A general method for evaluation of diffusion constants,dilute-solution viscoelasticity,and the dielectric property of a rigid macromolecule with an arbitrary configuration. I

As the first part of the study on relaxational behavior of rigid macromolecules in solution, a general method for evaluating diffusion constants of a rigid macromolecule with an arbitrary configuration in a viscous solvent is presented. The evaluation is made by modeling the molecule with a rigid assembly of Stokes spheres. The diffusion constants are expressed by a 6 × 6 matrix which consists of translational, rotational, and cross constants, each being a 3 × 3 submatrix. Procedures of numerical calculation are presented for a given configuration of the molecule, together with some exemplifying results for the rigid rod, rigid sphere, and myosin molecule. The results for the rod and the sphere are compared with analytical results.     

Bisintercalators of DNA with a rigid linker in an extended configuration.

A new class of DNA bisintercalators is reported in which phenanthridinium or acridinium rings are connected by rigid and extended linkers of varied length. Cross-linking of DNA by bisintercalation is inferred from the unwinding and folding of linear DNA induced by the compound; after ligation and removal of the bisintercalator, superhelical circles, catenanes, and knots that bear an imprint of the bisintercalator are observed. These novel bisintercalators are of interest because they can be used to probe the organization of DNA in three-dimensional space, especially near sites of replication, recombination, or topoisomerase action, where two duplexes must be in close proximity.     

Enzyme-linked immunoassay. II. A simple method for synthesis of the rabbit antibody-beta-D-galactosidase complex and its general applicability.

Rabbit anti-human IgG antibody was mildly reduced in the presence of 10 mM 2-mercaptoethylamine and then coupled to beta-D-galactosidase [EC 3.2.1.23] from Escherichia coli using N,N'-o-phenylenedimaleimide. Human IgG and the anti-human IgG antibody-beta-D-galactosidase complex were successively adsorbed on Sepharose 4B binding rabbit anti-human IgG antibody. In this way amounts of human IgG as small as 5X10(-15) moles could be measured by determining the activity of beta-D-galactosidase bound to the Sepharose.     

A method for determining the dielectric constant and the conductivity of membrane-bounded particles of biological relevance.

Numerical assessment is made regarding Pauly and Schwan's theory which describes the dielectric behavior of a suspension of "shell spheres" as a model of biological membrane-bounded particles. The results indicate that approximate expressions of the theory may give rise to serious errors when applied to particles smaller than about 1 mum in diameter. With a view to performing analysis according to a general expression of the theory, some of the characteristic responses of dielectric parameters upon changes in phase parameters are examined with particular reference to some numerical ranges of biological interest. On this basis a simplified and systematic procedure is proposed for estimating the phase parameters of particles whose shell phase can be regarded as non-conductive. As the application of the procedure proposed, a set of dielectric data of a synaptosome suspension is analyzed, so that the following three phase parameters are successfully determined: membrane capacitance (or shell phase dielectric constant), interval phase conductivity and internal phase dielectric constant. Some limitations of the procedure are discussed for the cases of conducting shells and small particles.     

A method for determining the dielectric constant and the conductivity of membrane-bounded particles of biological relevance

Numerical assessment is made regarding Pauly and Schwan's theory which describes the dielectric behaviour of a suspension of “shell spheres” as a model of biological membrane-bounded particles. The results indicate that approximate expressions of the theory may give rise to serious errors when applied to particles smaller than about 1 Μm in diameter. With a view to performing analysis according to a general expression of the theory, some of the characteristic responses of dielectric parameters upon changes in phase parameters are examined with particular reference to some numerical ranges of biological interest. On this basis a simplified and systematic procedure is proposed for estimating the phase parameters of particles whose shell phase can be regarded as non-conductive. As the application of the procedure proposed, a set of dielectric data of a synaptosome suspension is analyzed, so that the following three phase parameters are successfully determined: membrane capacitance (or shell phase dielectric constant), internal phase conductivity and internal phase dielectric constant. Some limitations of the procedure are discussed for the cases of conducting shells and small particles.     

A novel spectroscopic titration method for determining the dissociation constant and stoichiometry of protein-ligand complex.

We offer a new titration protocol for determining the dissociation constant and binding stoichiometry of protein-ligand complex, detectable by spectroscopic methods. This approach neither is limited to the range of protein or ligand concentrations employed during titration experiment nor relies on precise determinations of the titration "endpoint," i.e., the maximal signal changes upon saturation of protein by ligand (or vice versa). In this procedure, a fixed concentration of protein (or ligand) is titrated by increasing volumes of a stock ligand (or protein) solution, and the changes in the spectroscopic signal are recorded after each addition of the titrant. The signal for interaction between protein and ligand first increases, reaches a maximum value, and then starts decreasing due to dilution effect. The volume of the titrant required to achieve the maximum signal changes is utilized to calculate the dissociation constant and the binding stoichiometry of the protein-ligand complex according to the theoretical relationships developed herein. This procedure has been tested for the interaction of avidin with a chromophoric biotin analogue, 2-(4'-hydroxyazobenzene)benzoic acid by following the absorption signal of their interaction at 500 nm. The widespread applicability of this procedure to protein-ligand complexes detected by other spectroscopic techniques and its advantages over conventional methods are discussed.     

A quick method for the determination of inhibition constants.

The inhibition constant Ki in the common case of competitive inhibition can be obtained by simple comparison of progress curves in the presence and in the absence of inhibitor. The difference between the times taken for the concentration of substrate to fall to the same value is used to obtain Ki. The procedure to use when the product inhibits is described. When there is mixed inhibition, reactions at different substrate concentrations are used to obtain both inhibition constants.     

Multicenter evaluation of Neo-Sensitabs, a standardized diffusion method for yeast susceptibility testing

The agar diffusion method Neo-Sensitabs for sensitivity testing, was evaluated with 33 reference strains by fourteen laboratories. Tablets with 5-fluorocytosine, amphotericin B, nystatin, fluconazole, itraconazole, ketoconazole and tioconazole were used on Shadomy modified medium. These tests classify each strain as susceptible, intermediate or resistant to all tested antifungals by measuring the inhibition zone diameters. Intra and interlaboratory reproducibility was studied. Neo-Sensitabs sensitivity for fungi was easy to perform and reliable method with a reproducibility of 97.1% and superior to other commercialized methods, being specially interesting for antifungal susceptibility in vitro testing of triazole derivatives fluconazole and itraconazole.     

A general and sensitive chemical method for sequencing the glycosyl residues of complex carbohydrates

This paper describes a new glycosyl-sequencing method. This method was made possible by the ability to fractionate complex mixtures of peralkylated oligosaccharides by reversed-phase, high-pressure liquid chromatography. The fractionation ability of the reversed-phase system allows the isolation and subsequent unambiguous identification by g.l.c.-m.s. of disaccharides, almost all trisaccharides, and, in some cases, tetrasaccharides generated by successive partial acid hydrolysis, reduction, and ethylation of a permethylated, complex carbohydrate. As these small oligosaccharides overlap within the unhydrolyzed, complex carbohydrate, the oligosaccharide sequences may be pieced together, and, with the glycosyl-linkage composition of the intact complex carbohydrate, can be used to determine the glycosyl sequence of the complex carbohydrate. The details of the sequencing method are illustrated by the elucidation of the glycosyl sequences of three complex carbohydrates. These examples demonstrate the wide variety of complex carbohydrates whose structures can be ascertained by the new sequencing technique. Two of the examples are the commercially available polysaccharides, lichenan and xanthan, whose structures have already been reported. The other example is a nonasaccharide derived from xyloglucan, a structural polymer of plant cell-walls. The glycosyl residues of the complex carbohydrates studied include hexosyl, deoxyhexosyl, pentosyl, glycosyluronic, and pyruvic acetal-substituted hexosyl residues. It will be demonstrated that the new glycosyl-sequencing technique is not compromise by the presence, in the carbohydrate to be analyzed, of glycosyl linkages possessing very different acid labilities. Two major advantages of this sequencing technique are that it is relatively rapid and that it requires only milligram quantities of carbohydrate.     

Verification of a monogenic hypothesis on pedigrees with an arbitrary structure selected for proband. II. The problem of prognosis

Criteria of fitness of monogenic hypothesis based on the accuracy of prediction of individuals' phenotypes are proposed. These criteria made it possible to differentiate the pedigrees in heterogeneous samples. The accuracy of genetic counseling is discussed. The method is illustrated on the example of the familial hypercholesterinemia in man.     

A simple method for finding explicit analytic transition densities of diffusion processes with general diploid selection

The transition density function of the Wright-Fisher diffusion describes the evolution of population-wide allele frequencies over time. This function has important practical applications in population genetics, but finding an explicit formula under a general diploid selection model has remained a difficult open problem. In this article, we develop a new computational method to tackle this classic problem. Specifically, our method explicitly finds the eigenvalues and eigenfunctions of the diffusion generator associated with the Wright-Fisher diffusion with recurrent mutation and arbitrary diploid selection, thus allowing one to obtain an accurate spectral representation of the transition density function. Simplicity is one of the appealing features of our approach. Although our derivation involves somewhat advanced mathematical concepts, the resulting algorithm is quite simple and efficient, only involving standard linear algebra. Furthermore, unlike previous approaches based on perturbation, which is applicable only when the population-scaled selection coefficient is small, our method is nonperturbative and is valid for a broad range of parameter values. As a by-product of our work, we obtain the rate of convergence to the stationary distribution under mutation-selection balance.     

A general method for studying the secretion of macromolecules by single cells: II. A simplified procedure with enhanced sensitivity

A simple, sensitive precipitin-type single-cell secretion assay is described and applied to the study of immunoglobulin-secreting cells. Evidence is presented that its efficiency is comparable to that achieved with reverse hemolytic plaque techniques. Also described is a modification of the simplified procedure which possesses substantially increased sensitivity. Enhanced sensitivity is achieved through the use of monoclonal rheumatoid factors which preferentially react with rabbit or human immunoglobulins that are incorporated into immune complexes as a result of interaction with antigen. Addition of rheumatoid factor to the agarose-cell mixture leads to additional crosslinking of immune complexes that form around active cells, thereby increasing the probability of forming a detectable precipitate. The application of this procedure to the detection of cells producing T-cell products is also discussed.     

A thermal-variation method for analysing the rate constants of the Michaelis--Menten mechanism.

By analysing the variations of saturation velocity and Michaelis constant with temperature and invoking the mathematical constraint represented by the Arrhenius equation, it becomes possible to estimate k+2 and indistinguishably k+1 and k-1 for the Michaelis--Menten mechanism of one-substrate enzyme reactions. Distinction between k+1 and k-1 may be obtained through the determination of isotopic rate effects. This procedure thus provides a basis for evaluating all three rate constants of the one-substrate mechanism, and disproves the suggestion that k+1 and k-1 are intrinsically unobtainable from steady-state kinetic measurements.     

Kinetics, binding constant, and activation energy of the 48-kDa protein-rhodopsin complex by extra-metarhodopsin II

We have found that the 48-kDa protein (or S-antigen 48k) of the rod photoreceptor enhances the light-induced formation of the photoproduct metarhodopsin II (MII) from prephosphorylated rhodopsin. The effect is analogous to the known enhancement of MII (extra-MII) that results from selective interaction of MII with G-protein. We have determined some parameters of the MII-48k interaction by measuring the extra-MII absorption change induced by the 48-kDa protein. The amplitude saturation yields a dissociation constant for the MII-48k complex on the order of 50 nM. At the technical limit of these measurements, 13.7 degrees C and 12 microM 48-kDa protein, we find a rate of 2.3 s-1 for formation of the 48k-MII complex. Extrapolation of these values to cellular conditions yields an occupation time of phosphorylated MII by 48k less than 200 ms. This is short compared to estimated rates of phosphorylation. The temperature dependence of the MII-48k formation rate is very high (Q10 for 5 degrees C/15 degrees C = 9-10). The related Arrhenius activation energy (165 kJ mol-1) is correspondingly high and indicates a considerable transient chemical change during the binding process.     

A general method for the purification of restriction enzymes.

An abbreviated procedure has been developed for the purification of restriction endonucleases. This procedure uses chromatography on phosphocellulose and hydroxylapatite and results in enzymes of sufficient purity to permit their use in the sequencing, molecular cloning, and physical mapping of DNA.