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PubMed2000

Actin-latrunculin A structure and function. Differential modulation of actin-binding protein function by latrunculin A.

Latrunculin A is used extensively as an agent to sequester monomeric actin in living cells. We hypothesize that additional activities of latrunculin A may be important for its biological activity. Our data are consistent with the formation of a 1:1 stoichiometric complex with an equilibrium dissociation constant of 0.2 to 0.4 micrometer and provide no evidence that the actin-latrunculin A complex participates in the elongation of actin filaments. Profilin and latrunculin A bind independently to actin, whereas binding of thymosin beta(4) to actin is inhibited by latrunculin A. Potential implications of this differential effect on actin-binding proteins are discussed. From a structural perspective, if latrunculin A binds to actin at a site that sterically influences binding by thymosin beta(4), then the observation that latrunculin A inhibits nucleotide exchange on actin implies an allosteric effect on the nucleotide binding cleft. Alternatively, if, as previously postulated, latrunculin A binds in the nucleotide cleft of actin, then its ability to inhibit binding by thymosin beta(4) is a surprising result that suggests that significant allosteric changes affect the thymosin beta(4) binding site. We show that latrunculin A and actin form a crystalline structure with orthorhombic space group P2(1)2(1)2(1) and diffraction to 3.10 A. A high resolution structure with optimized crystallization conditions should provide insight regarding these remarkable allosteric properties.

Actins↗
PubMed1977

Can one mix two non-specific antisera and obtain a specific radioimmunoassay?

Sekihara et al. have proposed that it is possible to combine two antisera, each of which is unsatisfactory for a clinical radioimmunoassay due to cross-reactivity problems, and obtain an assay which is of sufficiently good specificity for practical application. The present report provides a theoretical analysis of this problem in a "reduced" case of minimal complexity. We assume infinitesimal concentration of tracer, equilibrium of reactants, perfect separation of bound and free, and that each of the two antisera contain only a single class of antibody sites which can bind to the desired ligand or to a cross-reacting species. Numerical methods are used to generate "ideal" dose response curves. The specificity is evaluated by three criteria: 1) as the ratio of ligand concentrations resulting in 10 or 50% reduction of binding of labeled ligand to antibody, i.e. %B/BO = 90 or 50%; 2) the %B/BO or %B/T at an arbitrary dose level; or 3) the apparent amount of ligand present, for an arbitrary dose of crossreacting ligand. Results indicate that mixing of two nonspecific antisera (each cross-reacting with a different ligand) results in a radioimmunoassay system with a specificity intermediate between that obtained with either of the antisera used alone. Whether this will provide a "satisfactory" assay depends on the purposes for which it is intended, the expected concentrations of cross-reacting ligands, etc. Computer simulation studies may be utilized to select the optimal ratio of the two antisera being used for the assay.

Androstenediols↗
PubMed1978

Automated data processing and radioassays.

Radioassays include (1) radioimmunoassays, (2) competitive protein-binding assays based on competition for limited antibody or specific binding protein, (3) immunoradiometric assay, based on competition for excess labeled antibody, and (4) radioreceptor assays. Most mathematical models describing the relationship between labeled ligand binding and unlabeled ligand concentration have been based on the law of mass action or the isotope dilution principle. These models provide useful data reduction programs, but are theoretically unfactory because competitive radioassay usually is not based on classical dilution principles, labeled and unlabeled ligand do not have to be identical, antibodies (or receptors) are frequently heterogenous, equilibrium usually is not reached, and there is probably steric and cooperative influence on binding. An alternative, more flexible mathematical model based on the probability or binding collisions being restricted by the surface area of reactive divalent sites on antibody and on univalent antigen has been derived. Application of these models to automated data reduction allows standard curves to be fitted by a mathematical expression, and unknown values are calculated from binding data. The vitrues and pitfalls are presented of point-to-point data reduction, linear transformations, and curvilinear fitting approaches. A third-order polynomial using the square root of concentration closely approximates the mathematical model based on probability, and in our experience this method provides the most acceptable results with all varieties of radioassays. With this curvilinear system, linear point connection should be used between the zero standard and the beginning of significant dose response, and also towards saturation. The importance is stressed of limiting the range of reported automated assay results to that portion of the standard curve that delivers optimal sensitivity. Published methods for automated data reduction of Scatchard plots for radioreceptor assay are limited by calculation of a single mean K value. The quality of the input data is generally the limiting factor in achieving good precision with automated as it is with manual data reduction. The major advantages of computerized curve fitting include: (1) handling large amounts of data rapidly and without computational error; (2) providing useful quality-control data; (3) indicating within-batch variance of the test results; (4) providing ongoing quality-control charts and between assay variance.

Electronic Data Processing↗
PubMed1996

Improving methods of chordal-sparing mitral valve replacement--Part III: Optimal direction for artificial chordae.

BACKGROUND AND AIMS OF THE STUDY: The optimal direction to preserve artificial chordae tendineae (CT) during mitral valve replacement (MVR) is not known, especially in regard to the response to inotropic stimulation which simulates exercise conditions. METHODS: Using a non-distorting isovolumic balloon technique, we compared left ventricular (LV) systolic and diastolic mechanics in 11 dogs in a control state (no chordal sparing) and with four different methods of chordal preservation: posterior, anterior, oblique (anterior papillary muscle chordae directed anteriorly and others posteriorly, the direction which theoretically augments LV systolic twist), and counter-oblique (counter, chordae preserved in directions opposite to oblique). RESULTS: Before dobutamine, delta Emax from the control was: 0.32 +/- 0.82, 0.10 +/- 0.43, 0.64 +/- 1.07, and 0.51 +/- 0.78 (anterior, posterior, oblique, and counter method, respectively). With dobutamine (3 mg/kg/min), delta Emax (mmHg/ml) was: 0.41 +/- 1.21, -0.13 +/- 0.75, 0.59 +/- 0.82*, and -0.34 +/- 0.71. Before dobutamine, delta LV stiffness (Sd, mmHg/ml) was -0.01 +/- 0.09, -0.02 +/- 0.12, 0.02 +/- 0.10, and 0.01 +/- 0.12; with dobutamine it was 0.01 +/- 0.09, 0.00 +/- 0.15, 0.03 +/- 0.15, and -0.06 +/- 0.11. Similarly, before dobutamine delta LV equilibrium volume (Veq) was -1.2 +/- 3.8, -0.3 +/- 3.0, -0.7 +/- 2.7, and -0.2 +/- 3.5, whereas with dobutamine zeta eq was -0.1 +/- 1.1, -0.4 +/- 0.8, 0.6 +/- 1.7, and -0.4 +/- 1.1. (Mean +/- S.D.; *p = 0.005 posterior and counter by ANOVA; p = NS (< 0.06) versus counter and posterior by ANOVA). CONCLUSIONS: The oblique method enhanced systolic LV function both with and without dobutamine, while a tendency towards better diastolic LV function (Veq) was observed with dobutamine. The anterior method was next best in preserving systolic function, both with and without dobutamine. LV diastolic function tended to deteriorate with dobutamine in the posterior group. Systolic function with the counter method deteriorated with dobutamine. These results warrant further study in an ejecting model to investigate LV systolic and diastolic mechanics with the oblique method of CT preservation, including interactions with LV systolic twist and diastolic recoil.

Animals↗
PubMed2004

p-benzoquinone-benzene clusters as potential nanomechanical devices: a theoretical study.

The equilibrium structures and binding energies of the benzene complexes of p-benzoquinones (PBQ) and its negatively charged anionic species (PBQ- and PBQ2-) have been investigated theoretically using second-order Møller-Plesset calculations. While neutral p-benzoquinone-benzene clusters (PBQ-Bz) prefer to have a parallel displaced geometry (P-c), CH...pi interactions (T-shaped geometries) prevail in the di-anionic PBQ-benzene (PBQ2- -Bz) complexes (T-e2-). Studies on dianionic p-benzoquinone-benzene clusters showed that two nonbonded intermolecular interactions compete in the most stable conformation. One is H-bonding interaction (C-H...O type) between carbonyl oxygen of p-benzoquinone and one of the hydrogen atoms of benzene, and the other is a pi-H interaction between pi-electron cloud of PBQ2- and another hydrogen atom of benzene. Blueshifted H-bonds were observed in T-shaped clusters. The changes in the geometrical preference of PBQ-Bz complex upon addition of electrons would be useful in designing optimized molecular mechanical devices based on the edge-to-face and face-to-face aromatic interactions.

Journal Article↗
PubMed2001

Acid-base physiology.

Acid-base homeostasis involves chemical and physiologic processes responsible for the maintenance of the acidity of body fluids at levels that allow optimal function of the whole individual. The chemical processes represent the first line of defense to an acid or alkali load and include the extracellular and intracellular buffers, whereas the physiologic processes modulate acid-base composition by changes in cellular metabolism and by adaptive responses in the excretion of volatile acids by the lungs and fixed acids by the kidneys. The need for the existence of multiple mechanisms involved in acid-base regulation stems from the critical importance of the hydrogen ion (H+) concentration on the operation of many cellular enzymes and function of vital organs, most prominently the brain and the heart. The task imposed on the mechanisms that maintain acid-base homeostasis is large, since metabolic pathways are continuously consuming or producing H+, and the daily load of waste products for excretion in the form of volatile and fixed acids is substantial. We review the determinants of the acidity of body fluids, the mechanisms that maintain normal acid-base composition, and the overall defense to disruption in acid-base equilibrium. Specific topics include an examination of the scales of acidity, buffer systems, intracellular acid-base regulation, excretion of acids, alkali and acid loading, and normal acid-base composition. The limitations of arterial blood sampling in the assessment of acid-base status are also evaluated.

Acid-Base Equilibrium↗
PubMedDate not supplied

A link-segment model of upright human posture for analysis of head-trunk coordination.

Sensory-motor control of upright human posture may be organized in a top-down fashion such that certain head-trunk coordination strategies are employed to optimize visual and/or vestibular sensory inputs. Previous quantitative models of the biomechanics of human posture control have examined the simple case of ankle sway strategy, in which an inverted pendulum model is used, and the somewhat more complicated case of hip sway strategy, in which multisegment, articulated models are used. While these models can be used to quantify the gross dynamics of posture control, they are not sufficiently detailed to analyze head-trunk coordination strategies that may be crucial to understanding its underlying mechanisms. In this paper, we present a biomechanical model of upright human posture that extends an existing four mass, sagittal plane, link-segment model to a five mass model including an independent head link. The new model was developed to analyze segmental body movements during dynamic posturography experiments in order to study head-trunk coordination strategies and their influence on sensory inputs to balance control. It was designed specifically to analyze data collected on the EquiTest (NeuroCom International, Clackamas, OR) computerized dynamic posturography system, where the task of maintaining postural equilibrium may be challenged under conditions in which the visual surround, support surface, or both are in motion. The performance of the model was tested by comparing its estimated ground reaction forces to those measured directly by support surface force transducers. We conclude that this model will be a valuable analytical tool in the search for mechanisms of balance control.

Biomechanical Phenomena↗
PubMed2006

Tracer monitored titrations: measurement of total alkalinity.

We introduce a new titration methodology, tracer monitored titration (TMT), in which analyses are free of volumetric and gravimetric measurements and insensitive to pump precision and reproducibility. Spectrophotometric monitoring of titrant dilution, rather than volume increment, lays the burden of analytical performance solely on the spectrophotometer. In the method described here, the titrant is a standardized mixture of acid-base indicator and strong acid. Dilution of a pulse of titrant in a titration vessel is tracked using the total indicator concentration measured spectrophotometrically. The concentrations of reacted and unreacted indicator species, derived from Beer's law, are used to calculate the relative proportions of titrant and sample in addition to the equilibrium position (pH) of the titration mixture. Because the method does not require volumetric or gravimetric additions of titrant, simple low-precision pumps can be used. Here, we demonstrate application of TMT for analysis of total alkalinity (A(T)). High-precision, high-accuracy seawater A(T) measurements are crucial for understanding, for example, the marine CaCO3 budget and saturation state, anthropogenic CO2 penetration into the oceans, calcareous phytoplankton blooms, and coral reef dynamics. We present data from 286 titrations on three types of total alkalinity standards: Na2CO3 in 0.7 mol kg x soln(-1) NaCl, NaOH in 0.7 mol kg x soln(-1) NaCl, and a seawater Certified Reference Material (CRM). Based on Na2CO3 standards, the accuracy and precision are +/-0.2 and +/-0.1% (4 and 2 micromol kg x soln(-1) for A(T) approximately 2100-2500 micromol kg x soln(-1), n = 242), using low-precision solenoid pumps to introduce sample and titrant. Similar accuracy and precision were found for analyses run 42 days after the initial experiments. Excellent performance is achieved by optimizing the spectrophotometric detection system and relying upon basic chemical thermodynamics for calculating the equivalence point. Although applied to acid-base titrations in this paper, the approach should be generally applicable to other types of titrations.

Journal Article↗
PubMed2001

Acyclic forms of [1-(13)C]aldohexoses in aqueous solution: quantitation by (13)C NMR and deuterium isotope effects on tautomeric equilibria.

High-resolution (13)C NMR spectra (150 MHz) have been obtained on the complete series of D-aldohexoses (D-allose 1, D-altrose 2, D-galactose 3, D-glucose 4, D-gulose 5, D-idose 6, D-mannose 7, D-talose 8) selectively labeled with (13)C at C1 in order to detect and quantify the percentages of acyclic forms, and to measure and/or confirm percentages of furanoses and pyranoses, in aqueous solution. Aldehyde and hydrate signals were detected for all aldohexoses, and percentages of these forms at 30 degrees C ranged from 0.006 to 0.7% (hydrate) and 0.0032 to 0.09% (aldehyde). Aldehyde percentages are largest for the altro, ido, and talo configurations, ranging from 0.01 to 0.09%; the ido configuration yielded the most hydrate (0.74%). Hydrate/aldehyde ratios vary with aldohexose configuration, ranging from 1.5 to 13, with gluco exhibiting the smallest ratio and gulo the largest. (2)H Equilibrium isotope effects (EIEs) on aldohexose anomerization were measured in D-galactose 3 and D-talose 8 selectively (13)C- and (2)H-labeled at C1 and H1. The (2)H isotope effect on (13)C chemical shift, and broadband (1)H- and (2)H-decoupling, were exploited to permit simultaneous observation and quantitation of the protonated and deuterated molecules in NMR samples containing equimolar mixtures of D-[1-(13)C]aldose and D-[1-(13)C; 1-(2)H]aldose. Small (2)H EIEs were observed for 8, but were undetectable for 3. These results suggest that configuration at C2 influences the magnitude of the (2)H isotope effect at H1 and/or that the observed effect cannot be reliably interpreted due to complications arising from the involvement of acyclic aldehyde forms as intermediates in the interconversion of cyclic forms. The observed (2)H isotope effects on aldohexose tautomeric equilibria provide new insights into the important question of whether (2)H substitutions can alter aldofuranose ring conformation, and lead to the identification of an optimal (2)H- and (13)C-substituted 2-deoxyribofuranose isotopomer on which to investigate this potential effect.

Carbon Isotopes↗
PubMed2006

Complementary use of simulations and molecular-thermodynamic theory to model micellization.

Molecular-thermodynamic descriptions of micellization in aqueous media can be utilized to model the self-assembly of surfactants possessing relatively simple chemical structures, where it is possible to identify a priori what equilibrium position they will adopt in the resulting micellar aggregate. For such chemical structures, the portion of the surfactant molecule that is expected to be exposed to water upon aggregate self-assembly can be identified and used as an input to the molecular-thermodynamic description. Unfortunately, for many surfactants possessing more complex chemical structures, it is not clear a priori how they will orient themselves within a micellar aggregate. In this paper, we present a computational approach to identify what portions of a surfactant molecule are hydrated in a micellar environment through the use of molecular dynamics simulations of such molecules at an oil/water interface (modeling the micelle core/water interface). The local environment of each surfactant segment is determined by counting the number of contacts of each segment with the water and oil molecules. After identifying the hydrated and the unhydrated segments of the surfactant molecule, molecular-thermodynamic modeling can be performed to predict: (i) the free-energy change associated with forming a micellar aggregate, (ii) the critical micelle concentration (CMC), and (iii) the optimal shape and size of the micellar aggregate. The computer simulation results were found to be sensitive to the atomic charge parameters utilized during the simulation runs. Two different methods of assigning atomic charges were tested, and the computer simulation and molecular-thermodynamic modeling results obtained using both sets of atomic charges are presented and compared. The combined computer simulation/molecular-thermodynamic modeling approach presented here is validated first by implementing it in the case of anionic (sodium dodecyl sulfate, SDS), cationic (cetyltrimethylammonium bromide, CTAB), zwitterionic (dodecylphosphocholine, DPC), and nonionic (dodecyl poly(ethylene oxide), C12E8) surfactants possessing relatively simple chemical structures and verifying that good predictions of CMCs and micelle aggregation numbers are obtained. In the case of C12E8, the challenges and limitations associated with simulating a single, polymeric E8 moiety at the oil/water interface to model its behavior at the micelle/water interface are discussed. Subsequently, the combined modeling approach is implemented in the case of the anionic surfactant 3-hydroxy sulfonate (AOS) and of the nonionic surfactant decanoyl-n-methylglucamide (MEGA-10), which possess significantly more complex chemical structures. The good predictions obtained for these two surfactants indicate that the combined computer simulation/molecular-thermodynamic modeling approach presented here extends the range of applicability of molecular-thermodynamic theory to allow modeling of the micellization behavior of surfactants possessing more complex chemical structures.

Micelles↗
PubMed1999

Intraoperative myocardial protection: current trends and future perspectives.

BACKGROUND: The results of contemporary coronary artery bypass graft surgery (CABG) are excellent. However, recently changing trends in the population at risk have necessitated new measures to minimize perioperative morbidity and mortality. METHODS: We reviewed cardioplegic innovations developed, evaluated, and currently employed at the Toronto Hospital. In addition, we conducted an evaluation of novel cardioplegic formulations, with an eye towards future clinical applications. RESULTS: At the Toronto Hospital, we demonstrated that blood provided better protection than crystalloid cardioplegia. Subsequently, we found that a terminal infusion of warm blood cardioplegia repleted myocardial adenosine triphosphate (ATP) levels and improved postoperative ventricular function. Recently, we reported that tepid (29 degrees C) cardioplegia reduced lactate and acid production during cardioplegic arrest, and improved postoperative ventricular function. Combining antegrade and retrograde cardioplegic delivery reduced lactate production, preserved ATP stores, and improved metabolic recovery after cross-clamp release. Cardioplegic flows of at least 200 mL/min were required to washout detrimental metabolic end-products and improve ventricular function. To further optimize myocardial protection, attempts have been made to harness the beneficial effects of ischemic preconditioning using adenosine. Similarly, insulin cardioplegia has been employed in order to enhance ventricular performance by stimulating early postoperative aerobic metabolism. Finally L-arginine, a nitric oxide donor has been demonstrated to be beneficial in experimental studies and may represent a further option for the enhancement of intraoperative myocardial protection. CONCLUSIONS: Despite continued improvements in cardioplegic techniques, low output syndrome following high-risk CABG remains an ongoing concern. The development of novel additives with various protective properties may provide added protection, allowing for a reduction morbidity and mortality following CABG.

Acid-Base Equilibrium↗
PubMed2006

Bioavailability of trace metals to aquatic microorganisms: importance of chemical, biological and physical processes on biouptake.

An important challenge in environmental biogeochemistry is the determination of the bioavailability of toxic and essential trace compounds in natural media. For trace metals, it is now clear that chemical speciation must be taken into account when predicting bioavailability. Over the past 20 years, equilibrium models (free ion activity model (FIAM), biotic ligand model (BLM)) have been increasingly developed to describe metal bioavailability in environmental systems, despite the fact that environmental systems are always dynamic and rarely at equilibrium. In these simple (relatively successful) models, any reduction in the available, reactive species of the metal due to competition, complexation or other reactions will reduce metal bioaccumulation and thus biological effects. Recently, it has become clear that biological, physical and chemical reactions occurring in the immediate proximity of the biological surface also play an important role in controlling trace metal bioavailability through shifts in the limiting biouptake fluxes. Indeed, for microorganisms, examples of biological (transport across membrane), chemical (dissociation kinetics of metal complexes) and physical (diffusion) limitation can be demonstrated. Furthermore, the organism can employ a number of biological internalization strategies to get around limitations that are imposed on it by the physicochemistry of the medium. The use of a single transport site by several metals or the use of several transport sites by a single metal further complicates the prediction of uptake or effects using the simple chemical models. Finally, once inside the microorganism the cell is able to employ a large number of strategies including complexation, compartmentalization, efflux or the production of extracellular ligands to minimize or optimize the reactivity of the metal. The prediction of trace metal bioavailability will thus require multidisciplinary advances in our understanding of the reactions occurring at and near the biological interface. By taking into account medium constraints and biological adaptability, future bioavailability modeling will certainly become more robust.

Bacteria↗
PubMed2004

Hybrid diatomics-in-molecules-based quantum mechanical/molecular mechanical approach applied to the modeling of structures and spectra of mixed molecular clusters Arn(HCl)m and Arn(HF)m.

A new hybrid QM/DIM approach aimed at describing equilibrium structures and spectroscopic properties of medium size mixed molecular clusters is developed. This methodology is applied to vibrational spectra of hydrogen chloride and hydrogen fluoride clusters with up to four monomer molecules embedded in argon shells Arn(H(Cl/F))m (n = 1-62, m = 1-4). The hydrogen halide complexes (QM part) are treated at the MP2/aug-cc-pVTZ level, while the interaction between HX molecules and Ar atoms (MM part) is described in terms of the semiempirical DIM methodology, based on the proper mixing between neutral and ionic states of the system [Grigorenko et al., J. Chem. Phys. 104, 5510 (1996)]. A detailed analysis of the resulting topology of the QM/DIM potential energy (hyper-)surface in the triatomic subsystem Ar-HX reveals more pronounced nonadditive atomic induction and dispersion contributions to the total interaction energy in the case of the Ar-HCl system. An extension of the original analytical DIM-based potential in the frame of the present model as well as the current limitations of the method are discussed. A modified algorithm for the gradient geometry optimization, along with partly analytical force constant matrix evaluation, is developed to treat large cages of argon atoms around molecular clusters. Calculated frequency redshifts of HX stretching vibrations in the mixed clusters relative to the isolated hydrogen-bonded complexes are in good agreement with experimental findings.

Journal Article↗
PubMed1991

Characterization of functional calcitonin gene-related peptide receptors on rat lymphocytes.

Calcitonin gene-related peptide (CGRP), a vasoactive neuropeptide present in peripheral neurons, is released at local sites of inflammation. In these studies specific high affinity adenylyl cyclase linked CGRP receptors were characterized on rat lymphocytes. The distribution, affinity, and specificity of CGRP receptors was analyzed by radioligand binding. 125I-[His10]CGRP binding to rat lymphocytes was rapid, reaching equilibrium by 20 to 30 min at 22 degrees C, and dependent on cell concentration. The dissociation constants, Kd, for the CGRP receptor on purified T and B lymphocytes are 0.807 +/- 0.168 nM and 0.387 +/- 0.072 nM and the densities are 774 +/- 387 and 747 +/- 244 binding sites/cell, respectively. Competition binding studies determined that rat CGRP inhibits 125I-[His10]CGRP binding to lymphocytes with the highest affinity (Ki = 0.192 +/- 0.073) followed by human CGRP and the CGRP receptor antagonist CGRP8-37. 125I-[His10]CGRP binding to rat lymphocytes was not inhibited by the neuropeptides substance P, calcitonin, or neuropeptide Y. Lymphocyte CGRP receptor proteins were identified by affinity labeling by using disuccinimidyl suberate to covalently cross-link 125I-[His10]CGRP to its receptor. Specifically labeled CGRP binding proteins visualized by SDS-PAGE analysis had molecular masses of 74.5 and 220 kDa. A third high molecular mass protein band which did not penetrate the gel was also observed. In functional studies, CGRP stimulated a rapid, sustained increase in cAMP with an ED50 of approximately 8 pM. In experiments comparing optimal concentrations of isoproterenol, a beta 2-adrenergic agonist, and CGRP, intracellular cAMP elevation after isoproterenol treatment returned to basal levels by 30 min, whereas cAMP was still elevated at 60 min after CGRP treatment. The response to CGRP was specific in that it could be completely blocked by CGRP8-37. The presence of high affinity functional CGRP receptors on T and B lymphocytes provides evidence for a modulatory role for CGRP in regulating lymphocyte function.

Adenylyl Cyclases↗
PubMed2005

A control theory approach to the analysis and synthesis of the experimentally observed motion primitives.

Recent experiments on frogs and rats, have led to the hypothesis that sensory-motor systems are organized into a finite number of linearly combinable modules; each module generates a motor command that drives the system to a predefined equilibrium. Surprisingly, in spite of the infiniteness of different movements that can be realized, there seems to be only a handful of these modules. The structure can be thought of as a vocabulary of "elementary control actions". Admissible controls, which in principle belong to an infinite dimensional space, are reduced to the linear vector space spanned by these elementary controls. In the present paper we address some theoretical questions that arise naturally once a similar structure is applied to the control of nonlinear kinematic chains. First of all, we show how to choose the modules so that the system does not loose its capability of generating a "complete" set of movements. Secondly, we realize a "complete" vocabulary with a minimal number of elementary control actions. Subsequently, we show how to modify the control scheme so as to compensate for parametric changes in the system to be controlled. Remarkably, we construct a set of modules with the property of being invariant with respect to the parameters that model the growth of an individual. Robustness against uncertainties is also considered showing how to optimally choose the modules equilibria so as to compensate for errors affecting the system. Finally, the motion primitive paradigm is extended to locomotion and a related formalization of internal (proprioceptive) and external (exteroceptive) variables is given.

Animals↗
PubMed2003

Protein binding in antiretroviral therapies.

There is marked variability in the extent to which the three classes of antiretroviral (ARV) drugs bind to plasma proteins (<5 to >99%). Protease inhibitors (PIs), with the exception of indinavir, are more than 90% protein bound, mainly to alpha1-acid glycoprotein (AAG). Efavirenz, a nonnucleoside reverse transcriptase inhibitor (NNRTI), is more than 99% bound, mainly to albumin. Nucleoside reverse transcriptase inhibitors (NRTIs) are not highly protein bound. The pharmacological activity of ARV drugs is dependent on unbound drug entering cells that harbor the human immunodeficiency virus (HIV). There has been concern that changes in protein binding could impact on antiviral activity and management. However, for PIs and NNRTIs, and for many drugs given orally, altered plasma binding would not be expected to influence the average exposure to unbound (active) drug after chronic oral dosing. Nevertheless, there will be a change in the relationship between total and unbound concentrations that will be important if, as part of therapeutic drug monitoring, the total rather than the unbound drug is measured. Measuring drug concentrations that are needed to inhibit different HIV strains (wild type and drug resistant) in vitro could also cause confusion because most methods employ bovine serum in the assay medium, and unbound concentrations are not directly measured. Estimating unbound drug concentrations in human plasma and in incubation media can be highly method dependent and thus may affect the calculated IC50 (the concentration of drug that results in 50% inhibition of viral replication). Because inhibitory quotients (IQs = C(trough)/IC50) are becoming part of pharmacokinetic/pharmacodynamic (PK/PD) analyses of clinical trial data, the strengths and weaknesses of the methods used for the determination of unbound drug concentration in plasma and in vitro systems--ultracentrifugation, ultrafiltration, and equilibrium dialysis--need to be understood. Consensus on standard procedures must be reached. In June 2002, a panel of experts assembled by the Forum for Collaborative HIV Research met in Washington, DC, to review the basic principles of protein binding of ARV drugs, and to discuss the impact that changes in plasma protein binding may have on the PKs and activity of ARV drugs as well as on therapeutic drug monitoring. The purpose of the meeting was to discuss the following topics: (1) basic principles of protein binding and how changes in binding can impact on drug PKs and drug exposure in vivo, (2) variability in plasma protein binding among patients taking ARV drugs, (3) the impact of HIV infection and concomitant diseases on the extent of plasma protein binding, (4) the likelihood of clinically relevant drug interactions at the level of plasma protein binding, (5) the evidence that measuring unbound concentrations of ARV drugs in the plasma of patients gives more meaningful information than total drug concentration and, therefore, should be considered in routine therapeutic drug monitoring of ARV agents, (6) optimal method(s) for measuring the unbound concentration of drugs in vitro (for IC50 determination) and in vivo, and (7) future studies that need to be considered to fully understand the importance of plasma protein binding in therapeutic drug monitoring. This report summarizes the topics discussed at this meeting. It guides the reader through the discussions that allowed the panel to formulate a series of statements regarding the significance of plasma protein binding of ARV drugs when studied in vitro and in vivo. The roundtable participants also identified research priorities that are important for understanding the sources of inter- and intraindividual variability in protein binding in patients. These include obtaining data on unbound as well as on total concentrations in PK studies; looking at variants of AAG and whether they differ in binding affinity; and emphasizing the importance of developing a standard procedure for drug susceptibility assays used to determine IC50 values.

Animals↗
PubMed1997

Reappraisal of indications and limitations of oxime therapy in organophosphate poisoning.

1 In vitro studies with human erythrocyte acetylcholinesterase (AChE) and the mouse diaphragm model were performed to unravel the various microscopic reaction parameters that contribute to the dynamic equilibrium of AChE inhibition, ageing and reactivation. These data may help to define more precisely the indications and limitations of oxime therapy in organophosphate (OP) poisoning. 2 Diethylphosphoryl-AChE resulting from intoxications with parathion, chlorpyrifos, chlorfenvinphos, diazinon and other OPs is characterized by slow spontaneous reactivation and low propensity for ageing. This kind of phosphorylated enzyme is particularly susceptible to reactivation by oximes. 3 None of the oximes tested (pralidoxime, obidoxime, HI 6 and HLö 7) can be regarded as a universally suitable reactivator. Obidoxime turned out to be the most potent and most efficacious oxime in reactivating AChE inhibited by various classes of OP insecticides and tabun. Obidoxime, however, was inferior to HI 6 against soman, sarin, cyclosarin and VX. Pralidoxime was generally less potent. 4 The kinetic data of reactivation established for diethylphosphoryl-AChE of human red cells indicate that the usually recommended dosage to attain a plasma concentration of 4 micrograms/ml does not permit exploitation of the full therapeutic potential of the oximes, in particular of pralidoxime. However, in suicidal mega-dose poisoning, oximes, even at optimal plasma concentrations, may be unable to cope with the fast re-inhibition of reactivated AChE in the first days following intoxication. 5 It is suggested that oximes be administered by continuous infusion following an initial bolus dose as long as reactivation can be expected and until permanent clinical improvement is achieved.

Acetylcholinesterase↗
PubMed1986

Homogeneous apoenzyme reactivation immunoassay for thyroxin-binding globulin in serum.

In this automated apoenzyme reactivation immunoassay system (Ames Optimate) for thyroxin-binding globulin (TBG), the sample and N6-aminohexylflavin adenine dinucleotide-labeled TBG react sequentially with antiserum. Then apoglucose oxidase is added to combine with the free fraction and generate glucose oxidase activity, which is measured colorimetrically. The assay requires 100 microL of sample and covers the clinically significant range for TBG (less than 2.5 to 55 mg/L). The first result is obtained in 16 min; assay of 29 samples and their blanks is completed in less than 1 h. The lower limit of detection is about 2.5 mg/L. Between-assay CVs (n = 9) were less than 9%, within-assay CVs (n = 5) were less than 6%, and analytical recovery of TBG was 103-112%. Reagents are stable at 4 degrees C for at least five months. Results by this method for serum TBG (y) compared well with those determined by radioimmunoassay (x): y = 1.029x--0.352 (r = 0.990, n = 49, Syx = 1.165 mg/L). In addition, with 39 other sera the ratio of total thyroxin (by RIA) to TBG compared well with free thyroxin measured by equilibrium dialysis (r = 0.930) and the free thyroxin index (r = 0.970).

Apoenzymes↗