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PubMedDate not supplied

Hydrolysis and redox factors affecting analysis of common phenolic marker compounds in botanical extracts and finished products.

Many of the marker compounds analyzed in herbal products are redox-active phenolic molecules, which are commonly found in plants as components of glycosides and starch polymers. Variability in degree of sample hydrolysis can occur due to differences in water content, pH, and temperature. Sonication versus shaking during extraction can also influence hydrolysis and oxidation of sensitive compounds. Some traditional botanical extract marker compounds are esters and glycosides of phenolics such as echinacoside from Echinacea while others are free phenolics, such as quercetin from glycosides in Ginkgo. Optimizing hydrolysis conditions maximizes free quercetin levels, but lowers echinacoside levels. Furthermore, acidic hydrolysis conditions mimic stomach conditions encountered by oral supplements and protect resulting free phenolics from oxidation. Oxidative degradation of botanical phenolic markers can be initiated by light, sonication, oxygen, basic pH conditions, heat, redox-active solvents, and formulation additives. Some phenolic markers reversibly cycle through multiple oxidation states creating a formula-specific equilibrium of oxidation states. Finished product formulations that include easily oxidized phenolics, carbonates, phosphates, and transition metals affect sample hydrolysis degree and redox equilibria, and quantitation. By recognizing and controlling hydrolysis and oxidation variables, more accurate and rugged methods can be developed allowing for improved botanical standardization and finished product analysis.

Hydrolysis↗
PubMed1984

Direct comparison of the rates of internalization and degradation of covalent receptor-insulin complexes in 3T3-L1 adipocytes. Internalization of occupied receptors is not the rate-limiting step in receptor-hormone complex degradation.

Insulin receptors on the surface of 3T3-L1 adipocytes were photolabeled using the iodinated analog, B29-lysine-substituted N-[N'-(2-nitro-4-azidophenyl)glycyl]insulin. Under optimal labeling conditions (below 15 degrees C), greater than 95% of the labeled receptor remained on the cell surface prior to incubation at 37 degrees C. When the labeled monolayers were returned to their normal culture environment (37 degrees C), the covalent receptor-insulin complexes were rapidly internalized at initial rates equivalent to 130-170% of labeled surface receptor/h. Internalization of the complexes proceeded to an equilibrium or end point distribution of 40% internal receptor and 60% cell-surface receptor. Under the several labeling conditions tested, covalent receptor-insulin complexes were degraded in an apparent first order process at 37 degrees C with half-lives between 5 and 7 h. This rate was equivalent to only 10% of the labeled receptor being degraded per h and was 13-17-fold slower than the initial rate of labeled receptor internalization. This study directly demonstrates that the initial rate of internalization of covalent receptor-insulin complexes is not the rate-limiting step in their degradation in 3T3-L1 adipocytes. Furthermore, 3T3-L1 adipocytes are unable to internalize all of the labeled surface receptor, suggesting that two classes of internalization competent and incompetent receptor may exist or that an equilibrium distribution of internal and cell-surface receptor is established by the relative rates of internalization and recycling of labeled receptor.

Adipose Tissue↗
PubMed1990

[Suitable variables for the evaluation of combined acid-base disorders: residual anions, buffer base sera, proteins and delta bases].

In 254 findings of the acid-base balance ions and proteins, the authors evaluated mutual relations between the acid-base balance and calculation from the anion column. Contrary to the commonly used anion gap value, the authors used concurrently for evaluation buffer base sera, serum protein values and residual anions. The logical result of summarization of these three parameters is the creation of a new term, delta bases, which correlates closely with the resulting metabolic acid-base situation. The optimal relationship of the mentioned parameters to the value of base excess is described by the following equation: BE = 0.805*BBS--0.852*RA--0.204*SProt--15.8 which is consistent with the theoretical value of delta bases.

Acid-Base Equilibrium↗
PubMed2004

Capillary electrophoresis studies on the aggregation process of beta-amyloid 1-42 and 1-40 peptides.

The possibility to monitor, in solution, the steps of beta-amyloid (Abeta) nucleation and therefore to describe this dynamic process by using capillary electrophoresis and under optimized experimental conditions is described. Striking differences in the electrophoretic patterns of Abeta 1-42 and Abeta 1-40 over time are here shown, and different aggregation states are elucidated, which reflect the very diverse oligomerization behavior of two very similar peptides. The isolation of one aggregated species of high molecular weight by ultracentrifugation allowed us to assess its role as toxic oligomer. The perturbation of the existing equilibrium among the identified species by the addition of small molecules can in principle interfere with the aggregation process of the peptides and ultimately prevent the plaque formation in vitro.

Amyloid beta-Peptides↗
PubMed1979

Synthesis and removal of phenylalanine ammonia-lyase activity in illuminated discs of potato tuber parenchyme.

(1) The synthesis and removal of phenylalanine ammonia-lyase (EC 4.3.1.5) in illuminated discs of potato (Solanum tuberosum cv King Edward) tuber tissue has been investigated by density labelling with deuterium (2H) from deuterium oxide (2H2O) followed by centrifugation to equilibrium in a CsC1 density gradient. (2) Temporal changes in enzyme level have been described in terms of the equation (dE/dt) = ks-kdE where (dE/dt) is the rate of change of enzyme level per unit of tissue (E) with respect to time (t), ks is the rate constant for synthesis of the enzyme and kd is the rate constant for the removal of active enzyme. (3) The optimal concentration of 2H2O was determined by analysis of the relationship between 2H2O concentration, development of enzyme activity and the magnitude of the increase in buoyant density of phenylalanine ammonia-lyase. A concentration of 2H2O of about 40% (v/v) was found to be optimal, allowing achievement of maximal or near maximal increases in the buoyant density of the enzyme without inhibition of the development of enzyme activity, thereby circumventing the major drawback of 2H2O as a source of density label. (4) The overlapping distribution profiles of enzyme activity after density gradient centrifugation were resolved by an iterative method of best fit which allows estimation of the proportions of pre-existing, unlabelled enzyme and newly synthesised, labelled enzyme at the end of the labelling period. This technique has been developed to obtain the rate constants for enzyme synthesis and for removal of active enzyme throughout the period of rapid change in enzyme level. (5) It is demonstrated that the initial rapid increase in phenylalanine ammonia-lyase activity in illuminated discs reflects an increase in the rate constant for enzyme synthesis in the absence of activation of pre-existing enzyme and in the absence of removal of active enzyme. The abrupt transition to a phase of decline in enzyme activity is caused by (a) a reduction in the rate constant for enzyme synthesis and (b) a dramatic increase in the rate constant for removal of active enzyme. The subsequent stabilisation of the enzyme is caused by decay of both rate constants to relatively low levels. (6) The results are consistent with hypothesis that rapid modulation of enzyme levels during tissue differentiation is achieved by simultaneous changesin the rate constants for both enzyme synthesis and for removal of active enzyme.

Ammonia-Lyases↗
PubMed2006

B3LYP/6-311++G** study of structure and spin-spin coupling constant in methyl 2-O-sulfo-alpha-L-iduronate.

Structures of three most stable conformers ((1)C4, (4)C1, (2)S0) of methyl 2-O-sulfo-alpha-L-iduronate monosodium salt have been analyzed by DFT using the B3LYP/6-311++G** method. The optimized geometries confirmed the influence of both 2-O-sulfate and carboxylate groups upon the pyranose ring geometry. The computed energies showed that the chair (1)C4 form is the most stable one. Time-averaged DFT-calculated proton-proton and proton-carbon spin-spin coupling constants agree with the experimental data and indicate that only two chair forms contribute to the conformational equilibrium of methyl 2-O-sulfo-alpha-L-iduronate monosodium salt. The influence of the charged groups upon the magnitudes of spin-spin coupling constants is also discussed.

Carbohydrate Conformation↗
PubMed2006

Determination of paracetamol in pure and pharmaceutical dosage forms by pulse perturbation technique.

A new procedure for kinetic determination of paracetamol in pharmaceuticals is proposed. The method is based on potentiometric monitoring of the concentration perturbations of the matrix reaction system being in a stable non-equilibrium stationary state close to the bifurcation point. In the case considered as the matrix system, the Bray-Liebhafsky oscillatory reaction is used. The response of the matrix system to the perturbations by different concentrations of paracetamol is followed by a Pt-electrode. Proposed method relies on the linear relationship between maximal potential shift, DeltaEm, and the logarithm of added paracetamol amounts. It is obtained in optimized experimental conditions for variable amounts of paracetamol in the range 0.0085 and 1.5 micromol. The sensitivity and precision of proposed method were quite good (0.0027 micromol as the limit of detection and 2.4% as R.S.D.). Some aspects of possible chemical interactions between paracetamol and matrix are discussed. Applicability of the proposed method to the direct determination of paracetamol in pharmaceutical formulations was demonstrated.

Acetaminophen↗
PubMed2004

A diffusion quantum Monte Carlo study of geometries and harmonic frequencies of molecules.

This article describes an approach in determination of equilibrium geometries and harmonic frequencies of molecules by the Ornstein-Uhlenbeck diffusion quantum Monte Carlo method based on the floating spherical Gaussians. In conjunction with a projected and renormalized Hellmann-Feynman gradient and an electronic energy at variational Monte Carlo and diffusion quantum Monte Carlo, respectively, the quasi-Newton algorithm implemented with the Broyden-Fletcher-Goldfarb-Shanno updated Hessian was used to find the optimized molecular geometry. We applied this approach to N2 and H2O molecules. The geometry and harmonic frequencies calculated were consistent with some sophisticated ab initio calculated values within reasonable statistical uncertainty.

Journal Article↗
PubMed1982

Gonadotropin-releasing hormone stimulation of luteinizing hormone release: A ligand-receptor-effector model.

A divalent antibody conjugate of a pure antagonist of gonadotropin-releasing hormone (GnRH) behaved as an agonist--i.e., released luteinizing hormone (LH) from pituitary cultures. Release was measured over a wide range of conjugate concentrations; it rose to a maximum of 66% of the LH released by the optimal concentration of GnRH and declined to basal levels at very high concentrations. This behavior was modeled on the assumption that the antibody conjugate, A, can react with a receptor, R, to form a complex, A . R2. This dimer then can react with a quiescent effector, E (e.g., a closed Ca2+ ion channel), to form A . R2 . E, which contains activated effector and leads to cellular responses. The equilibrium equations governing the behavior of this model were derived, solved, and found to yield a good fit to the experimental data. Consideration of our data in this model system, and of other available data describing the behavior of ligands in other cells, suggests that the present model may be of wide applicability.

Gonadotropin-Releasing Hormone↗
PubMed2006

Transmembrane helix packing of ErbB/Neu receptor in membrane environment: a molecular dynamics study.

Dimerization or oligomerization of the ErbB/Neu receptors are necessary but not sufficient for initiation of receptor signaling. The two intracellular domains must be properly oriented for the juxtaposition of the kinase domains allowing trans-phosphorylation. This suggests that the transmembrane (TM) domain acts as a guide for defining the proper orientation of the intracellular domains. Two structural models, with the two helices either in left-handed or in right-handed coiling have been proposed as the TM domain structure of the active receptor. Because experimental data do not distinguish clearly helix-helix packing, molecular dynamics (MD) simulations are used to investigate the energetic factors that drive Neu TM-TM interactions of the wild and the oncogenic receptor (Val664/Glu mutation) in DMPC or in POPC environments. MD results indicate that helix-lipid interactions in the bilayer core are extremely similar in the two environments and raise the role of the juxtamembrane residues in helix insertion and helix-helix packing. The TM domain shows a greater propensity to adopt a left-handed structure in DMPC, with helices in optimal position for strong inter-helical Hbonds induced by the Glu mutation. In POPC, the right-handed structure is preferentially formed with the participation of water in inter-helical Hbonds. The two structural arrangements of the Neu(TM) helices both with GG4 residue motif in close contact at the interface are permissible in the membrane environment. According to the hypothesis of a monomer-dimer equilibrium of the proteins it is likely that the bilayer imposes structural constraints that favor dimerization-competent structure responsible of the proper topology necessary for receptor activation.

Cell Membrane↗
PubMed1995

The control of childhood viral infections by pulse vaccination.

Pulse vaccination, the repeated application of vaccine over a defined age range, is gaining prominence as a strategy for the elimination of childhood viral infections such as measles and polio. However, unlike routine or continuous mass infant immunization, epidemiological understanding of this control method is in its infancy. This paper develops initial work by Agur et al. (1993) using simple steady-state and age-structured dynamic models to extend the theory of the mechanism of action of pulse vaccination, and to explore the relationship between the maximum permitted interval between pulses and key epidemiological, demographic and vaccination variables. Initially, a conceptual model is presented to illustrate the principles of pulse vaccination and to make comparison with routine immunization procedures. An ordinary differential equation model, which assumes homogeneous mixing, is then used to derive equilibrium expressions for the pulse interval in relation to (i) different demographic profiles, (ii) population growth characteristics (stationary or exponentially increasing), (iii) combined routine and pulse immunization, and (iv) the age range vaccinated. Finally, simulations using age-structured compartmental deterministic models illustrate complex epidemiological dynamics associated with pulse vaccination, particularly where there is age heterogeneity in contact rates in the population. The resultant uncertainty in defining an optimal pulse interval raises concerns of a practical nature.

Age Factors↗
PubMed2002

Propagation of intense short laser pulses in the atmosphere.

The propagation of short, intense laser pulses in the atmosphere is investigated theoretically and numerically. A set of three-dimensional (3D), nonlinear propagation equations is derived, which includes the effects of dispersion, nonlinear self-focusing, stimulated molecular Raman scattering, multiphoton and tunneling ionization, energy depletion due to ionization, relativistic focusing, and ponderomotively excited plasma wakefields. The instantaneous frequency spread along a laser pulse in air, which develops due to various nonlinear effects, is analyzed and discussed. Coupled equations for the power, spot size, and electron density are derived for an intense ionizing laser pulse. From these equations we obtain an equilibrium for a single optical-plasma filament, which involves a balancing between diffraction, nonlinear self-focusing, and plasma defocusing. The equilibrium is shown to require a specific distribution of power along the filament. It is found that in the presence of ionization a self-guided optical filament is not realizable. A method for generating a remote spark in the atmosphere is proposed, which utilizes the dispersive and nonlinear properties of air to cause a low-intensity chirped laser pulse to compress both longitudinally and transversely. For optimally chosen parameters, we find that the transverse and longitudinal focal lengths can be made to coincide, resulting in rapid intensity increase, ionization, and white light generation in a localized region far from the source. Coupled equations for the laser spot size and pulse duration are derived, which can describe the focusing and compression process in the low-intensity regime. More general examples involving beam focusing, compression, ionization, and white light generation near the focal region are studied by numerically solving the full set of 3D, nonlinear propagation equations.

Journal Article↗
PubMed2006

Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems.

BACKGROUND: Aberrant signaling by ErbB-2 (HER 2, Neu), a member of the human Epidermal Growth Factor (EGF) receptor family, is associated with an aggressive clinical behaviour of carcinomas, particularly breast tumors. Antibodies targeting the ErbB-2 pathway are a preferred therapeutic option for patients with advanced breast cancer, but a worldwide deficit in the manufacturing capacities of mammalian cell bioreactors is foreseen. METHODS: Herein, we describe a multi-platform approach for the production of recombinant Single chain Fragments of antibody variable regions (ScFvs) to ErbB-2 that involves their functional expression in (a) bacteria, (b) transient as well as stable transgenic tobacco plants, and (c) a newly developed cell-free transcription-translation system. RESULTS: An ScFv (ScFv800E6) was selected by cloning immunoglobulin sequences from murine hybridomas, and was expressed and fully functional in all the expression platforms, thereby representing the first ScFv to ErbB-2 produced in hosts other than bacteria and yeast. ScFv800E6 was optimized with respect to redox synthesis conditions. Different tags were introduced flanking the ScFv800E6 backbone, with and without spacer arms, including a novel Strep II tag that outperforms conventional streptavidin-based detection systems. ScFv800E6 was resistant to standard chemical radiolabeling procedures (i.e. Chloramine T), displayed a binding ability extremely similar to that of the parental monovalent Fab' fragment, as well as a flow cytometry performance and an equilibrium binding affinity (Ka approximately 2 x 10(8) M(-1)) only slightly lower than those of the parental bivalent antibody, suggesting that its binding site is conserved as compared to that of the parental antibody molecule. ScFv800E6 was found to be compatible with routine reagents for immunohistochemical staining. CONCLUSION: ScFv800E6 is a useful reagent for in vitro biochemical and immunodiagnostic applications in oncology, and a candidate for future in vivo studies.

Journal Article↗
PubMed1988

Reconstitution of hormone-responsive detergent-solubilized follicle stimulating hormone receptors into liposomes.

An FSH receptor-enriched fraction that responds to exogenous FSH by activation of adenylate cyclase was prepared by ultrafiltration of sucrose density gradient-purified light membranes derived from bovine calf testes homogenates and solubilized with Triton X-100. To further confirm the functional nature of the detergent-solubilized FSH receptor, the extract was incorporated by lipid hydration into large multilamellar vesicles composed of dioleoyl phosphatidylcholine and cholesterol, 2:1 molar ratio. Receptor incorporation was determined by measurement of specific binding of [125I] human FSH ([125I] hFSH). Substitution of dioleoyl phosphatidylcholine with dipalmitoyl phosphatidylcholine or increasing the cholesterol concentration of the vesicles reduced specific binding of [125I]hFSH. Under conditions favoring optimal incorporation of the receptor, specific binding of [125I]hFSH was time and temperature dependent and saturable when increasing concentrations of radioligand were added to a constant amount of proteoliposomes. Reconstituted proteoliposomes bound 1600 fmol FSH/mg protein with an affinity of 3.54 x 10(9) M-1. Inhibition of [125I] hFSH binding by hFSH was comparable to that seen with the membrane-bound receptor (ED50 = 10 ng). Equilibrium binding studies with [3H]Gpp(NH)p indicated that a single class of high affinity GTP binding sites with an association constant (Ka) of 3.33 x 10(7) m-1 which bound 2.19 fmol [3H]Gpp(NH)p/mg protein had also been incorporated into the proteoliposomes. Addition of FSH induced a 2-fold stimulation of [3H]Gpp(NH)p binding, supporting our earlier studies suggesting that the detergent-solubilized FSH receptor is complexed to the G protein. Of particular significance in the present study was the observation that both NaF and FSH stimulated cAMP production in the reconstituted system. In addition to belonging to a class of membrane receptors functionally and physically associated with G protein, this observation suggests that FSH receptors in bovine calf testicular membranes may be associated, at least in part, with adenylate cyclase as well.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1980

Acyl-CoA synthetase in rat liver peroxisomes. Computer-assisted analysis of cell fractionation experiments.

The subcellular distribution of the acyl coenzyme A synthetases of rat liver was reinvestigated in order to determine whether part of this activity occurs in peroxisomes. Rat liver was fractionated by differential centrifugation and by equilibrium density centrifugation. Acyl-CoA synthetase was assayed using a new, simple extraction procedure on three substrates: palmitate, laurate, and octanoate. Comparison of the resulting synthetase distributions with the distributions of marker enzymes for peroxisomes, mitochondria, and endoplasmic reticulum demonstrated the presence of some synthetase activity in each of the three organelles. These trimodal synthetase distributions were evaluated quantitatively by means of a computer program that calculated optimal linear combinations of marker enzymes using a least squares criterion. Peroxisomes were found to contain 7% of the liver's palmitoyl-CoA synthetase activity and 6% of its lauroyl-CoA synthetase activity, but no demonstrable octanoyl-CoA synthetase activity. The remainder of these activities are divided between the mitochondria and endoplasmic reticulum, in a manner that agrees with previous studies. The chain length specificity of the synthetase(s) of each organelle appears to be unique. The absolute activity of the peroxisomal palmitoyl-CoA synthetase is sufficient to maintain maximal peroxisomal beta-oxidation. Clofibrate treatment of the rats caused a 2.6- to 3.1-fold increase in the liver's total acyl-CoA synthetase activities. The subcellular distribution was not greatly affected by this drug treatment.

Animals↗
PubMed1993

Mechanism of putrescine transport in human pulmonary artery endothelial cells.

Effective lung repair requires optimal replication of critical cell populations in the lung. Endogenous polyamines such as putrescine, spermidine and spermine play important roles in cell proliferation and differentiation, and may arise due to intracellular synthesis or transport into the cell. To determine the mechanism of polyamine transport in lung endothelial cells, the uptake of putrescine in human pulmonary artery endothelial cells was examined. Putrescine (7 nM) uptake into the cells approached equilibrium at 1 hr and was inhibited by methylglyoxal bis(guanylhydrazone). Kinetic studies revealed that uptake occurred via both a high- and low-affinity system. The effect of several amines (700 microM) on the 15-min uptake of putrescine was examined and a rank order of inhibition was determined: methylglyoxal bis(guanylhydrazone) > putrescine > spermine > spermidine > gentamicin > mepiperphenidol. alpha-Aminoisobutyric acid, a prototype system A amino acid, and tetraethylammonium, an organic cation, had no effect. N-ethylmaleimide inhibited transport 71%, whereas dinitrophenol did not. A reduction in temperature from 37 degrees C to 5 degrees C resulted in a 42% decrease in putrescine transport. Additionally, removing fetal bovine serum from the uptake medium reduced transport 38%. These data indicate that human pulmonary artery endothelial cells possess a specific transport system for polyamines. An improved understanding of this pathway in pulmonary endothelial cells may permit development of strategies to facilitate growth and repair of this critical cell population.

Amines↗
PubMed2004

An optimized algorithm for flux estimation from isotopomer distribution in glucose metabolites.

MOTIVATION: Analysis of the conversion of (13)C glucose within the metabolic network allows the evaluation of the biochemical fluxes in interconnecting metabolic pathways. Such analyses require solving hundreds of equations with respect to individual isotopomer concentrations, and this assumes applying special software even for constructing the equations. The algorithm, proposed by others could be improved. METHOD: A C-code linked to the program written in Mathematica (Wolfram Research Inc.), constructs and solves differential equations for all isotopomer concentrations, using the general enzyme characteristics (K(m), equilibrium constant, etc.). This code uses innovative algorithm of determination for the isotopomers-products, thus essentially decreasing the computation time. Feasible metabolic fluxes are provided by the parameters of enzyme kinetics found from the data fitting. RESULTS: The software effectively evaluates metabolic fluxes based on the measured isotopomer distribution, as was illustrated by the analysis of glycolysis and pentose phosphate cycle. The mechanism of transketolase and transaldolase catalysis was shown to induce a specific kind of isotopomer re-distribution, which, despite the significance of its effect, usually is not taken into account. AVAILABILITY: The software could be freely downloaded from the site: http://bq.ub.es/bioqint/label_distribution/.

Algorithms↗
PubMed2000

A two-parameter model of cell membrane permeability for multisolute systems.

A two-parameter model of cell osmotic response (F. W. Kleinhans, 1998, Cryobiology 37, 271-289) is expanded for multisolute systems. The cell water volume W and intracellular osmolalities of N solutes are related as W[1 + L(p)RTSigma(N)(i=1)(M(i)/P(i))] = W(0)[1 + L(p)RTSigma(N)(i=1)(M(0)i)/P(i))], where M(i) is the intracellular osmolality of the ith solute (i = 1 ellipsis N), P(i) is the membrane permeability of the ith solute, L(p) is the membrane hydraulic conductivity, R is the gas constant, T is the absolute temperature, and the subscript "0" denotes the initial values at time zero. The above formula allows calculating the final (equilibrium) volume when all entities are permeable. Simple algebraic expressions for calculation of the number and magnitude of transient maximum volume excursions are presented. These simple expressions can all be calculated by hand on a pocket calculator. Practical examples of one-, two-, and three-solute systems are discussed. Special attention has been given to situations when systems contain an impermeable component. All formulas are simple to use for optimization of variety of cryobiological protocols. Application of the theory for optimization of addition and dilution of a permeable cryoprotectant is also discussed.

Animals↗