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The salivary peroxidase system: thermodynamic, kinetic and antibacterial properties.

Enzymes are specific catalysts which optimize the rates of reactions vital to living organisms. If the oxidation of thiocyanate (which occurs, in vivo, in human saliva and salivary glands) is a vital reaction for the maintenance of oral health, then the enzyme (salivary peroxidase), which catalyzes this reaction, should function optimally under in vivo conditions. Studies have shown that salivary peroxidase maintains the SCN- oxidation reaction in an apparent state of dynamic equilibrium, that the kinetic properties of the enzyme provide optimum rates of reaction under in vivo conditions, and that the antibacterial properties of the products of the reaction are optimum under those conditions where unlimited bacterial metabolism provides the greatest threat to host tissues. The evidence indicates that the salivary peroxidase enzyme has evolved in such a way as to maximize its protective value in the oral cavity.

Bacteria↗

On the design of BSB neural associative memories using semidefinite programming.

This article is concerned with the reliable search for optimally performing BSB (brain state in a box) neural associative memories given a set of prototype patterns to be stored as stable equilibrium points. By converting and/or modifying the nonlinear constraints of a known formulation for the synthesis of BSB-based associative memories into linear matrix inequalities, we recast the synthesis into semidefinite programming problems and solve them by recently developed interior point methods. The validity of this approach is illustrated by a design example.

Association↗

Purification and properties of squid mantle adenylate kinase. Role of NADH in control of the enzyme.

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) from the mantle muscle of the squid, Loligo pealeii, was purified over 170-fold to homogeneity as judged by polyacrylamide and starch gel electrophoresis. The tissue contains a single isozyme of adenylate kinase, the enzyme from cytoplasmic and mitochondrial compartments (90 and 10% of total activity, respectively) being identical in physical and kinetic properties. Molecular weight was found to be 27,000 +/- 400. The enzyme shows a pH optimum of 8.2 in the forward (APD utilizing) and 7.4 in the reverse direction. Michaelis constants for ADP, ATP, and AMP are 0.70, 0.13, and 0.15 mM, respectively, with optimal Mg2+:adenylate ratios being 1:2 for ADP and 1:1 for ATP. A comparison of mass action ratios with the equilibrium constant indicated that squid adenylate kinase is held out of equilibrium in resting, but not active, muscle. A search for metabolic modulators of adenylate kinase revealed that NADH (Ki of 0.1 mM) was the only modulator which exerted a significant effect within its in vivo concentration range. The data presented indicate that NADH inhibition is the factor maintaining adenylate kinase in a nonequilibrium state in resting muscle and that release of this inhibition can serve to integrate adenylate kinase into the known scheme of intermediary metabolism in this tissue. A sharp drop in NADH levels at the onset on muscular work co-ordinates that activation of aerobic metabolism in this tissue and allows adenylate kinase to return to equilibrium function. At equilibrium, the enzyme can function to ampligy the concentration of AMP, a potent activator and deinhibitor of key glycolytic and Krebs cycle enzymes. The effect of modulators of adenylate kinase in preventing denaturation by heat or proteolysis revealed that NADH and substrates induced conformational changes in the enzyme which rendered it less susceptible to denaturation. The conformation state induced by NADH differed from that induced by substrate.

Adenylate Kinase↗

[How do antioxidants work?].

Reactive oxygen species, formed by incomplete reduction of molecular oxygen, may cause oxidative stress in the organism and be involved in the pathogenesis of many diseases. A number of anti-oxidants are necessary to counteract the harmful effects. Some antioxidants are enzymes which catalyze the breakdown of reactive oxygen species, some act by chelating transition metals, which makes them non-reactive, while chain-breaking antioxidants act by halting the cascade of free radical reactions. The effect of redox active antioxidants depends on their redox status. In some circumstances antioxidants can have pro-oxidant effects. The mechanisms may be reductive release of metals, accumulation of the oxidized form of redox active antioxidants, or merely an unfavourable equilibrium between various antioxidants. At present there is no basis for recommending the prophylactic use of commercially produced antioxidants. In particular, the optimal combinations of antioxidants are not yet known. If the aim is to improve the antioxidant defence of the body, our advice is still a mixed diet and regular physical activity.

Antioxidants↗

Effect of the nonpeptide neurotrophic compound SR 57746A on the phenotypic survival of purified mouse motoneurons.

1. Neurotrophic factors have been used for the treatment of several neurodegenerative diseases. However, their use is limited by their inability to cross the blood-brain barrier, their short half life and their side effects. SR 57746A is a new orally active compound that exhibits in vivo and in vitro neurotrophic effects in several experimental models. 2. We show here that SR 57746A (1 microM) increases the phenotypic survival of embryonic purified mouse motoneurons in vitro to the same extent as brain-derived neurotrophic factor (100 ng ml-1), and increases the outgrowth and number of their neurites. It acts in a dose-dependent manner up to 1 microM which is the optimal concentration. Above this concentration, its neurotrophic effect decreases. 3. Genistein (10 microM), a protein tyrosine kinase inhibitor, also increases the phenotypic survival and differentiation of mouse motoneurons. It does not act in a synergistic or additive manner with SR 57746A. However, at concentrations equal or superior to 25 microM, it decreases the survival of motoneurons. This suggests that the neurotrophic effect of genistein is due to a favourable alteration of equilibrium between phosphorylated and dephosphorylated states of proteins involved in survival and differentiation of motoneurons. 4. Like genistein, SR 57746A should be used at a critical concentration (1 microM) to exert its optimal effects. Since SR 57746A does not act synergistically with genistein, it is likely that its mechanism of action involves a pathway similar to that affected by this tyrosine kinase inhibitor. 5. At the present time, SR 57746A is the only orally active compound and the only synthetic compound shown to be active on motoneurons in vitro. It should thus be considered as a good candidate for the treatment of motoneuron diseases.

Animals↗

Abnormal force patterns for multidirectional postural responses in patients with Parkinson's disease.

This study tests the hypothesis that the basal ganglia are involved in optimizing postural responses for changes in perturbation direction and stance width. We compared the patterns of horizontal and vertical ground reactive forces under each leg in response to eight directions of surface translation in Parkinson's disease (PD) subjects and age-matched control subjects standing with both narrow and wide stance. Although passive reactive forces were larger, active forces were weaker and in abnormal directions for subjects with PD. Unlike the control subjects, who corrected their postural equilibrium in response to lateral and diagonal-lateral perturbations primarily with their loaded limbs, the PD subjects used both legs more symmetrically to recover equilibrium. PD subjects also did not change the magnitude or direction of reactive forces when initial stance width changed. These results support the hypothesis that the basal ganglia are important for optimizing automatic postural response patterns for changes in perturbation direction and for initial stance conditions.

Aged↗

Dietary recommendations for obese patients with chronic kidney disease.

For optimal management of chronic kidney disease (CKD), dietary modification should be an integral part of patient care. Dietary considerations for obese patients with CKD are numerous and complicated and involve modification of intake of calories, protein, fat, phosphorus, and electrolytes. General principles for dietary management of obese patients include (1) ensuring adequate monitoring of nutritional status through assessment of diet, nutrition-related laboratory parameters, and anthropometrics; (2) creation of an individualized diet plan that meets clinical guidelines and has favorable effects on obesity-related conditions such as blood pressure and lipids; (3) careful attention to patients' food choices, portion size, and food-preparation methods; (4) recommending adjustment of overall energy intake to promote weight loss, yet maintain good nutritional status; and (5) modification of diet as the patient's nutritional status changes and CKD progresses. The basic objectives of dietary modification are to lighten the excretory load of products of metabolism and to help the kidney maintain normal equilibrium of the body's internal environment. Dietary modifications must be individualized and appropriate to the stage of CKD. This review describes dietary factors important in optimizing nutritional status of obese patients with CKD. Additionally, current clinical practice guidelines and strategies for meeting them are discussed.

Calcium, Dietary↗

[Metabolic changes after whole gut washout with an electrolyte solution (author's transl)].

Colonic lavage with physiological saline causes undesirable side effects. A solution as first described by Hewitt was modified and used clinically for whole gut lavage. In comparison with the original solution an unchanged high fluid resorption of 22% of the total was seen. However, changes in acid-base and electrolyte balance were less than after washout with normal saline. Hypokalaemia or metabolic acidosis were not seen any longer. The modified solution thus seems more suitable than saline but does not yet fulfill all the requirements of an optimal solution.

Acid-Base Equilibrium↗

The structure and properties of 7-(2-oxoethyl)guanine: a model for a key DNA alkylation product of vinyl chloride.

Recent laboratory studies have shown that the major in-vivo DNA alkylation product of vinyl chloride is the 7-(2-oxoethyl) derivative of guanine, and the suggestion has been made that this derivative is responsible for the carcinogenicity of vinyl chloride. Some nuclear magnetic resonance evidence indicates that this alkylation product may be in equilibrium with a cyclic hemiacetal; this would affect the hydrogen bonding between guanine and cytosine. In order to help elucidate the properties of this key DNA alkylation product, we have computed the structure and properties of the same derivative of the isolated guanine molecule, i.e., 7-(2-oxoethyl)guanine. An ab initio self-consistent-field molecular orbital procedure was used. In this paper, we present the optimized (STO-3G) structure of 7-(2-oxoethyl)guanine, and the STO-5G energy, atomic charges and dipole moment. We found very little tendency for the formation of a cyclic hemiacetal; the equilibrium constant was estimated to be about 10(-9).

DNA Damage↗

Optimization of efficiency in the glyoxalase pathway.

A quantitative kinetic model for the glutathione-dependent conversion of methylglyoxal to D-lactate in mammalian erythrocytes has been formulated, on the basis of the measured or calculated rate and equilibrium constants associated with (a) the hydration of methylglyoxal, (b) the specific base catalyzed formation of glutathione-(R,S)-methylglyoxal thiohemiacetals, (c) the glyoxalase I catalyzed conversion of the diastereotopic thiohemiacetals to (S)-D-lactoylglutathione, and (d) the glyoxalase II catalyzed hydrolysis of (S)-D-lactoylglutathione to form D-lactate and glutathione. The model exhibits the following properties under conditions where substrate concentrations are small in comparison to the Km values for the glyoxalase enzymes: The overall rate of conversion of methylglyoxal to D-lactate is primarily limited by the rate of formation of the diastereotopic thiohemiacetals. The hydration of methylglyoxal is kinetically unimportant, since the apparent rate constant for hydration is (approximately 500-10(3))-fold smaller than that for formation of the thiohemiacetals. The rate of conversion of methylglyoxal to (S)-D-lactoylglutathione is near optimal, on the basis that the apparent rate constant for the glyoxalase I reaction (kcatEt/Km congruent to 4-20 s-1 for pig, rat, and human erythrocytes) is roughly equal to the apparent rate constant for decomposition of the thiohemiacetals to form glutathione and methylglyoxal [k(obsd) = 11 s-1, pH 7]. The capacity of glyoxalase I to use both diastereotopic thiohemiacetals, versus only one of the diastereomers, as substrates represents a 3- to 6-fold advantage in the steady-state rate of conversion of the diastereomers to (S)-D-lactoylglutathione.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Design, synthesis, and conformational dynamics of a gated molecular basket.

We have developed a synthesis and examined the conformational behavior and recognition properties of dynamic molecular containers 1-3. As follows from the 1H NMR dilution, diffusion NMR, and vapor pressure osmometry measurements, compound 1 has a low affinity for intermolecular aggregation and is mostly present in monomeric form in dilute chloroform solutions. Inspecting the O-H chemical shift resonances of 1, 3, and model compound 4 as a function of temperature afforded the deltadelta/deltaT coefficients of 17.0, 17.3, and 4.7 ppb K(-1), respectively. In combination with the results from variable temperature 1H NMR and IR measurements, the existence of conformers of 1 and 3 in equilibrium, each having a different extent of hydrogen bonding, was confirmed. Molecular mechanics calculations suggested 1a as the most favorable conformation, with three additional conformers, 1b, 1c, and 1d, populating local energy minima. Further optimization of each of the four conformers using semiempirical PM3 and ab initio (HF/6-31G) methods allowed a determination of their relative free energies and the corresponding Boltzmann population distributions which were heavily weighted toward 1a. A computed composite IR spectrum of a fraction-weighted mixture of the conformers of 1 reproduced the experimentally observed IR spectrum in its structural features, leading to a conclusion that conformer 1a indeed dominates the equilibrium. The egg-shaped cavity of 1 (136.6 angstroms3) is complementary in size, shape, and electrostatic potential to chloroform (74.9 angstroms3). A single-crystal X-ray study of 2 revealed a disordered chloroform molecule positioned inside the cavitand along its C3 axis.

Journal Article↗

Physicochemical parameters affecting the charcoal adsorption assay for quantitative retinoid-binding measurement.

The different parameters affecting the accuracy and reliability of the dextran-coated charcoal adsorption assay for characterization of retinoic acid receptors ligand binding activity were investigated. Using dextran-coated charcoal (DCC) at a final 10 mg/ml concentration, an efficient adsorption of free [3H]retinoic acid was observed with a yield in the range 99.2 to 99.8% for ligand concentrations varying from 10(-9) to 10(-4) M. Nonspecific adsorption of retinoic acid reached 50% to polystyrene and silanized glass and 70% to uncoated glass. Results obtained by the DCC method and by gel-filtration assay were correlated; however, the DCC assay appeared easier to perform and gave more reproducible results. When a careful measurement of free retinoid concentration was performed, the apparent equilibrium dissociation constant (KD) of retinoic acid was 3.1 +/- 0.4 nM and the KD of CD367, a synthetic retinoid, was 1.8 +/- 0.3 nM. Optimal pH for the binding of [3H]retinoic acid or [3H]CD367 was in the range 7.5 to 8.5. Under the conditions described for the adsorption assay, bound retinoid measurement was linearly related to the protein concentration between 0.05 and 0.25 mg/ml. At a lower protein concentration, addition of bovine serum albumin exerted a stabilizing effect on retinoid binding, allowing an accurate measurement of the number of specific binding sites. Using retinoic acid as ligand, bacterial extracts often resulted in a level of nonspecific binding in the range 10-25%. It could be lowered (4-10%) when resorting to [3H]CD367.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗

Development of rapid one-step immunochromatographic assay.

An analytical system for a one-step immunoassay has been constructed using the concept of immunochromatography. The system employed two different antibodies that bound distinct epitopes of an analyte molecule: an antibody labeled with a signal generator (e.g., colloidal gold), which was placed in the dry state at a predetermined site on a glass-fiber membrane, and another antibody immobilized on the surface of a nitrocellulose membrane. Three membranes, one with the tracer, one with immobilized antibody, and a cellulose membrane as the absorbent of medium (in a sequence from the bottom), were attached to a plastic film and cut into strips. Aqueous medium containing analyte absorbed from the bottom end of the immunostrip dissolved the labeled antibody, and the antigen-antibody binding complex formed was transported into the next nitrocellulose membrane by the flow caused by capillary action. The complex subsequently reacted with the immobilized antibody, which generated a signal in proportion to the analyte concentration. The convective mass transfer of the immunoreactant to the binding partner allowed the assay to be performed with no handling of reagents. The reaction, however, was carried out under nonequilibrium conditions, which resulted in decreased sensitivity as compared with assays performed in an equilibrium mode (e.g., ELISA). To minimize such sacrifice, major factors that control system performance were identified and the system was then devised under optimal conditions.

Animals↗

Organic and inorganic components in estuarine colloids: implications for sorption and transport of pollutants.

In colloids isolated from Chesapeake Bay and its subestuaries the concentrations of Al, Fe, and a number of trace elements were determined to vary with the clay mineral fraction that was most abundant in freshwater samples collected during the winter. The elements As, Ba, Sb, and Zn, however, increased with increasing organic content, indicating a covariance with the organic component. Organic analyses for amino acids (proteins), carbohydrates, and lipids indicate that these biopolymers comprised 4 to 22%, 20 to 60%, and less than 1%, respectively, of the colloidal organic carbon in these samples. The results are significant because amino acids and carbohydrates contain oxygen, nitrogen, and sulfur functional groups capable of reacting with trace metals and organic pollutants. The sorption properties of several neutral hydrophobic organic compounds, including PAHs and herbicides, and several aromatic amines were investigated using the estuarine colloidal material. The effects of several environmental variables on these sorption properties were determined. The results indicate that colloids have the capacity to sorb and transport relatively insoluble pollutants that otherwise might remain immobile in the environment. Colloidal organic matter in natural water systems (lakes, rivers, estuaries and the oceans, as well as groundwater) may serve as substrates for the sorption or binding of organic contaminants. Although most of the data has been developed using neutral hydrophobic organic compounds, data also exist for selected polar compound groups such as aromatic amines. The chemical behavior of these compound groups in interaction with DOM appears to have some similarity to their chemical interaction with sediments and soils. Partitioning constants are linear, except for polar compounds, and appear to be closely correlated to fundamental compound properties such as solubilities and octanol-water partition coefficients. Even though a variety of methods were used to collect the colloids for study and to determine partition coefficients, no single method appears to be superior. Ultrafiltration techniques appear to be optimal for collecting the colloids, since this method subjects the materials to less extreme chemical conditions than the preparation of humic extracts, whereas equilibrium techniques such as dialysis and head space analysis appear to yield the best results for partitioning constants. However, each of these techniques has limitations with respect to the classes of compounds that can be studied successfully.

Colloids↗

Characterization of the interaction constants for the binding of two unlabelled ligands to the sites of a receptor. An experimental strategy.

The model describing the binding of three ligands to the sites of a receptor is theoretically studied in order to characterize the binding interaction between two unlabelled ligands. For this, the binding of a third labelled ligand, the affinity of which is dependent on the presence of the unlabelled ligands, must be measured in different conditions. This paper describes an experimental strategy leading to an accurate determination of the equilibrium parameters of the model. This strategy, which assumes that the model is compatible with the data, rests on the determination of the optimal experimental conditions, i.e. the choice of the ligand concentrations which minimize the error of the parameter estimates. For this purpose, a computer program which runs on a microcomputer under the MS-DOS operating system has been elaborated on the basis of the D-optimization criterion. This work shows that such a computer analysis is essential to the determination of the optimal experimental design, even when very simple biochemical systems are considered.

Ligands↗

Characterization of human platelet UDPglucose-collagen glucosyltransferase using a new rapid assay.

A rapid and specific assay has been developed for UDPglucose-collagen glucosyltransferase (UDPglucose: 5-hydroxylysine-collagen glucosyltransferase, EC 2.4.1.66) using galactosylhydroxylysine (Gal-Hyl) as acceptor. Studies with intact human platelets and isolated plasma membranes indicated that about 5--10% of the total activity was surface bound and the rest was of cytoplasmic origin. The two forms of the enzyme had similar broad pH optima (6.5--8.0), Km values for UDPglucose (5 muM) and Gal-Hyl (approx. 4 mM) and for optimal manganese concentrations (25 mM). The soluble form of the enzyme was purified 80-fold. The reaction mechanism was determined as being rapid equilibrium random BiBi + dead end complex or ordered BiBi with UDPglucose being the first substrate to bind. Using Gal-Hyl bound in purified alpha 1 chain of chick skin collagen, a Km value three orders of magnitude less (2 muM) was found than for free Gal-Hyl and the manganese requirement decreased to 2 mM. These results suggest that the binding to the enzyme of Gal-Hyl in the collagen molecule is enhanced by the presence of the protein portion so that the enzyme may be capable of recognizing not only the carbohydrate side chains but also the primary structure of collagen.

Animals↗

EMG assisted optimization: a hybrid approach for estimating muscle forces in an indeterminate biomechanical model.

There are two basic approaches to estimate individual muscle forces acting on a joint, given the indeterminacy of moment balance equations: optimization and electromyography (EMG) assisted. Each approach is characterized by unique advantages and liabilities. With this in mind, a new hybrid method which combines the advantages of both of these traditional approaches, termed 'EMG assisted optimization' (EMGAO), was described. In this method, minimal adjustments are applied to the individual muscle forces estimated from EMG, so that all moment equilibrium equations are satisfied in three dimensions. The result is the best possible match between physiologically observed muscle activation patterns and the predicted forces, while satisfying the moment constraints about all three joint axes. Several forms of the objective function are discussed and their effect on individual muscle adjustments is illustrated in a simple two-dimensional example.

Algorithms↗

Kinetic and equilibrium studies in removing lead ions from aqueous solutions by natural sepiolite.

The capacity of sepiolite for the removal of lead ions from aqueous solution was investigated under different experimental conditions. The Langmuir and Freundlich equations, which are in common use for describing sorption equilibrium for wastewater-treatment applications, were applied to data. The constants and correlation coefficients of these isotherm models for the present system at different conditions such as pH, temperature and particle size were calculated and compared. The equilibrium process was well described by the Langmuir isotherm model and the maximum sorption capacity was found to be 93.4 mg/g for the optimal experimental condition. The thermodynamic parameters (DeltaG(o), DeltaH(o) and DeltaS(o)) for lead sorption on the sepiolite were also determined from the temperature dependence. The influences of specific parameters such as the agitation speed, particle size and initial concentration for the kinetic studies were also examined. The sorption kinetics were tested for first order reversible, pseudo-first order and pseudo-second order reaction and the rate constants of kinetic models were calculated. The best correlation coefficients were obtained using the pseudo-second order kinetic model, indicating that lead uptake process followed the pseudo-second order rate expression.

Environmental Pollutants↗