Globule Phases Research Articles

Cyclosporine release and distribution in ophthalmic emulsions determined by pulsatile microdialysis

An in vitro release test based on pulsatile microdialysis (PMD) is presented for the purpose of measuring the release of cyclosporine from ophthalmic emulsions, along with a method to determine the drug distribution within the oil-rich globule, surfactant-rich micelle and aqueous phases of the emulsion formulation. Compositionally equivalent formulations containing 0.05% cyclosporine were prepared with similar physical parameters (globule size, viscosity, surface tension zeta potential, osmolality, pH) but made with different manufacturing conditions. Emulsions were made by ultrasonication, using different ultrasonication times (22–49 min) and temperatures (50–82 °C). Formulations were stored at room temperature (20 °C) and PMD was performed under two conditions, one in which the receiving medium temperature was 20 °C, and another in which the receiving medium temperature was 35 °C to mimic the temperature change expected when a drop of formulation is administered to the eye. The PMD release data were taken at release times of 20, 40, 60, 90, 120, 180, 300 and 600 s. All experiments showed a qualitatively similar release pattern, with a rapid initial rate of drug release (Release-1) for the first few minutes, followed by a much slower release (Release-2). In addition, imposing a sudden temperature change on the formulation was observed to affect the release, with some formulations releasing faster into receiver media at 35 °C than at 20 °C, while others released faster into 20 °C than 35 °C receiver media. The drug distribution was also calculated from PMD release data into 20 °C receiver media using a novel release kinetics model. The drug distribution varied among the formulations, with 54–77% of the cyclosporine in the oil phase of the emulsions. PMD is a promising method to evaluate how manufacturing-induced differences affect the distribution and release kinetics of cyclosporine within the emulsion formulation.

Phase Transition of Catenated DNA Networks in Poly(ethylene glycol) Solutions.

Conformational phase transitions of macromolecules are an important class of problems in fundamental polymer physics. While the conformational phase transitions of linear DNA have been extensively studied, this feature of topologically complex DNA remains unexplored. We report herein the polymer-and-salt-induced (Ψ) phase transition of 2D catenated DNA networks, called kinetoplasts, using single-molecule fluorescence microscopy. We observe that kinetoplasts can undergo a reversible transition from the flat phase to the collapsed phase in the presence of NaCl as a function of the crowding agent poly(ethylene glycol). The nature of this phase transition is tunable through varying ionic strengths. For linear DNA, the coexistence of coil and globule phases was attributed to a first order phase transition associated with a double well potential in the transition regime. Kinetoplasts, however, navigate from the flat to the collapsed phase by passing through an intermediate regime, characterized by the coexistence of a multipopulation with varying shapes and sizes. Conformations of individual molecules in the multipopulation are long-lived, which suggests a rugged energy landscape.

Collapse transition in polymer models with multiple monomers per site and multiple bonds per edge.

We present results from extensive Monte Carlo simulations of polymer models where each lattice site can be visited by up to K monomers and no restriction is imposed on the number of bonds on each lattice edge. These multiple monomer per site (MMS) models are investigated on the square and cubic lattices, for K=2 and 3, by associating Boltzmann weights ω_{0}=1, ω_{1}=e^{β_{1}}, and ω_{2}=e^{β_{2}} to sites visited by 1, 2, and 3 monomers, respectively. Two versions of the MMS models are considered for which immediate reversals of the walks are allowed (RA) or forbidden (RF). In contrast to previous simulations of these models, we find the same thermodynamic behavior for both RA and RF versions. In three dimensions, the phase diagrams, in space β_{2}×β_{1}, are featured by coil and globule phases separated by a line of Θ points, as thoroughly demonstrated by the metric ν_{t}, crossover ϕ_{t}, and entropic γ_{t} exponents. The existence of the Θ lines is also confirmed by the second virial coefficient. This shows that no discontinuous collapse transition exists in these models, in contrast to previous claims based on a weak bimodality observed in some distributions, which indeed exists in a narrow region very close to the Θ line when β_{1}<0. Interestingly, in two dimensions, only a crossover is found between the coil and globule phases.

Monte Carlo simulations of polymers with nearest- and next nearest-neighbor interactions on square and cubic lattices

We study a generalized interacting self-avoiding walk (ISAW) model with nearest- and next nearest-neighbor (NN and NNN) interactions on square and cubic lattices. In both dimensions, the phase diagrams show coil and globule phases separated by continuous transition lines. Along these lines, we calculate the metric νt, crossover ϕt and entropic γt exponents, all of them in good agreement with the exact values of the Θ universality class. Therefore, the introduction of NNN interactions does not change the class of the ISAW model, which still exists even for repulsive forces. The growth parameters μt are shown to change monotonically with temperature along the Θ-lines. In the square lattice, the Θ-line has an almost linear behavior, which was not found in the cubic one. Although the region of repulsive NNN interactions, with attractive NN ones, leads to stiff polymers, no evidence of a transition to a crystalline phase was found.

Temperature-Pressure Stability and T-P Phase Diagram of Parvalbumin

Fish allergens can trigger life-threatening reactions in predisposed individuals, which is especially important in countries where seafood is an integrate part of the diet. The major fish allergen parvalbumins are abundant in the white muscle of many fish species. Parvalbumin belongs to the family of EF-hand proteins and has a globular shape containing six helical parts and two active Ca2+ binding sites.High pressure is known to unfold proteins. We performed high pressure FTIR experiments, to explore the T-p phase diagram of cod parvalbumin. This method allows following the secondary structure of the protein as well as the Ca2+ binding to the aspartic glutamic acid residues.The infrared spectrum of parvalbumin is characteristic for the helical conformation, in agreement with the crystal structure, but without adding Ca2+ we found that the sample does not adopt the fully ordered structure. A marked transition in the structure of the parvalbumin was observed with the central point of 0.5 GPa (at room temperature). We assign this change to a native-molten globule transition on the basis of the position of the amide I band of the infrared spectrum. This transition was reversible, which also supports the lack of complete unfolding.Temperature experiments show a two-step unfolding of the protein. Above 45°C we have molten globule kind of loosened structure, and the complete unfolding occurs above 70°C. The unfolding is accompanied with aggregation of the protein. The aggregation can however be prevented by relatively low pressure of 0.2 GPa.Using the above phase transition points the T-p phase diagram of parvalbumin was determined in presence of Ca2+. It shows a very complex pattern, containing two different molten globule phases besides the native, unfolded and aggregated ones.

Discontinuous molecular dynamics simulation study of polymer collapse

Discontinuous molecular dynamics simulations were used to study the coil-globule transition of a polymer in an explicit solvent. Two different versions of the model were employed, which are differentiated by the nature of monomer-solvent, solvent-solvent, and nonbonded monomer-monomer interactions. For each case, a model parameter lambda determines the degree of hydrophobicity of the monomers by controlling the degree of energy mismatch between the monomers and solvent particles. We consider a lambda-driven coil-globule transition at constant temperature. The simulations are used to calculate average static structure factors, which are then used to determine the scaling exponents of the system in order to determine the theta-point values lambda(theta) separating the coil from the globule state. For each model we construct coil-globule phase diagrams in terms of lambda and the particle density rho. Additionally, we explore for each model the effects of varying the range of the attractive interactions on the phase boundary separating the coil and globule phases. The results are analyzed in terms of a simple Flory-type theory of the collapse transition.

Knots in Globule and Coil Phases of a Model Polyethylene

We examine the statistics of knots with numerical simulations of a simplified model of polyethylene. We can simulate polymers of up to 1000 monomers (each representing roughly three CH(2) groups), at a range of temperatures spanning coil (good solvent) and globule (bad solvent) phases. We quantify the abundance of knots in the globule phase and in confined polymers, and their rarity in the swollen phase. Since our polymers are open, we consider (and test) various operational definitions for knots, which are rigorously defined only for closed chains. We also associate a typical size with individual knots, which are found to be small (tight and localized) in the swollen phase but large (loose and spread out) in the dense phases.

Equilibrium and kinetic phenomena in a stiff homopolymer and possible applications to DNA

We study the bead-and-spring model of a stiff chain using a self-consistent mean-field approach. For high stiffness parameter the system may undergo a transition to the phase in which the globule acquires a toruslike shape. The phase diagram of the model contains one second- and two first-order transitions meeting at a bicritical point. The stability of the toroidal conformation and scalings of the torus geometry are analyzed. We investigate different kinetic regimes after an instantaneous quench between the extended coil, torus and the spherical globule phases. The kinetic laws that govern these conformational changes are obtained.

HETEROPOLYMER FOLDING ON A APE-100 SUPERCOMPUTER

We discuss a model of protein folding, the IMP model of a random heteropolymer. We discuss the thermodynamic behavior of a linear heteropolymer in which the interaction between different monomers contains a quenched random component. We show the existence, along with the usual coil and globule phases, of a new folded phase, characterized by long relaxation times and by the existence of few stable states. We discuss further relevant features of the model. We describe massively parallel simulations of this model on the APE-100 supercomputer.

Random self-interacting chains: a mechanism for protein folding

The authors investigate by Monte Carlo simulations the thermodynamic behaviour of a linear heteropolymer in which the interaction between different monomers contains a quenched random component. They show the existence, along with the usual coil and globule phases, of a new folded phase, characterized by long relaxation times and by the existence of few stable states.

Gel electrophoretic analysis of proteins in human milk and colostrum

1. 1. Protein patterns of skim milk and fat globule phases were relatively unchanging throughout 9 months lactation, the only notable differences being a rise in lysozyme and a decline up to 6 months with increase thereafter in lactoferrin. 2. 2. Profound changes in protein patterns of the breast secretion were observed during the first week postpartum. 3. 3. True colostrum, in which peptides of sIgA dominate the protein patterns, exists for at most the 2 initial days of secretion. 4. 4. Proteins of mature milk are not coordinated to appear in the colostral secretion simultaneously. A band corresponding to α-lactalbumin is present from the initial secretion; that for β-casein emerges approximately 2 days layer.

Membrane Distribution and Carotenoid Content in Human Colostrum

SummaryDaily colostrum samples from 5 donors were fractionated into fat globules, non‐lipid phase (skim milk), and cells. These and portions of the intact samples were assayed for protein, phospholipid and cholesterol, the latter two serving as criteria of membrane. The intact samples were also analyzed for total lipid and carot‐enoids, and skim milk phases for protein patterns by gel electrophoresis. Membrane material was highest in the first two daily samples with substantial amounts in both fat globule and skim milk phases; quantities declined over the 5‐day sampling period, particularly in the skim milk. Membrane in the cell fraction was &lt;1% of total. In first colostrum there was more than twice as much membrane material as in mature milk (&gt;1 month of lactation), and seven times as much in the skim milk phase. The protein pattern and carotenoid content of colostrum changed markedly during the sampling period. The elevated carotenoids in colostrum are attributed to mobilization of lipids stored in the breast. Based on pigmentation and density, colostrum contains two kinds of fat globules.

Membrane distribution and carotenoid content in human colostrum.

Daily colostrum samples from 5 donors were fractionated into fat globules, non-lipid phase (skim milk), and cells. These and portions of the intact samples were assayed for protein, phospholipid and cholesterol, the latter two serving as criteria of membrane. The intact samples were also analyzed for total lipid and carotenoids, and skim milk phases for protein patterns by gel electrophoresis. Membrane material was highest in the first two daily samples with substantial amounts in both fat globule and skim milk phases; quantities declined over the 5-day sampling period, particularly in the skim milk. Membrane in the cell fraction was less than 1% of total. In first colostrum there was more than twice as much membrane material as in mature milk (greater than 1 month of lactation), and seven times as much in the skim milk phase. The protein pattern and carotenoid content of colostrum changed markedly during the sampling period. The elevated carotenoids in colostrum are attributed to mobilization of lipids stored in the breast. Based on pigmentation and density, colostrum contains two kinds of fat globules.

A method for isolation of milk fat globules.

The traditional procedure for isolating milk fat globules involves repeated cycles of centrifuging to obtain globules and redispersion of them in fresh buffer to eliminate other milk components. We have evaluated a simpler, less manipulative method whereby globules are centrifuged out of the milk and through an overlying buffer layer. Human milk samples ranging from 0.1 to 35 ml were centrifuged at 1500 X g for 20 min after deposition under a suitable quantity of buffer. This yielded purified globules, in less time, which could be dispersed more satisfactorily than those by the traditional procedure. Protein, phospholipid and cholesterol contents of globules by the two methods were quite similar. A lower protein content (10.4 vs 13.2 mg/g of lipid) was characteristic of globules prepared by the multiple wash method. However, large differences could not be seen in gel electrophoresis patterns of the proteins. By using plastic centrifuge tubes, tube freezing and cleavage just below the globule layer enables clean separation of globule and nonglobule phases for analysis of milk component distributions. Macro (5 to 35 ml of sample) and micro (200 microliter or less) versions of the method are described. Limited trials showed that the method can be applied satisfactorily to cow's and goat's milks, but for highly pure globules a deeper buffer column than that used with human milk is required because of their much higher casein content.

Influence of Pasteurization Before and After Separation of Cream on the Oxidative Stability of Ripened Cream Butter

Ripened cream butter manufactured from cream separated from pasteurized milk (80 C for 15 sec) was more susceptible to oxidative changes than the corresponding butter manufactured from cream which was separated from milk at 50 C and the cream subsequently pasteurised (80 C for 15 sec). The propensity to oxidation is related to changes in copper distribution between the serum and fat globule phases and also to the relative ratio of serum to fat at the time of pasteurization.