Phase separation in aqueous solutions of lens gamma-crystallins: special role of gamma s.

We have studied liquid-liquid phase separation in aqueous ternary solutions of calf lens gamma-crystallin proteins. Specifically, we have examined two ternary systems containing gamma s--namely, gamma IVa with gamma s in water and gamma II with gamma s in water. For each system, the phase-separation temperatures (Tph (phi)) alpha as a function of the overall protein volume fraction phi at various fixed compositions alpha (the "cloud-point curves") were measured. For the gamma IVa, gamma s, and water ternary solution, a binodal curve composed of pairs of coexisting points, (phi I, alpha 1) and (phi II, alpha II), at a fixed temperature (20 degrees C) was also determined. We observe that on the cloud-point curve the critical point is at a higher volume fraction than the maximum phase-separation temperature point. We also find that typically the difference in composition between the coexisting phases is at least as significant as the difference in volume fraction. We show that the asymmetric shape of the cloud-point curve is a consequence of this significant composition difference. Our observation that the phase-separation temperature of the mixtures in the high volume fraction region is strongly suppressed suggests that gamma s-crystallin may play an important role in maintaining the transparency of the lens.

Characteristics of virus-specific CD8+ T cells in the liver during the control and resolution phases of influenza pneumonia

Dissection of the primary and secondary response to an influenza A virus established that the liver contains a substantial population of CD8+ T cells specific for the immunodominant epitope formed by H-2Db and the influenza virus nucleoprotein peptide fragment NP366–374 (DbNP366). The numbers of CD8+ DbNP366+ cells in the liver reflected the magnitude of the inflammatory process in the pneumonic lung, though replication of this influenza virus is limited to the respiratory tract. Analysis of surface phenotypes indicated that the liver CD8+ DbNP366+ cells tended to be more “activated” than the set recovered from lymphoid tissue but generally less so than those from the lung. The distinguishing characteristic of the lymphocytes from the liver was that the prevalence of the CD8+ DbNP366+ set was always much higher than the percentage of CD8+ T cells that could be induced to synthesize interferon γ after short-term, in vitro stimulation with the NP366–374 peptide, whereas these values were generally comparable for virus-specific CD8+ T cells recovered from other tissue sites. Also, the numbers of apoptotic CD8+ T cells were higher in the liver. The results overall are consistent with the idea that antigen-specific CD8+ T cells are destroyed in the liver during the control and resolution phases of this viral infection, though this destruction is not necessarily an immediate process.

Lipidic cubic phases: A novel concept for the crystallization of membrane proteins

Understanding the mechanisms of action of membrane proteins requires the elucidation of their structures to high resolution. The critical step in accomplishing this by x-ray crystallography is the routine availability of well-ordered three-dimensional crystals. We have devised a novel, rational approach to meet this goal using quasisolid lipidic cubic phases. This membrane system, consisting of lipid, water, and protein in appropriate proportions, forms a structured, transparent, and complex three-dimensional lipidic array, which is pervaded by an intercommunicating aqueous channel system. Such matrices provide nucleation sites (“seeding”) and support growth by lateral diffusion of protein molecules in the membrane (“feeding”). Bacteriorhodopsin crystals were obtained from bicontinuous cubic phases, but not from micellar systems, implying a critical role of the continuity of the diffusion space (the bilayer) on crystal growth. Hexagonal bacteriorhodopsin crystals diffracted to 3.7 Å resolution, with a space group P63, and unit cell dimensions of a = b = 62 Å, c = 108 Å; α = β = 90° and γ = 120°.

Equilibration rates in lipid monolayers

A theoretical analysis is given for the rate of change of domain sizes in lipid monolayers at the air–water interface. The calculation is applicable to liquid domains formed from binary mixtures of lipids that form two coexisting liquid phases. Under conditions where the two lipid molecules have approximately equal areas, the equilibration rate does not involve macroscopic hydrodynamic flow in the subphase but rather depends on the diffusion coefficient of the lipid molecules. The calculation shows that the equilibration rate in binary mixtures of cholesterol and phosphatidylcholine is remarkably slow, the radius of a typical 20-μm diameter domain changing by as little as a part in a million per second. Under these circumstances, equilibration times of the order of days or weeks are expected. Even with such long times, the final state reached by the monolayer will in general be a state of metastable equilibrium, rather than true equilibrium.

Stripe phases in high-temperature superconductors

Stripe phases are predicted and observed to occur in a class of strongly correlated materials describable as doped antiferromagnets, of which the copper-oxide superconductors are the most prominent representatives. The existence of stripe correlations necessitates the development of new principles for describing charge transport and especially superconductivity in these materials.

Two Genetically Separable Phases of Growth Inhibition Induced by Blue Light in Arabidopsis Seedlings1

High fluence-rate blue light (BL) rapidly inhibits hypocotyl growth in Arabidopsis, as in other species, after a lag time of 30 s. This growth inhibition is always preceded by the activation of anion channels. The membrane depolarization that results from the activation of anion channels by BL was only 30% of the wild-type magnitude in hy4, a mutant lacking the HY4 BL receptor. High-resolution measurements of growth made with a computer-linked displacement transducer or digitized images revealed that BL caused a rapid inhibition of growth in wild-type and hy4 seedlings. This inhibition persisted in wild-type seedlings during more than 40 h of continuous BL. By contrast, hy4 escaped from the initial inhibition after approximately 1 h of BL and grew faster than wild type for approximately 30 h. Wild-type seedlings treated with 5-nitro-2-(3-phenylpropylamino)-benzoic acid, a potent blocker of the BL-activated anion channel, displayed rapid growth inhibition, but, similar to hy4, these seedlings escaped from inhibition after approximately 1 h of BL and phenocopied the mutant for at least 2.5 h. The effects of 5-nitro-2-(3-phenylpropylamino)-benzoic acid and the HY4 mutation were not additive. Taken together, the results indicate that BL acts through HY4 to activate anion channels at the plasma membrane, causing growth inhibition that begins after approximately 1 h. Neither HY4 nor anion channels appear to participate greatly in the initial phase of inhibition.

Direct structure analysis in protein electron crystallography: crystallographic phases for halorhodopsin to 6-A resolution.

The crystal structure of halorhodopsin was determined in (centrosymmetric) projection to 6-A resolution by direct methods that use only the amplitudes of the electron diffraction pattern. A multisolution technique was used to generate initial 15-A-resolution basis sets, and after selection of the best phase set (by the closest match of magnitude of Eobs and magnitude of Ecalc), annealing of individual reflections was used to improve its accuracy. The Sayre equation was then used to expand the phase terms to 10 A, followed again by phase annealing. A final expansion with the Sayre equation enlarged this corrected phase set to 6 A. When the condition of density flatness was used to locate the best phase solution after each extension, a final structure could be observed that was quite similar to the one found earlier by analysis of electron micrographs.