A continuous spectrophotometric assay method for peptidylarginine deiminase type 4 activity

A simple, continuous spectrophotometric assay for peptidylarginine deiminase (PAD) is described. Deimination of peptidylarginine results in the formation of peptidylcitrulline and ammonia. The ammonia released during peptidylarginine hydrolysis is coupled to the glutamate-dehydrogenase-catalyzed reductive amination of alpha-ketoglutarate to glutamate and reduced nicotinamide adenine dinucleotide (NADH) oxidation. The disappearance of absorbance at 340nm due to NADH oxidation is continuously measured. The specific activity obtained by this new protocol for highly purified human PAD is comparable to that obtained by a commonly used colorimetric procedure, which measures the ureido group of peptidylcitrulline by coupling with diacetyl monoxime. The present continuous spectrophotometric method is highly sensitive and accurate and is thus suitable for enzyme kinetic analysis of PAD. The Ca(2+) concentration for half-maximal activity of PAD obtained by this method is comparable to that previously obtained by the colorimetric procedure.     

Carboxymethylated liver alcohol dehydrogenase. Transient kinetic studies and effect of substrate structure on alcohol oxidation.

1. The rate constants for NADH binding and dissociation for carboxymethylated alcohol dehydrogenase have been determined and compared to those for the native enzyme. 2. Steady-state and transient kinetic experiments have shown that the hydrogen transfer step is rate-determining for oxidation of ethanol by carboxymethylated alcohol dehydrogenase. The rate constant of 0.19 s-1 is considerably slower than that for the native enzyme. 3. The steady-state parameter, V/[E], was obtained for each of a series of alcohols and correlated with the Taft sigma parameter. The linear relationship obtained indicates that the same step, hydrogen transfer, is rate-determining for all the alcohols. The sigma value obtained is the same as for the native enzyme; the implications of this for the mechanism of hydrogen transfer are discussed.     

The interspike interval of a cable model neuron with white noise input

The firing time of a cable model neuron in response to white noise current injection is investigated with various methods. The Fourier decomposition of the depolarization leads to partial differential equations for the moments of the firing time. These are solved by perturbation and numerical methods, and the results obtained are in excellent agreement with those obtained by Monte Carlo simulation. The convergence of the random Fourier series is found to be very slow for small times so that when the firing time is small it is more efficient to simulate the solution of the stochastic cable equation directly using the two different representations of the Green's function, one which converges rapidly for small times and the other which converges rapidly for large times. The shape of the interspike interval density is found to depend strongly on input position. The various shapes obtained for different input positions resemble those for real neurons. The coefficient of variation of the interspike interval decreases monotonically as the distance between the input and trigger zone increases. A diffusion approximation for a nerve cell receiving Poisson input is considered and input/output frequency relations obtained for different input sites. The cases of multiple trigger zones and multiple input sites are briefly discussed.     

Information obtained in cladistic analysis.

The application of elementary equations from information theory to the elements involved in cladistic analysis is formalized mathematically. An equation is derived that quantifies the amount of information obtained by constructing a cladogram from a cladistic data matrix. Given particular conditions, the amount of information obtained increases monotonically with increases of the number of taxa involved and, so, may be used directly as a comparative measure of species richness for sister groups; in general, however, the amount of information obtained is related to the distribution of character states on the cladogram(s) deduced. An example is presented in which clades representing 11 phyla in the animal kingdom are compared in terms of information yielded. The amount of information obtained is consistent for different numbers of taxa and characters used in classifications. Speculative evolutionary explanations are presented for differences of information yielded among the phyla analyzed.     

Exciton interactions in synthetic porphyrin dimers

The Soret absorption spectra of six synthetic rigid porphyrin dimers whose crystal structures have been determined are simulated using simple exciton theory. The objective is to test the validity of the point dipole and associated approximations; the electronic interaction parameters are thus calculated using data obtained from the monomer spectra, with no adjustable parameters. Satisfactory agreement between theory and experiment is obtained for one class of dimers but not for a second. This poses a challenge for semiempirical electronic structure methods as to whether improvements over the point dipole calculations can be obtained.     

Optimal Homotopy Asymptotic Method for Flow and Heat Transfer of a Viscoelastic Fluid in an Axisymmetric Channel with a Porous Wall

In this article, an approximate analytical solution of flow and heat transfer for a viscoelastic fluid in an axisymmetric channel with porous wall is presented. The solution is obtained through the use of a powerful method known as Optimal Homotopy Asymptotic Method (OHAM). We obtained the approximate analytical solution for dimensionless velocity and temperature for various parameters. The influence and effect of different parameters on dimensionless velocity, temperature, friction factor, and rate of heat transfer are presented graphically. We also compared our solution with those obtained by other methods and it is found that OHAM solution is better than the other methods considered. This shows that OHAM is reliable for use to solve strongly nonlinear problems in heat transfer phenomena.     

The spatial spread and final size of models for the deterministic host-vector epidemic

A disease is considered which is transferred between two populations, termed hosts and vectors. The disease is transmitted solely from infected vector to uninfected host and from infected host to uninfected vector. Two models are formulated in which infectious individuals are introduced at time t = 0 into the populations of susceptibles, thus triggering an epidemic through those populations. Conditions are established for a major epidemic to occur, and the final size of the epidemic is obtained for these models when no spatial aspect is considered. When a spatial aspect is included in the models, again the condition for a major epidemic is obtained. The pandemic theorem is proved rigorously, giving a lower bound for the proportion of each population, at each point, who eventually suffer the epidemic. The behavior a long way from the initial focus of infection is also rigorously obtained.     

Use of holographic laser interferometry to study the diffusion of polymers in gels

The aim of this study was to demonstrate the potential for holographic interferometry to be used for diffusion studies of large molecules in gels. The diffusion and partitioning of BSA (67,000 g/mol) and pullulans (5,900-112,000 g/mol) in agarose gel were investigated. The gel diffusion coefficients obtained for BSA were higher when distilled water was used as a solvent compared to those obtained with 0.1 M NaCl as the solvent. Furthermore, the gel diffusion coefficient increased with increasing BSA concentration. The same trend was found for liquid BSA diffusion coefficients obtained by DLS. BSA partition coefficients obtained at different agarose gel concentrations (2-6%, w/w) decreased slightly with increasing gel concentration. However, all BSA gel diffusion coefficients measured were significantly lower than those in pure solvent and they decreased with increasing agarose concentration. The gel diffusion coefficients obtained for pullulans decreased with increasing pullulan molecular weight. The same effect from increased molecular weight was seen in the liquid diffusion coefficients measured by DLS. The pullulan partition coefficients obtained decreased with increasing molecular weight. However, pullulans with a larger Stokes' radius than BSA had partition coefficients that were higher or approximately the same as BSA. This implied that the pullulan molecules were more flexible than the BSA molecules. The results obtained for BSA in this study agreed well with other experimental studies. In addition, the magnitude of the relative standard deviation was acceptable and in the same range as for many other methods. The results thereby obtained showed that holographic interferometry is a suitable method for studying diffusion of macromolecules in gels.     

Computational fluid dynamics in abdominal aorta bifurcation: non-Newtonian versus Newtonian blood flow in a real case study

Hemodynamic in abdominal aorta bifurcation was investigated in a real case using computational fluid dynamics. A Newtonian and non-Newtonian (Walburn-Schneck) viscosity models were compared. The geometrical model was obtained by 3D reconstruction from CT-scan and hemodynamic parameters obtained by laser-Doppler. Blood was assumed incompressible fluid, laminar flow in transient regime and rigid vessel wall. Finite volume-based was used to study the velocity, pressure, wall shear stress (WSS) and viscosity throughout cardiac cycle. Results obtained with Walburn-Schneck’s model, during systole, present lower viscosity due to shear thinning behavior. Furthermore, there is a significant difference between the results obtained by the two models for a specific patient. During the systole, differences are more pronounced and are preferably located in the tortuous regions of the artery. Throughout the cardiac cycle, the WSS amplitude between the systole and diastole is greater for the Walburn-Schneck’s model than for the Newtonian model. However, the average viscosity along the artery is always greater for the non-Newtonian model, except in the systolic peak. The hemodynamic model is crucial to validate results obtained with CFD and to explore clinical potential.     

Histone-specific acetyltransferase from the rat liver nuclei

Certain properties of histone-specific acetyltransferases A, B and C, obtained from the rat liver are determined. pH optimum for enzyme A is within the range of 7.5-8.5, for B--7.8 and for C--7.5. The maximal activity for enzymes A, B and C is observed with the 60 micrograms/ml concentration of the substrate. The activity is inhibited by N-maleimide, iodacetamide and chloromercuribenzoate. The results obtained show that a number of similar properties are typical of the above enzymes.     

HOW TO ESTIMATE AND USE THE VARIANCE OF d' FROM DIFFERENCE TESTS

d' is an estimate of δ, a measure of the degree of sensory difference between two products, that can be obtained easily using tables, from the proportion of difference tests performed correctly. Tables of δ are available for the 2-AFC, 3-AFC, triangular and duo-trio tests. Tables for calculating the variance of d' for these tests are provided in this paper. They can be used for comparison of d's, especially for those obtained from different difference tests. A simple procedure is described here for computing values for the variance of d'. Having obtained the variance, confidence intervals for d' can be obtained, tests of significance for d' can be made as well as tests of whether two or more d's are significantly different. The formula and tables for the number of judgments required for the estimation of δ are given also in this paper.     

The derivation of certain pandemic bounds

Exact results have previously been obtained concerning the spread of infection in continuous space contact models describing a class of multitype epidemics. The Pandemic Theorem gave a lower bound for the spatial final size. A discrete space model is considered. A simpler, more direct proof based on an infinite matrix formulation of the final size equations is used to obtain the pandemic result for this model. An upper bound is obtained, which is valid for both continuous and discrete space models. This enables a limiting result to be obtained for the spatial final size when the amount of initial infection tends to zero.     

A novel mass spectrometric procedure to rapidly determine the partial structure of heparin fragments

The molecular weight and degree of sulfation has been obtained for di-, tetra- and hexasaccharide fragments of heparin obtained by enzymatic depolymerization of porcine mucosal heparin. The sodium salt form of the sulfated oligosaccharide is adsorbed onto an immobilized cationic surfactant film which is inserted directly into the mass spectrometer. Analyses are routinely obtained on 25-50 microgram samples in less than an hour. This approach provides rapid confirmatory structural information that is complementary to existing methodologies.     

An approximate solution for a transient two-phase stirred tank bioreactor with nonlinear kinetics

The derivation of an approximate solution method for models of a continuous stirred tank bioreactor where the reaction takes place in pellets suspended in a well-mixed fluid is presented. It is assumed that the reaction follows a Michaelis-Menten-type kinetics. Analytic solution of the differential equations is obtained by expanding the reaction rate expression at pellet surface concentration using Taylor series. The concept of a pellet's dead zone is incorporated; improving the predictions and avoiding negative values of the reagent concentration. The results include the concentration expressions obtained for (a) the steady state, (b) the transient case, imposing the quasi-steady-state assumption for the pellet equation, and (c) the complete solution of the approximate transient problem. The convenience of the approximate method is assessed by comparison of the predictions with the ones obtained from the numerical solution of the original problem. The differences are in general quite acceptable.     

Evaluation of the time course of neurotransmitter release from the measured PSC and MPSC

A method for obtaining delay histograms for the time course of neurotransmitter release is presented. The delay histogram is derived from the measured psc (or the sum of several psc's) and the mpsc (obtained experimentally or otherwise) by means of a simple, quick, mathematical procedure. The procedure may be automated for the greater part. No approximation of the mpsc shape is performed, and the method is applicable to all quantal contents. For low and medium quantal contents, the delay histograms obtained by the method are compared to those obtained by direct analysis. A reasonable agreement is achieved. An experiment of high quantal content, for which direct analysis is impossible, is then analysed using the new method. Difficulties which may arise when applying the procedure and methods to overcome them are discussed at length. Other methods are set forth in the Discussion.     

On some problems of random nets

The probability problems connected with random nets are restated as probabilities of drawings from an urn containing black and white balls. A partial difference equation is obtained and its solution is given. For large nets a series expression is obtained for the connectivity γ, and this is shown to be equivalent to the transcendental equation obtained by R. Solomonoff and A. Rapport (1951).     

Rapid Methods for Extracting Autolysins from Bacillus subtilis

Two procedures are described for the extraction of autolysins from whole cells. One method uses 5 M LiCl at 4 C. The amount of enzyme obtained by this method is six times more than that obtained by autolysis of cell walls and fourteen times more than that obtained by extracting cell walls with LiCl. With the other method, cells are extracted with 2% Triton X-100. This is less efficient than the LiCl method but yields about one-half the amount of enzyme obtained by cell wall autolysis and about the same amount as obtained by extracting cell walls with salt. Both procedures yield autolysin with multiple pH optima. Autolysins can be extracted from several bacterial species by either the LiCl or the detergent method. The data suggest that these techniques have sufficient sensitivity to detect small differences in autolytic activity among mutants and various organisms and are also suitable for large-scale isolation of autolysin for purification and characterization studies.     

VI. The concurrent estimation of the major monoamine metabolites in human and non-human primate brain by HPLC with fluorescence and electrochemical detection

A simple and sensitive method for the concurrent determination of the monoamine metabolites MHPG, DOPAC, HVA and 5HIAA in brain samples is described. After solvent extraction at acid pH, the metabolites are separated by HPLC on a C₁₈ reversed phase column using phosphate buffers. Detection and quantification are achieved using fluorescence and electrochemical detection in series. The method is applied to control samples of divers areas of human and non-human primate brain, and the distribution of results agrees well with those obtained by existing methods. The concentrations found also agreed well with literature values, and, for 5HIAA and DOPAC, with results obtained on parallel samples analysed by fluorimetry and by GC. Results for HVA however are higher than those obtained by GC, but agree well with literature values obtained by fluorimetry and by GCMS.     

A force field for naproxen

A united-atom potential model for naproxen suitable for molecular dynamics (MD) simulation has been developed. The charge distribution is approximated by point charges obtained from ab initio calculations using the CHELPG method. Also the intramolecular interactions such as bond and angle vibration, and the torsion potential are obtained from ab initio calculations. The dispersive interaction contribution is taken from the literature. By MD simulation using a naproxen film in slap geometry, the temperature dependence of the density, surface tension and self-diffusion coefficient as well as the melting temperature for the developed potential model are obtained.