Kinetics and mechanism of the lamellar gel/lamellar liquid-crystal and lamellar/inverted hexagonal phase transition in phosphatidylethanolamine: a real-time X-ray diffraction study using synchrotron radiation

A study of the kinetics and mechanism of the thermotropic lamellar gel/lamellar liquid-crystalline and lamellar/inverted hexagonal phase transition in dihexadecylphosphatidylethanolamine (DHPE) at various hydration levels has been carried out. Measurements were made by using a real-time X-ray diffraction method at the Cornell High Energy Synchrotron Source. This represents an extension of an earlier study concerning the lamellar gel/lamellar liquid-crystalline phase transition in dipalmitoylphosphatidylcholine [Caffrey, M., & Bilderback, D. H. (1984) Biophys. J. 45, 627-631]. With DHPE, the chain-melting and the nonbilayer transitions were examined under active heating and passive cooling conditions by using a temperature jump to effect phase transformation. Measurements were made at hydration levels ranging from 0% to 60% (w/w) water, and in all cases, the transitions were found to be repeatable, be reversible, and have an upper bound on the transit times (time required to complete the transition) of less than or equal to 3 s. The shortest transit time recorded for the chain-melting and lamellar/hexagonal transitions was less than 1 s. At 8% (w/w) water, the transit times were still on the order of seconds even though the transition does not involve the intermediate L alpha phase. Note, the measured transit times are gross values incorporating the intrinsic transit time in addition to the time required to heat or cool the sample through the transition temperature range and to supply or remove the latent heat of the transition. Regardless of the direction of the transition, both appear to be two state to within the sensitivity limits of the real-time method. From simultaneous wide- and low-angle measurements at the lamellar chain-melting transition, loss of long-range order in the lamellar gel phase appears to precede the chain-melting process. On the basis of the real-time X-ray diffraction measurements, a mechanism is proposed for the lamellar/hexagonal phase transition. The mechanism does not involve large or energetically expensive molecular rearrangements, leads directly to a hexagonal lattice coplanar with the lamellar phase, incorporates facile reversibility, repeatability, and cooperativity, accounts for an observed, apparent memory in the hexagonal phase of the original lamellar phase orientation, and is consistent with the experimental observation of a predominantly two-state transition. In conjunction with the kinetic measurements, the DHPE/water phase diagram was constructed. At and above 12% (w/w) water, the thermotropic transition sequence is L beta'/L alpha/HII.(ABSTRACT TRUNCATED AT 400 WORDS)     

Kinetics of the main phase transition of hydrated lecithin monitored by real-time X-ray diffraction.

A method is described for observing and recording in real-time x-ray diffraction from an unoriented hydrated membrane lipid, dipalmitoylphosphatidylcholine (DPPC), through its thermotropic gel/liquid crystal phase transition. Synchrotron radiation from the Cornell High Energy Synchrotron Source (Ithaca, New York) was used as an x-ray source of extremely high brilliance and the dynamic display of the diffraction image was effected using a three-stage image intensifier tube coupled to an external fluorescent screen. The image on the output phosphor was sufficiently intense to be recorded cinematographically and to be displayed on a television monitor using a vidicon camera at 30 frames X s1. These measurements set an upper limit of 2 s on the DPPC gel----liquid crystal phase transition and indicate that the transition is a two-state process. The real-time method couples the power of x-ray diffraction as a structural probe with the ability to follow kinetics of structural changes. The method does not require an exogenous probe, is relatively nonperturbing, and can be used with membranes in a variety of physical states and with unstable samples. The method has the additional advantage over its static measurement counterpart in that it is more likely to detect transiently stable intermediates if present.     

Kinetics and mechanism of the pressure-induced lamellar order/disorder transition in phosphatidylethanolamine: a time-resolved X-ray diffraction study

By using synchrotron radiation, a movie was made of the X-ray scattering pattern from a biological liquid crystal undergoing a phase transition induced by a pressure jump. The system studied includes the fully hydrated phospholipid dihexadecylphosphatidylethanolamine in the lamellar gel (L beta') phase at a temperature of 68 degrees C and a pressure of 9.7 MPa (1400 psig). Following the rapid release of pressure to atmospheric the L beta' phase transforms slowly into the lamellar liquid crystal (L alpha) phase. The pressure perturbation is applied with the intention of producing a sudden phase disequilibrium followed by monitoring the system as it relaxes to its new equilibrium condition. Remarkably, the proportion of sample in the L alpha phase grows linearly with time, taking 37 s to totally consume the L beta' phase. The time dependencies of radius, peak intensity, and width of the powder diffraction ring of the low-angle (001) lamellar reflections were obtained from the movie by image processing. The concept of an "effective pressure" is introduced to account for the temperature variations that accompany the phase transition and to establish that the observed large transit time is indeed intrinsic to the sample and not due to heat exchange with the environment. The reverse transformation, L alpha to L beta', induced by a sudden jump from atmospheric pressure to 9.7 MPa, is complete in less than 13 s. These measurements represent a new approach for studying the kinetics of lipid phase transitions and for gaining insights into the mechanism of the lamellar order/disorder transition.     

Peptidomic analysis of skin secretions demonstrates that the allopatric populations of Xenopus muelleri (Pipidae) are not conspecific

Mueller's clawed frog Xenopus muelleri (Peters 1844) occupies two non-contiguous ranges in east and west Africa. The phylogenetic relationship between the two populations is unclear and it has been proposed that the western population represents a separate species. Peptidomic analysis of norepinephrine-stimulated skin secretions from X. muelleri from the eastern range resulted in the identification of five antimicrobial peptides structurally related to the magainins (magainin-M1 and -M2), xenopsin-precursor fragments (XPF-M1) and caerulein-precursor fragments (CPF-M1 and -M2) previously found in skin secretions of other Xenopus species. A cyclic peptide (WCPPMIPLCSRF.NH₂) containing the RFamide motif was also isolated that shows limited structural similarity to the tigerinins, previously identified only in frogs of the Dicroglossidae family. The components identified in skin secretions from X. muelleri from the western range comprised one magainin (magainin-MW1), one XPF peptide (XPF-MW1), two peptides glycine-leucine amide (PGLa-MW1 and -MW2), and three CPF peptides (CPF-MW1, -MW2 and -MW3). Comparison of the primary structures of these peptides suggest that western population of X. muelleri is more closely related to X. borealis than to X. muelleri consistent with its proposed designation as a separate species. The CPF peptides showed potent, broad-spectrum activity against reference strains of bacteria (MIC 3-25 μM), but were hemolytic against human erythrocytes.     

A direct demonstration that the ethanol-induced transition of DNA is between the A and B forms: an X-ray diffraction study

DNA undergoes a reversible co-operative change in a number of its properties over a characteristic range of ethanol concentrations. This ethanol-induced transition of DNA has been studied by a number of groups, using circular dichroism as well as other assay techniques. There has been disagreement as to the species of DNA involved. We have used X-ray diffraction methods on DNA fibers exposed to an excess of solvent. The diffraction patterns serve as “fingerprints” to clearly show that the transition occurs between the B and A forms of DNA (at low and high ethanol concentrations, respectively) in agreement with the conclusions of some of the previous workers. At still higher ethanol concentrations, the diffraction pattern changes from that of the A form to that of a more disordered form.     

Mechanism of the lamellar/inverse hexagonal phase transition examined by high resolution x-ray diffraction

For the first time the electron density of the lamellar liquid crystalline as well as of the inverted hexagonal phase could be retrieved at the transition temperature. A reliable decomposition of the d-spacings into hydrophobic and hydrophilic structure elements could be performed owing to the presence of a sufficient number of reflections. While the hydrocarbon chain length, d(C), in the lamellar phase with a value of 14.5 A lies within the extreme limits of the estimated chain length of the inverse hexagonal phase 10 A < d(C) < 16 A, the changes in the hydrophilic region vary strongly. During the lamellar-to-inverse hexagonal phase transition the area per lipid molecule reduces by approximately 25%, and the number of water molecules per lipid increases from 14 to 18. On the basis of the analysis of the structural components of each phase, the interface between the coexisting mesophases between 66 and 84 degrees C has been examined in detail, and a model for the formation of the first rods in the matrix of the lamellar phospholipid stack is discussed. Judging from the structural relations between the inverse hexagonal and the lamellar phase, we suggest a cooperative chain reaction of rod formation at the transition midpoint, which is mainly driven by minimizing the interstitial region.     

A multigene phylogeny demonstrates that Tuber aestivum and Tuber uncinatum are conspecific

For almost 2 centuries it has been disputed whether Tuber aestivum and Tuber uncinatum constitute two different species of truffles. Molecular markers have been applied previously to contribute to resolving this question, coming to different conclusions. In this study, we address this question by analyzing the genetic structure of truffles assigned to either of the two putative species from a geographically broad sampling across Europe. We used an approach involving multigene phylogenies and coalescent analyses of nine regions from five genes. All tests conducted supported the conspecificity of Tuber aestivum and Tuber uncinatum.     

X-ray diffraction study on ordered, disordered and reconstituted intercellular lipid lamellar structure in stratum corneum

From small angle X-ray diffraction for the stratum corneum of hairless mouse, it was obtained that in the normal stratum corneum, the 1st, 2nd and 3rd order diffraction peaks for the intercellular lipid lamellar structure appear at 13.8, 6.87 and 4.59 nm, respectively and also a broad hump for the 4th order reflection appears as observed by the previous researchers. In the damaged stratum corneum prepared by the treatment of sodium dodecyl sulfate, these small-angle diffraction peaks disappear and only the broad maxima remain around the 1st, 2nd and 3rd order diffraction peaks. These facts indicate that in the normal stratum the lamellar structure is ordered and in the damaged stratum corneum the lamellar structure is disordered. Furthermore, in the reconstituted lamellar structure obtained by immersing into the dilute suspension of the mixture of ceramide 3, cholesterol and stearic acid, the 1st, 2nd and 3rd order diffraction peaks reappear at 13.3, 6.67 and 4.44 nm, respectively. This fact indicates that the reorganization of the ordered lamellar structure takes place by adding the mixture to the damaged stratum corneum.     

Kinetics of the lamellar-inverse hexagonal phase transition determined by time-resolved X-ray diffraction.

The kinetics of the lamellar (L alpha)-inverse hexagonal (HII) phase transition in diacylphosphatidylethanolamine (PE)--water systems were probed with time-resolved X-ray diffraction. Transition kinetics in the fast time regime (approximately 100 ms) were studied by initiating large temperature jumps (up to 30 degrees C) with a 50-ms electrical current pulse passed through a lipid-salt water dispersion, resulting in ohmic heating of the sample. Diffraction with a time resolution to 10 ms was acquired at the National Synchrotron Light Source. The time constant for the phase transition for 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was on the order of 100 ms for the largest temperature jumps recorded. Faster transition behavior was found for a 1,2-dielaidoyl-sn-glycero-3-PE mixture. The HII lattice parameters for both systems were seen to swell from an initial value commensurate with the lamellar lattice to the final equilibrium value. The rate of swelling was seen to be independent of the magnitude of the temperature jump. For small temperature jumps (less than 10 degrees C), the phase transition kinetics slow dramatically, and transition studies can readily be performed on a conventional rotating anode X-ray source. At 4 degrees C, a DOPE sample was observed to slowly convert to the hexagonal phase over the course of a week, with the decay in the lamellar intensity fitting a power law behavior over four decades of time. This power law behavior is shown to have interesting consequences to the determination of the phase transition temperature of lipid-water dispersions by conventional methods such as calorimetry.     

Atomic force microscopy imaging demonstrates that P2X2 receptors are trimers but that P2X6 receptor subunits do not oligomerize

P2X receptors are cation-selective channels activated by extracellular ATP. The architecture of these receptors is still not completely clear. Here we have addressed this issue by both chemical cross-linking and direct imaging of individual receptors by atomic force microscopy (AFM). Cross-linking of the P2X(2) receptor produced higher order adducts, consistent with the presence of trimers. The mean molecular volume of the receptor determined by AFM (409 nm(3)) also points to a trimeric structure. P2X(2) receptors bearing His(6) epitope tags were incubated with anti-His(6) antibodies, and the resultant complexes were imaged by AFM. For receptors with two bound antibodies, the mean angle between the antibodies was 123 degrees , again indicating that the receptor is a trimer. In contrast, cross-linking of the P2X(6) receptor did not produce higher order adducts, and the mean molecular volume of the receptor was 145 nm(3). We conclude that P2X(2) receptors are trimers, whereas the P2X(6) receptor subunits do not form stable oligomers.     

Structure of lamellar lipid domains and corneocyte envelopes of murine stratum corneum. An X-ray diffraction study

The lipid of the outermost layer of the skin is confined largely to the extracellular spaces surrounding the corneocytes of the stratum corneum where it forms a multilamellar adhesive matrix to act as the major permeability barrier of the skin. Knowledge of the molecular architecture of these intercellular domains is important for understanding various skin pathologies and their treatment, percutaneous drug delivery, and the cosmetic maintenance of the skin. We have surveyed by X-ray diffraction the structure of the intercellular domains and the extracted lipids of murine stratum corneum (SC) at 25, 45, and 70 degrees C which are temperatures in the vicinity of known thermal phase transitions [Rehfeld, S. J., & Elias, P. M. (1982) J. Invest. Dermatol. 79, 1-3]. The intercellular domains produce lamellar diffraction patterns with a Bragg spacing of 131 +/- 2 A. Lipid extracted from the SC and dispersed in excess water does not produce a simple lamellar diffraction pattern at any temperature studied, however. This and other facts suggest that another component, probably a protein, must be present to control the architecture of the intercellular lipid domains. We have also obtained diffraction patterns attributable to the protein envelopes of the corneocytes. The patterns suggest a beta-pleated sheet organizational scheme. No diffraction patterns were observed that could be attributed to keratin.     

Water-silica gel interactions,X-ray diffraction study at room and low temperature

X-ray diffraction experiments on water confined in silica gel powder hydrated at about 20% are presented and analyzed at room temperature and down to 77 K. The structural modification of confined water observed at second neighbors is due to the competition between the confinement effect and the water-silica interaction.     

Mitochondrial DNA barcoding detects some species that are real, and some that are not

Mimicry and extensive geographical subspecies polymorphism combine to make species in the ithomiine butterfly genus Mechanitis (Lepidoptera; Nymphalidae) difficult to determine. We use mitochondrial DNA (mtDNA) barcoding, nuclear sequences and amplified fragment length polymorphism (AFLP) genotyping to investigate species limits in this genus. Although earlier biosystematic studies based on morphology described only four species, mtDNA barcoding revealed eight well-differentiated haplogroups, suggesting the presence of four new putative 'cryptic species'. However, AFLP markers supported only one of these four new 'cryptic species' as biologically meaningful. We demonstrate that in this genus, deep genetic divisions expected on the basis of mtDNA barcoding are not always reflected in the nuclear genome, and advocate the use of AFLP markers as a check when mtDNA barcoding gives unexpected results.     

Infrared and X-ray diffraction study of the effect of protonation of DNA on its B-to-A transition

The influence of H+ on the secondary structure of DNA and on its B-to-A transition has been studied by employing X-ray diffraction and infrared spectroscopy. Helical parameters for DNA molecules with different degrees of protonation were determined. It was shown that H+ binding stabilizes the B-form of DNA in fibers over a wide range of water and inorganic salt content. Only 0.03 H+ bound per nucleotide is sufficient to prevent the B-to-A transition caused by decreasing relative humidity in DNA fibers containing 4% NaCl. The effectiveness of B-form stabilization by H+ is explained by changes in DNA-solvent molecule interactions, especially in the major groove of double helices.     

Kinetics and mechanism of the barotropic lamellar gel/lamellar liquid crystal phase transition in fully hydrated dihexadecylphosphatidylethanolamine: a time-resolved x-ray diffraction study using pressure jump.

The kinetics and mechanism of the barotropic lamellar gel (L beta')/lamellar liquid crystal (L alpha) phase transition in fully hydrated 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE) has been studied using time-resolved x-ray diffraction (TRXRD). The phase transition was induced by pressure jumps of varying amplitudes in both the pressurization and depressurization directions at controlled temperature (78 degrees C). Both low- and wide-angle diffracted x rays were recorded simultaneously in live time using an x-ray-sensitive image intensifier coupled to a CCD camera and Super-VHS videotape recorder. Such an arrangement allowed for the direct and quantitative characterization of the long- (lamellar repeat spacing) and short-range order (chain packing) during a kinetic experiment. The image-processed live-time x-ray diffraction data were fitted using a nonlinear least-squares model, and the parameters of the fits were monitored continuously throughout the transition. The pressure-induced transitions from the L alpha to the L beta' phase and from the L beta' to the L alpha phase was two-state (no formation of intermediates apparent during the transition) to within the sensitivity limits of the method. The corresponding transit time (the time during which both phases coexist) associated with the long- and short-range order of the pressurization-induced L alpha-to-L beta' phase transition decreased to a limiting value of approximately 50 ms with increasing pressure jump amplitude. This limiting value was close to the response time of the detector/recording system. Thus, the intrinsic transit time of this transition in fully hydrated DHPE at 78 degrees C was less than or equal to 50 ms. In contrast, the depressurization-induced L beta'-to-L alpha phase transition was slower, taking approximately 1 s to complete, and occurred with no obvious dependence of the transit time on pressure jump amplitude. In the depressurization jump experiment, the lipid responded rapidly to the pressure jump in the L beta' phase up to the rate-determining L beta'-to-L alpha transition. Such behavior was examined carefully, as it could complicate the interpretation of phase transition kinetic measurements.     

Evidence that Enzyme Polymorphisms are not Selectively Neutral

THE discovery of large amounts of electrophoretically-detectable genetic variation within natural populations has aroused considerable interest in the factors maintaining so much polymorphism. It has been proposed that these allozyme polymorphisms reflect the action of random processes^1–6, the polymorphic variation being of little or no selective significance. Alternative hypotheses have suggested that these polymorphisms may be maintained by a balance of selective forces^7–10.