Light-scattering studies on supercoil unwinding

It has been shown previously that supercoiled [unk]X174 bacteriophage intracellular DNA (mol.wt. 3.2x10(6)) with superhelix density, sigma=-0.025 (-12 superhelical turns) at 25 degrees C is best represented as a Y shape. In this work two techniques have been used to unwind the supercoil and study the changes in tertiary structure which result from changes in the secondary structure. The molecular weights from all experiments were in the range 3.2x10(6)+/-0.12x10(6). In experiments involving temperature change little change in the Y shape was observed between sigma=-0.027 (-13 superhelical turns, 14.9 degrees C) and sigma=-0.021 (-10 superhelical turns, 53.4 degrees C) as evidenced by the root-mean-square radius and the particle-scattering factor P(theta). However, at sigma=-0.0176 (-8 superhelical turns, 74.5 degrees C) the root-mean-square radius fell to between 60 and 70nm from 90nm indicating a large structural change, as did alterations in the P(theta) function. In experiments with the intercalating dye proflavine from values of bound proflavine of 0-0.06mol of dye/mol equiv. of nucleotide which correspond to values of sigma from -0.025 to -0.0004 (-12 to 0 superhelical turns) a similar transition was found when the superhelix density was changed by the same amount, and the molecule was shown to go through a further structural change as the unwinding of the duplex proceeded. At sigma=-0.018 (-9 superhelical turns) the structure was compatible with a toroid, and at sigma=-0.0004 it was compatible with a circle but at no point in the sequence of structure transitions was the structure compatible with the conventional straight interwound model normally visualized as the shape of supercoiled DNA.     

Light-scattering spectroscopic study of polysaccharide protein conjugate

By combining gel permeation chromatography (GPC) and light-scattering spectroscopy, including photon correlation and angular distribution of absolute scattered intensity, we were able to characterize immunologically active Haemophilus influenzae type b polysaccharide (HIB Ps) bovine serum albumin (BSA) conjugates in terms of equivalent hydrodynamic radius r_h ～ (6.2 ± 0.6) × 10² Å, apparent radius of gyration r_g ～ (5.4 ± 0.3) × 10² Å, apparent molecular weight M_w ～ (3.5 ± 0.4) × 10⁶ g/mol, and a second virial coefficient A₂ ～ (1.9 ± 0.3) × 10^?4 cm³ mol/g². We could study the effects of each of the processes in the conjugate formation according to the following procedure: BSA (dialysis, modification, fractionation) + HIB Ps → HIB Ps/BSA conjugate (conjugate formation, fractionation). Narrow distributions of HIB Ps BSA conjugate formation can be achieved using fractionated BSA.     

Light-scattering studies of pectic substances in aqueous solution

The behaviour of pectic substances in aqueous solution has been studied by the light-scattering technique. Pectic substances were found to be present as high molecular aggregates having strong, intermolecular interactions, and their behaviour depends upon the nature of the carbohydrate chain. Additives can cause dissociation of the polysaccharide aggregates, resulting, in some cases, in particles having similar weight-average ( _w) and number-average ( _n) molecular weights.     

Light-scattering and potentiometric-titration studies of poly-L-tyrosine in aqueous solution

Aqueous solutions of poly-L -tyrosine were studied by potentiometric titrations, light-scattering measurements, and infrared spectroscopy in order to characterize the conformational changes the macromolecules exhibit when the hydrogen-ion concentration of the solutions is varied. It was found that when the pH of the system at 25°C is lowered from a value of about 13 the poly-L -tyrosine molecules undergo a random-coil to β-structure conformation change at pH 11.5. It appears, that under the experimental conditions used, the formation of the β structure is an intramolecular process which involves the folding of the polymer backbone into several hairpins of antiparallel β structure. On further lowering of the pH of the solutions aggregation (pH 11.35) and subsequently precipitation (pH 11.2) takes place. Since the apparent pK of the polymer does not show a drastic change during the random-coil to β-structure conformation change, it was concluded, that the therinodynamic driving force for this conformation change is mainly due to the electrostatic effect of the partially charged side chains of the poly-L -tyrosine molecules.     

Light-scattering and infrared-spectrophotometric studies of chitin and chitin derivatives

A sample of chitin isolated from the shell of the crab Scylla serrata had, when in lithium thiocyanate solution, an average, weight-average molecular weight (1) of 1.036 x 10⁶ daltons, an intrinsic dissymmetry (2) of 1.93, and a Z-average radius of gyration (3) of 64.14 nm. Carboxymethylchitin and glycol chitin, in 0.5M sodium chloride, had, respectively, (1) 1.896 and 1.819 x 10⁶ daltons, (2) 3.25 and 4.31. and (3) 143.49 and 251.57 nm. They had similar degrees of polymerization, they underwent dissociation as the concentration of sodium chloride was increased to 2.5M, and the molecular packing of the chains was essentially side-by-side. Chitin in 5.55M lithium thiocyanate and carboxymethylchitin in 2.5M sodium chloride had similar degrees of polymerization. It is concluded that a small but significient number of the amino groups in the chitin molecule are not acetylated.     

Light-scattering studies of the monomer-dimer states of gramicidin A.

Measurements of the intensity and autocorrelation function of ligh quasielastically scattered by gramicidin A indicate that the molecules exist as dimers in methanol and dioxane and monomers in dimethyl sulfoxide when solute concentrations range from 18 to 50 mg/ml. This gives further evidence that gramicidin A can dimerize in low-polarity media such as the hydrocarbon part of the membrane bilayers.     

Light-scattering studies of cation-stimulated filament assembly of newborn rat epidermal keratin

Effects of CaCl2 on in vitro polymerization of keratin extracted from cornified cells of newborn rat were investigated by means of light-scattering and supramolecular structures. Elongation and parallel assembly of filaments occurred with addition of CaCl2 to dialyzed keratin solutions and was detected by an increase in light-scattering intensity. Nonfibrous aggregates which occurred in higher buffer concentrations and in lower pH were also recorded as intensity increased. MgCl2, ZnCl2, and GdCl3 demonstrated similar effects, but NaCl and KCl showed no effect.     

Light-scattering studies of bull spermatozoa. II. Interaction and concentration effects.

The complete autocorrelation function of the intensity fluctuations of laser light scattered from motile bull spermatozoa is shown to depend upon several factors not previously considered. Samples of bull spermatozoa generally contain a substantial proportion of dead cells, which give rise to slowly decaying components of the autocorrelation function. Whereas previous work has concentrated on the form of the fast decaying autocorrelation component, we are concerned here with the relative amplitude and shape of the slow autocorrelation component and the general form of the composite function. In principle, the relative amplitudes of the fast and slow components of the autocorrelation function can be used as an assay of the proportion of swimming cells. We show that this amplitude ratio depends upon cell concentration, scattering cell geometry, and scattering angle. A simple model is developed to explain these results on the basis of the asymmetry of light scattered from these cells, motile/immotile cell interactions, wall-swimming effects, and geotactic reorientation of dead cells.     

Light-scattering measurement of lipid-glycoprotein interaction

Light-scattering measurements of DHPC (1,2 dihehadecyl-sn-glycero-3-phosphatidylcholine) vesicles and complex DHPC-ovomucoid were used to follow the time course of the osmotic response after mixing with solutions containing various salts and neurtral molecules. The osmotic effects of these salts and neutral molecules. on pure DHPC vesicles and vesicles formed by the complex DHPC-ovomucoid can be used to characterize the membrane permeability under the conditions of the experiment. The half-permeation times were determined in the presence and absence of the ovomucoid. The presence of ovomucoid on the vesicles appears to increase the half-permeation times of positively charged salt ions in comparison to the negatively charged dipicrylamine, (DPA^-) lipophilic ion. This phenomenon is explained in terms of complex formation thereby diminishing the available negatively charged ionic groups which presumably are involved in the transition process across the lipid vesicles. The half-permeation times for neutral molecules did not change in the presence of ovomucoid. This indicates a mechanism of transition quite different from the one characteristic for the salt ions. On the basis of this evidence and the electrostatic bonding it is concluded that the ovomucoid is bound on the polar surface of the lipid vesicles.     

Light-scattering from detergent-complexed biological macromolecules

A theory is described for Rayleigh light-scattering from solutions of detergent-complexed macromolecules applicable to measurements carried out under conditions of Donnan equilibrium. The theory shows that when scattering measurements are made on detergent-solubilized macromolecules in the presence of detergent micelles the apparent Mr is dependent on the extent of detergent binding and effective charge on the detergent-macromolecule complex and the micellar charge and aggregation number. Equations are given for the apparent Mr of the macromolecule under limiting conditions of high salt and low salt concentration. Low-angle laser-light-scattering measurements were made on lysozyme complexed with sodium n-dodecyl sulphate both in the absence and in the presence of detergent micelles. These experimentally obtained data were used in conjunction with the detergent-binding isotherm to test the theory at high ionic strength. Light-scattering measurements were also made on detergent-saturated complexes as a function of ionic strength and pH. The results are in reasonable accord with both the qualitative and the quantitative predictions of the theory.     

Stopped-flow studies of myelin basic protein association with phospholipid vesicles and subsequent vesicle aggregation

When mixed with vesicles containing acidic phospholipids, myelin basic protein causes vesicle aggregation. The kinetics of this vesicle cross-linking by myelin basic protein was investigated by using stopped-flow light scattering. The process was highly cooperative, requiring about 20 protein molecules per vesicle to produce a measurable aggregation rate and about 35 protein molecules per vesicle to produce the maximum rate. The maximum aggregation rate constant approached the theoretical vesicle-vesicle collisional rate constant. Vesicle aggregation was second order in vesicle concentration and was much slower than protein-vesicle interaction. The highest myelin basic protein concentration used here did not inhibit vesicle aggregation, indicating that vesicle cross-linking occurred through protein-protein interactions. In contrast, poly(L-lysine)-induced vesicle aggregation was easily inhibited by increasing peptide concentrations, indicating that it did cross-link vesicles as a peptide monomer. The myelin basic protein:vesicle stoichiometry required for aggregation and the low affinity for protein dimerization suggested that multiple protein cross-links were needed to form a stable aggregate. Stopped-flow fluorescence was used to estimate the kinetics of myelin basic protein-vesicle binding. The half-times obtained suggested a rate constant that approached the theoretical protein-vesicle collisional rate constant.     

Meta-analysis of genetic association studies

Meta-analysis, a statistical tool for combining results across studies, is becoming popular as a method for resolving discrepancies in genetic association studies. Persistent difficulties in obtaining robust, replicable results in genetic association studies are almost certainly because genetic effects are small, requiring studies with many thousands of subjects to be detected. In this article, we describe how meta-analysis works and consider whether it will solve the problem of underpowered studies or whether it is another affliction visited by statisticians on geneticists. We show that meta-analysis has been successful in revealing unexpected sources of heterogeneity, such as publication bias. If heterogeneity is adequately recognized and taken into account, meta-analysis can confirm the involvement of a genetic variant, but it is not a substitute for an adequately powered primary study.