Fabrication of Asymmetric Phosphatidylserine-Containing Lipid Vesicles: A Study on the Effects of Size, Temperature, and Lipid Composition.

The asymmetric distribution of lipids in plasma membranes is closely related to the physiological functions of cells. To improve our previous approach in fabricating asymmetric vesicles, we defined a parameter, asymmetric degree, in this work and investigated the effects of vesicle size, incubation temperature, and lipid composition on the formation process of asymmetric phosphatidylserine (PS)-containing lipid vesicles. The results indicate that all of the three factors have marked but different effects on the time-dependent asymmetric degree of the vesicles as well as the flip and flop rate constants of the PS lipids. However, only vesicle size and PS content show significant influence on the maximal asymmetric degree of the vesicles, while the incubation temperature exhibits negligible effect. This work not only deepens our understanding on the packing property of PS molecules in self-assembled membranes and the formation mechanism of asymmetric vesicles but also practically provides a solution to regulate the asymmetric degree of the PS-containing vesicles using the established kinetic equation. In addition, the method would facilitate researches related to asymmetric vesicles or reconstruction of biological membranes.

Controllable engineering of asymmetric phosphatidylserine-containing lipid vesicles using calcium cations.

A novel method to produce controllable asymmetric lipid vesicles using Ca2+ is reported. The enrichment of negatively charged phosphatidylserine (PS) molecules in the inner leaflet is found not due to charge-charge attraction, but rather a modulation effect on the occupying size of the headgroups of PS molecules.

Two-State or Non-Two-State? An Excess Spectroscopy-based Approach to Differentiate the Existing Forms of Molecules in Liquids Mixtures.

Characterization/identification of the clusters/associates in liquids has long been a challenging topic. In this paper, we report a method to identify molecules with two different existing forms in a binary liquid solution. In this so-called two-state situation, the excess infrared spectra of a vibration mode of the respective molecule will show identical band shape if the other component is transparent in the region. More conveniently, the positions of the positive peak, negative peak, and zero-value will be seen to be fixed with varying compositions of the binary system. In the case of non-two-state mixtures, for example the mere solvation of solute by solvent, those positions will be variable. The conclusions are supported/demonstrated by computational simulation and experiments on two binary systems, D2O-H2O and C6F5I-cyclo-C6H12.

Complexation of Lysozyme with Sodium Poly(styrenesulfonate) via the Two-State and Non-Two-State Unfoldings of Lysozyme.

To provide an in-depth understanding of the complexation mechanism of protein and polyelectrolyte, a heating-cooling-reheating protocol was employed to study the unfolding and refolding behaviors of a model protein, lysozyme, in the presence of a negatively charged polyelectrolyte, sodium poly(styrenesulfonate) (PSS). It was found that, with elevated PSS concentration, a new state (state I) was first formed via a "two-state" conversion process and this state could further convert to a completely unfolded state (state II) via a "non-two-state" conversion. This non-two-state conversion process occurs without the coexistence of states I and II but involves the formation of various intermediate unfolded protein structures. Different from the pure lysozyme that exhibited refolding upon cooling from its heat-denatured state, lysozyme in state I could undergo unfolding upon heating but no refolding upon cooling, while lysozyme in state II did not undergo unfolding or refolding upon thermal treatments. In addition, the effects of ionic strength and molecular weight of polyelectrolyte on the unfolding and refolding behaviors of lysozyme were also investigated. The present work provides a better understanding of the principles governing protein-polyelectrolyte interactions and may have implications for the fabrication of biocolloids and biofilms.

Comparative study of halogen- and hydrogen-bond interactions between benzene derivatives and dimethyl sulfoxide.

The halogen bond, similar to the hydrogen bond, is an important noncovalent interaction and plays important roles in diverse chemistry-related fields. Herein, bromine- and iodine-based halogen-bonding interactions between two benzene derivatives (C6 F5 Br and C6 F5 I) and dimethyl sulfoxide (DMSO) are investigated by using IR and NMR spectroscopy and ab initio calculations. The results are compared with those of interactions between C6 F5 Cl/C6 F5 H and DMSO. First, the interaction energy of the hydrogen bond is stronger than those of bromine- and chlorine-based halogen bonds, but weaker than iodine-based halogen bond. Second, attractive energies depend on 1/r(n) , in which n is between three and four for both hydrogen and halogen bonds, whereas all repulsive energies are found to depend on 1/r(8.5) . Third, the directionality of halogen bonds is greater than that of the hydrogen bond. The bromine- and iodine-based halogen bonds are strict in this regard and the chlorine-based halogen bond only slightly deviates from 180°. The directional order is iodine-based halogen bond>bromine-based halogen bond>chlorine-based halogen bond>hydrogen bond. Fourth, upon the formation of hydrogen and halogen bonds, charge transfers from DMSO to the hydrogen- and halogen-bond donors. The CH3 group contributes positively to stabilization of the complexes.

Demixing and crystallization of DODAB in DPPC-DODAB binary mixtures.

The crystallization mechanism of one lipid component within multicomponent lipid mixtures remains unclear. To shed light on this issue, we studied the demixing and crystallization behaviors of a binary lipid system using neutral dipalmitoylphosphatidylcholine (DPPC) and cationic dioctadecyldimethylammonium bromide (DODAB) as model molecules. The results indicate that when DODAB is no more than equimolar (e.g., DPPC/DODAB = 2/1 and 1/1), DPPC is miscible with DODAB and hinders the crystallization of DODAB, and the samples undergo reversible gel-fluid phase transitions upon heating and cooling. However, when DODAB is dominant in the mixture (DPPC/DODAB = 1/2), cooling of the mixed fluid phase results in the formation of a DODAB-rich gel domain and a DPPC-DODAB mixed gel domain. Such phase-separated mixed gels can undergo further demixing and crystallization, producing a DODAB-rich crystalline domain and a DPPC-rich tilted gel domain upon prolonged (or plus low-temperature) incubation. Besides, evidence has been given that the crystallized DODAB-rich domain remains in the same lipid bilayer as the DPPC-rich domain. All the three binary lipid mixtures can hold large amounts of water in the lipid interlamellar regions, allowing the incorporation of a large number of water-soluble substances such as DNA or proteins, which can be used for the fabrication of functional biofilms and biomaterials. Influences of water content and salt concentration on the phase structures (e.g., repeat distances) of the binary mixtures have also been studied.

Partial molecular cloning of the JHK retrovirus using gammaretrovirus consensus PCR primers.

The JHK virus (JHKV) was previously described as a type C retrovirus that has some distinctive ultrastructural features and replicates constitutively in a human B-lymphoblastoid cell line, JHK-3. In order to facilitate the cloning of sequences from JHKV, a series of partially degenerate consensus retroviral PCR primers were created by a data-driven design approach based on an alignment of 14 diverse gammaretroviral genomes. These primers were used in the PCR amplification of purified JHK virion cDNA, and ana lysis of the resulting amplified sequence indicates that the JHKV is in the murine leukemia virus (MLV) family. The JHK sequence is nearly identical to the corresponding region of the Bxv-1 endogenous mouse retrovirus (GenBank accession AC115959) and distinct from XMRV. JHKV gag-specific amplification was demonstrated with nucleic acids from uncultivated, frozen, peripheral blood mononuclear cells (PBMCs) of the index patient, but not in PBMCs from nine healthy blood donors. Unlike earlier reports, in which MLV-like sequences were identified in human source material, which may have been due to murine contamination, budding retrovirions were demonstrated repeatedly by electron microscopy in uncultivated lymphocytes of the index patient that were morphologically identical in their development to the virions in the JHK-3 cells, and immunological evidence was obtained that the index patient produced IgG antibodies that bound to the budding viral particles in patient PBMCs and in the JHK-3 cells. These data indicate that the patient had been infected by JHKV, lending significance to the demonstration of JHKV amplicons in nucleic acids of the patient's PBMCs. In future studies, the PCR primer sets described herein may expand the detection of an amplifiable subset of viruses related to MLV.

Stepwise ordering of imidazolium-based cationic surfactants during cooling-induced crystallization.

Surfactants bearing imidazolium cations represent a new class of building blocks in molecular self-assembly. These imidazolium-based cationic surfactants can exhibit various morphologies during phase transformations. In this work, we studied the self-assembly and phase behavior of 1-hexadecyl-3-methylimidazolium chloride (C(16)mimCl) aqueous dispersions (0.5-10 wt %) by using isothermal titration calorimetry, differential scanning calorimetry, synchrotron small- and wide-angle X-ray scattering, freeze-fracture electron microscopy, optical microscopy, electrical conductance, and Fourier transform infrared spectroscopy. It was found that C(16)mimCl in aqueous solutions can form two different crystalline phases. At higher C(16)mimCl concentrations (>6 wt %), the initial spherical micelles convert directly to the stable crystalline phase upon cooling. At lower concentrations (0.5 or 1 wt %), the micelles first convert to a metastable crystalline phase upon cooling and then transform to the stable crystalline phase upon further incubation at low temperature. The electrical conductance measurement reveals that the two crystalline phases have similar surface charge densities and surface curvatures. Besides, the microscopic and spectroscopic investigations of the two crystalline phases suggest that the metastable crystalline phase has preassembled morphology and a preordered submolecular packing state that contribute to the final stable crystalline structure. The formation of a preordered structure prior to the final crystalline state deepens our understanding of the crystallization mechanisms of common surfactants and amphiphilic ionic liquids and should thus be widely recognized and explored.

Urea-nuclease treatment of concentrated retrovirions preserves viral RNA and removes polymerase chain reaction-amplifiable cellular RNA and DNA.

Cellular nucleic acids can interfere with the molecular cloning of retroviruses, a problem that is particularly serious with viruses propagated in lymphoblastoid cells that release large amounts of microvesicles and other cellular components. The approach taken to circumvent such problems involved first suspending viral pellets in water to allow any residual microvesicles to swell and perhaps lyse during overnight or longer incubation periods. Urea was then added to a concentration of 1.5-2.0 M to uncoil proteins that may protect nucleic acids from hydrolysis on the further addition of Micrococcal nuclease and ribonuclease A, both of which remain enzymatically active in molar urea solutions. The viral RNA was extracted and residual DNA removed by deoxyribonuclease I treatments. The utility of the method was demonstrated with two different retroviruses, a Moloney murine leukemia virus variant and Rous sarcoma virus, such that viral RNA thus purified was shown to be free of contamination by PCR-amplifiable cellular GAPDH mRNA and ribosomal RNA. This general approach should be applicable to viruses of any type in circumstances where contamination by cellular RNA and DNA poses a problem.

Molecular cloning, complete sequence, and biological characterization of a xenotropic murine leukemia virus constitutively released from the human B-lymphoblastoid cell line DG-75.

A previously undetected retrovirus has been isolated from the human Epstein-Barr virus (EBV)-negative, B-lymphoblastoid DG-75 cell line, widely used for EBV gene transfection studies. The complete 8207-base genome of the DG-75 retrovirus was molecularly cloned from viral mRNA and sequenced (Accession No. AF221065). Northern blot analysis with probes specific for the putative RU-5, gag, pol, and env regions identified a full-length viral RNA and spliced env mRNA. DG-75 viral RNA was isolated from the DG-75 cell sublines UW and KAR, but not from the HAD subline. The DG-75 retrovirus was isolated with primer-binding sites that match tRNA(Thr) and tRNA(Gln2). Homology searches revealed homology to (i) xenotropic NZB-9-1 env mRNA, (ii) Moloney-MLV pol region, and (iii) a truncated Evi-2 endogenous proviral sequence gag and pol region. Viral interference and infectivity assays confirmed the xenotropic nature of the DG-75 retrovirus. The DG-75 retrovirus is the first isolate of an exogenous xenotropic MLV in which the full-length genomic sequence has been characterized.