining. SR function was determined on the basis of [H]-ryanodine binding, the kinetics of calcium-induced calcium release, measured by quick filtration technique, and oxalate-supported calcium uptake. In working hearts, ranolazine significantly reduced oxygen consumption (P = 0.031), in the absence of significant changes in contractile performance, and decreased irreversible ischemic injury (P = 0.011), if administered either before ischemia-reperfusion (25.4% ± 4.7% vs. 42.7% ± 6.0%) or only at the time of reperfusion (20.2% ± 5.2% vs. 43.7% ± 9.9%). In SR experiments, treatment with ranolazine determined a significant reduction in [H]-ryanodine binding (P = 0.029), because of decreased binding site density (369 ± 9 vs. 405 ± 12 fmol/mg), and in the kinetics of SR calcium release (P = 0.011), whose rate constant was decreased, whereas active calcium uptake was not affected. Ranolazine effectiveness at reperfusion and its ability to module SR calcium release suggest that this drug might be particularly useful to induce cardioprotection during coronary revascularization interventions, although the relevance of the effects on calcium homeostasis remains to be determined.

Developmental dynamics of neurotensin binding sites in the human hypothalamus during the first postnatal year.

The aim of the present study was to determine a detailed mapping of neurotensin (NT) in the human hypothalamus, during the first postnatal year using an in vitro quantitative autoradiography technique and the selective radioligand monoiodo-Tyr3-NT. Ten human postmortem hypothalami obtained from control neonates and infants (aged from 2 h to 1 year of postnatal age) were used. The biochemical kinetics of the binding in all obtained in this study revealed that the binding affinity constants were of high affinity (in the nanomolar range) and did not differ significantly between all cases investigated. Furthermore, competition experiments show insensitivity to levocabastine and were in favor of the presence of the high affinity site of NT receptor. Autoradiographic distribution showed that NT binding sites were widely distributed throughout the rostrocaudal extent of the hypothalamus. However, the distribution of NT binding sites was not homogenous and regional variations exist. In general, the highest densities were mainly present in the anterior hypothalamic level, particularly in the preoptic area. High NT binding site densities are also present at the mediobasal hypothalamic level, particularly in the paraventricular, parafornical, and dorsomedial nuclei. At the posterior level, low to very low densities could be observed in all the mammillary complex subdivisions, as well as the posterior hypothalamic area. Although this topographical distribution is almost identical during the postnatal period analyzed, age-related variations exist in discrete structures of the hypothalamus. The densities were higher in neonates/less aged infants than older infants in preoptic area (medial and lateral parts). The developmental profile is characterized by a progressive decrease from the neonate period to 1 year of postnatal age with a tendency to reach adult levels. On the other hand, the low levels of NT binding sites observed in posterior hypothalamus did not vary during the first postnatal year. They contrast in that with the very high levels we reported previously in adult. In conclusion, the present study demonstrates the occurrence of high NT binding sites density in various structures in many regions in the human neonate/infant hypothalamus, involved in the control of neuroendocrine and/or neurovegetative functions.

Bio-solid-phase extraction/tandem mass spectrometry for identification of bioactive compounds in mixtures.

We describe a two-step column-based bioassay method with tandem mass spectrometric detection for rapid identification of bioactive species in mixtures. The first step uses an immobilized enzyme reactor (IMER) column interfaced to an electrospray ionization mass spectrometer (ESI-MS) to identify mixtures containing bioactive compounds (i.e., enzyme inhibitors), while the second step uses bioselective solid-phase extraction (bioSPE) columns to isolate compounds from "hit" mixtures, which are then identified online by data-dependent ESI-MS. IMER columns were prepared by entrapment of adenosine deaminase (ADA) into sol-gel derived monolithic silica columns, and used to perform a primary IMER screen of mixtures prepared from a bioactive library, which resulted in four apparent hit compounds. Such columns did not provide sufficient binding site density to allow bioSPE, and thus a new column format was developed using ADA that was covalently immobilized to monolithic silica capillary columns, providing ∼500-fold more protein binding sites than were present in columns containing entrapped proteins. Using the covalently linked ADA columns, bioactive mixtures identified by IMER were infused until a maximum total ion current was achieved, followed by washing with a buffer to remove unbound compounds. A harsh wash with 3% acetic acid eluted the strongly bound ligands and the resulting peak triggered data dependent MS/MS to identify the ligand, showing that two of the apparent hits were true ADA inhibitors and demonstrating the ability of this method to rapidly identify bioactive compounds in mixtures.

Distribution of neurotransmitter receptors and zinc in the pigeon (Columba livia) hippocampal formation: A basis for further comparison with the mammalian hippocampus

The avian hippocampal formation (HF) and mammalian hippocampus share a similar functional role in spatial cognition, but the underlying neuronal mechanisms allowing the functional similarity are incompletely understood. To understand better the organization of the avian HF and its transmitter receptors, we analyzed binding site densities for glutamatergic AMPA, NMDA, and kainate receptors; GABAA receptors; muscarinic M1 , M2 and nicotinic (nACh) acetylcholine receptors; noradrenergic α1 and α2 receptors; serotonergic 5-HT1A receptors; dopaminergic D1/5 receptors by using quantitative in vitro receptor autoradiography. Additionally, we performed a modified Timm staining procedure to label zinc. The regionally different receptor densities mapped well onto seven HF subdivisions previously described. Several differences in receptor expression highlighted distinct HF subdivisions. Notable examples include 1) high GABAA and α1 receptor expression, which rendered distinctive ventral subdivisions; 2) high α2 receptor expression, which rendered distinctive a dorsomedial subdivision; 3) distinct kainate, α2 , and muscarinic receptor densities that rendered distinctive the two dorsolateral subdivisions; and 4) a dorsomedial region characterized by high kainate receptor density. We further observed similarities in receptor binding densities between subdivisions of the avian and mammalian HF. Despite the similarities, we propose that 300 hundred million years of independent evolution has led to a mosaic of similarities and differences in the organization of the avian HF and mammalian hippocampus and that thinking about the avian HF in terms of the strict organization of the mammalian hippocampus is likely insufficient to understand the HF of birds.

Effect of meta-chlorobenzhydryl urea (m-ClBHU) on benzodiazepine receptor system in rat brain during experimental alcoholism.

Chronic alcohol intake induces neuroadaptive changes in benzodiazepine receptors modulating GABAA receptors that promote alcohol addiction. Analysis of benzodiazepine receptors in the brain of Wistar rats differing by alcohol preference has demonstrated that affinity of [(3)H]flunitrazepam and [(3)H]Ro5-4864 binding with membrane fraction was reduced, while the density of specific binding sites in the brain cortex of heavy drinking and low drinking rats was increased in comparison with rats nonpreferring alcohol. Administration of anticonvulsant meta-chlorobenzhydryl urea increased affinity of benzodiazepine receptors in the brain cortex of heavy drinking rats, which improved GABA neurotransmission in the brain of these animals and reduced alcohol consumption.

Expression of Adrenoceptor Subtypes in Preterm Piglet Heart Is Different to Term Heart

Preterm delivery increases the risk of inadequate systemic blood flow and hypotension, and many preterm infants fail to respond to conventional inotrope treatments. If the profile of cardiac adrenoceptor subtypes in the preterm neonate is different to that at term this may contribute to these clinical problems. This study measured mRNA expression of β1, β2, α1A, α2A and α2B-adrenoceptor subtypes by real time PCR in term (113d), preterm (91d) and preterm piglets (91d) exposed to maternal glucocorticoid treatment. Abundance of β-adrenoceptor binding sites in the left ventricle was measured using saturation binding assays. Relative abundance of β1-adrenoceptor mRNA in untreated preterm hearts was ∼50% of term abundance in both left and right ventricles (P<0.001). Trends in receptor binding site density measurements supported this observation (P = 0.07). Glucocorticoid exposure increased β1-adrenoceptor mRNA levels in the right ventricle of preterm hearts (P = 0.008) but did not alter expression in the left ventricle (P>0.1). Relative abundance of α1A-adrenoceptor mRNA was the same in preterm and term piglet hearts (P = >0.1) but was reduced by maternal glucocorticoid treatment (P<0.01); α2A-adrenoceptor mRNA abundance was higher in untreated and glucocorticoid exposed preterm piglet hearts than in term piglets (P<0.001). There was no difference between male and female piglets in mRNA abundance of any of the genes studied. In conclusion, there is reduced mRNA abundance of β1-adrenoceptors in the preterm pig heart. If this lower expression of β-adrenoceptors occurs in human preterm infants, it could explain their poor cardiovascular function and their frequent failure to respond to commonly used inotropes.

Critical body residues, Michaelis–Menten analysis of bioaccumulation, lethality and behaviour as endpoints of waterborne Ni toxicity in two teleosts

Traditionally, water quality guidelines/criteria are based on lethality tests where results are expressed as a function of waterborne concentrations (e.g. LC50). However, there is growing interest in the use of uptake and binding relationships, such as biotic ligand models (BLM), and in bioaccumulation parameters, such as critical body residue values (e.g. CBR50), to predict metal toxicity in aquatic organisms. Nevertheless, all these approaches only protect species against physiological death (e.g. mortality, failed recruitment), and do not consider ecological death which can occur at much lower concentrations when the animal cannot perform normal behaviours essential for survival. Therefore, we investigated acute (96h) Ni toxicity in two freshwater fish species, the round goby (Neogobius melanostomus) and rainbow trout (Oncorhynchus mykiss) and compared LC, BLM, and CBR parameters for various organs, as well as behavioural responses (spontaneous activity). In general, round goby were more sensitive. Ni bioaccumulation displayed Michaelis-Menten kinetics in most tissues, and round goby gills had lower Kd (higher binding affinity) but similar Bmax (binding site density) values relative to rainbow trout gills. Round goby also accumulated more Ni than did trout in most tissues at a given exposure concentration. Organ-specific 96h acute CBR values tended to be higher in round goby but 96h acute CBR50 and CBR10 values in the gills were very similar in the two species. In contrast, LC50 and LC10 values were significantly higher in rainbow trout. With respect to BLM parameters, gill log KNiBL values for bioaccumulation were higher by 0.4-0.8 log units than the log KNiBL values for toxicity in both species, and both values were higher in goby (more sensitive). Round goby were also more sensitive with respect to the behavioural response, exhibiting a significant decline of 63-75% in movements per minute at Ni concentrations at and above only 8% of the LC50 value; trout exhibited no clear behavioural response. Across species, diverse behavioral responses may be more closely related to tissue Ni burdens than to waterborne Ni concentrations. To our knowledge, this is the first study to link Ni bioaccumulation with behavioural endpoints. In future it would be beneficial to expand these analyses to a wider range of species to determine whether Ni bioaccumulation, specifically in the gills, gut and whole fish, may be a good predictor of behavioural changes from metal exposure; which in the wild can lead to ecological death.

Hallucinogenic 5-HT2AR agonists LSD and DOI enhance dopamine D2R protomer recognition and signaling of D2-5-HT2A heteroreceptor complexes

Dopamine D2LR-serotonin 5-HT2AR heteromers were demonstrated in HEK293 cells after cotransfection of the two receptors and shown to have bidirectional receptor–receptor interactions. In the current study the existence of D2L-5-HT2A heteroreceptor complexes was demonstrated also in discrete regions of the ventral and dorsal striatum with in situ proximity ligation assays (PLA). The hallucinogenic 5-HT2AR agonists LSD and DOI but not the standard 5-HT2AR agonist TCB2 and 5-HT significantly increased the density of D2like antagonist 3H-raclopride binding sites and significantly reduced the pKiH values of the high affinity D2R agonist binding sites in 3H-raclopride/DA competition experiments. Similar results were obtained in HEK293 cells and in ventral striatum. The effects of the hallucinogenic 5-HT2AR agonists on D2R density and affinity were blocked by the 5-HT2A antagonist ketanserin. In a forskolin-induced CRE-luciferase reporter gene assay using cotransfected but not D2R singly transfected HEK293 cells DOI and LSD but not TCB2 significantly enhanced the D2LR agonist quinpirole induced inhibition of CRE-luciferase activity. Haloperidol blocked the effects of both quinpirole alone and the enhancing actions of DOI and LSD while ketanserin only blocked the enhancing actions of DOI and LSD. The mechanism for the allosteric enhancement of the D2R protomer recognition and signalling observed is likely mediated by a biased agonist action of the hallucinogenic 5-HT2AR agonists at the orthosteric site of the 5-HT2AR protomer. This mechanism may contribute to the psychotic actions of LSD and DOI and the D2-5-HT2A heteroreceptor complex may thus be a target for the psychotic actions of hallunicogenic 5-HT2A agonists.

An autoradiographic study of neurotensin receptors in the human hypothalamus

The aim of the present investigation was to determine a detailed mapping of neurotensin (NT) in the human hypothalamus, the brain region involved in neuroendocrine control. For this, we investigated the presence and the distribution of neurotensin binding sites in the human hypothalamus, using an in vitro quantitative autoradiography technique and the selective radioligand monoiodo-Tyr3-neurotensin (2000Ci/mM). This study was performed on nine adult human postmortem hypothalami. We first determined the biochemical kinetics of the binding and found that binding affinity constants were of high affinity and do not differ significantly between all cases investigated. Our analysis of the autoradiographic distribution shows that NT binding sites are widely distributed throughout the rostrocaudal extent of the hypothalamus. However, the distribution of NT binding sites is not homogenous and regional variations exist. In general, the highest densities are mainly present in the anterior hypothalamic level, particularly in the preoptic region and the anterior boarding limit (i.e. the diagonal band of Broca). Important NT binding site densities are also present at the mediobasal hypothalamic level, particularly in the paraventricular, parafornical and dorsomedial nuclei. At the posterior level, relatively moderate densities could be observed in the mammillary complex subdivisions, apart from the supramammillary nucleus and the posterior hypothalamic area. In conclusion, the present study demonstrates the occurrence of high concentrations of NT binding sites in various structures in many regions in the human adult hypothalamus, involved in the control of neuroendocrine and/or neurovegetative functions.

Avidin as a model for charge driven transport into cartilage and drug delivery for treating early stage post-traumatic osteoarthritis

Local drug delivery into cartilage remains a challenge due to its dense extracellular matrix of negatively charged proteoglycans enmeshed within a collagen fibril network. The high negative fixed charge density of cartilage offers the unique opportunity to utilize electrostatic interactions to augment transport, binding and retention of drug carriers. With the goal of developing particle-based drug delivery mechanisms for treating post-traumatic osteoarthritis, our objectives were, first, to determine the size range of a variety of solutes that could penetrate and diffuse through normal cartilage and enzymatically treated cartilage to mimic early stages of OA, and second, to investigate the effects of electrostatic interactions on particle partitioning, uptake and binding within cartilage using the highly positively charged protein, Avidin, as a model. Results showed that solutes having a hydrodynamic diameter ≤10 nm can penetrate into the full thickness of cartilage explants while larger sized solutes were trapped in the tissue's superficial zone. Avidin had a 400-fold higher uptake than its neutral same-sized counterpart, NeutrAvidin, and >90% of the absorbed Avidin remained within cartilage explants for at least 15 days. We report reversible, weak binding (KD ∼ 150 μm) of Avidin to intratissue sites in cartilage. The large effective binding site density (NT ∼ 2920 μm) within cartilage matrix facilitates Avidin's retention, making its structure suitable for particle based drug delivery into cartilage.

Metal mixture toxicity to aquatic biota in laboratory experiments: Application of the WHAM-FTOX model

The WHAM-FTOX model describes the combined toxic effects of protons and metal cations towards aquatic organisms through the toxicity function (FTOX), a linear combination of the products of organism-bound cation and a toxic potency coefficient (αi) for each cation. Organism-bound, metabolically-active, cation is quantified by the proxy variable, amount bound by humic acid (HA), as predicted by the WHAM chemical speciation model. We compared published measured accumulations of metals by living organisms (bacteria, algae, invertebrates) in different solutions, with WHAM predictions of metal binding to humic acid in the same solutions. After adjustment for differences in binding site density, the predictions were in reasonable line with observations (for logarithmic variables, r2=0.89, root mean squared deviation=0.44), supporting the use of HA binding as a proxy. Calculated loadings of H+, Al, Cu, Zn, Cd, Pb and UO2 were used to fit observed toxic effects in 11 published mixture toxicity experiments involving bacteria, macrophytes, invertebrates and fish. Overall, WHAM-FTOX gave slightly better fits than a conventional additive model based on solution concentrations. From the derived values of αi, the toxicity of bound cations can tentatively be ranked in the order: H<Al<(Zn–Cu–Pb–UO2)<Cd. The WHAM-FTOX analysis indicates much narrower ranges of differences amongst individual organisms in metal toxicity tests than was previously thought. The model potentially provides a means to encapsulate knowledge contained within laboratory data, thereby permitting its application to field situations.

Simultaneous optimal experimental design for in vitro binding parameter estimation

Simultaneous optimization of in vitro ligand binding studies using an optimal design software package that can incorporate multiple design variables through non-linear mixed effect models and provide a general optimized design regardless of the binding site capacity and relative binding rates for a two binding system. Experimental design optimization was employed with D- and ED-optimality using PopED 2.8 including commonly encountered factors during experimentation (residual error, between experiment variability and non-specific binding) for in vitro ligand binding experiments: association, dissociation, equilibrium and non-specific binding experiments. Moreover, a method for optimizing several design parameters (ligand concentrations, measurement times and total number of samples) was examined. With changes in relative binding site density and relative binding rates, different measurement times and ligand concentrations were needed to provide precise estimation of binding parameters. However, using optimized design variables, significant reductions in number of samples provided as good or better precision of the parameter estimates compared to the original extensive sampling design. Employing ED-optimality led to a general experimental design regardless of the relative binding site density and relative binding rates. Precision of the parameter estimates were as good as the extensive sampling design for most parameters and better for the poorly estimated parameters. Optimized designs for in vitro ligand binding studies provided robust parameter estimation while allowing more efficient and cost effective experimentation by reducing the measurement times and separate ligand concentrations required and in some cases, the total number of samples.