Experimental support for tilt-dependent theory of biomembrane mechanics.

Recent simulations have indicated that the traditional model for topographical fluctuations in biomembranes should be enriched to include molecular tilt. Here we report the first experimental data supporting this enrichment. Utilizing a previously posited tilt-dependent model, a height-height correlation function was derived. The x-ray scattering from a liquid crystalline stack of oriented fluid phase lipid bilayers was calculated and compared with experiment. By fitting the measured scattering intensity, both the bending modulus K(c)=8.3±0.6×10⁻²° J and the tilt modulus K(θ)=95±7 mN/m were determined for DOPC lipid bilayers at 30 °C.

Exploiting intrinsic triangular geometry in relativistic (3)He+Au collisions to disentangle medium properties.

Recent results in d+Au and p+Pb collisions at RHIC and the LHC provide evidence for collective expansion and flow of the created medium. We propose a control set of experiments to directly compare particle emission patterns from p+Pb, d+Au, and ^{3}He+Au or t+Au collisions at the same sqrt[s_{NN}] . Using a Monte Carlo Glauber simulation we find that a ^{3}He or triton projectile, with a realistic wave function description, induces a significant intrinsic triangular shape to the initial medium. If the system lives long enough, this survives into a significant third-order flow moment v_{3} even with viscous damping. By comparing systems with one, two, and three initial hot spots, one could disentangle the effects from the initial spatial distribution of the deposited energy and viscous damping. These are key tools for answering the question of how small a droplet of matter is necessary to form a quark-gluon plasma described by nearly inviscid hydrodynamics.

Identification of fifteen novel mutations and genotype-phenotype relationship in Fabry disease.

Fabry disease is an X-linked recessive disorder caused by a deficiency in the lysosomal enzyme alpha-galactosidase A, which results in a progressive multisystem disease. Most families have private mutations and no general correlation between genotype and disease manifestations has been described to date. Forty-nine patients (47 males and 2 females) from 36 affected families were selected for the study. Their evaluation included clinical examination, identification of alpha-galactosidase A gene mutations and residual enzymatic activity. For mutation detection, each exon with flanking intronic sequences was amplified by polymerase chain reaction (PCR) from the patient's genomic DNA and sequenced. Analysis of the resulting sequences was conducted to identify structural defects in the gene. Each of the Fabry patients carried a mutation in the alpha-galactosidase A gene. Fifteen mutations were novel. They included missense mutations (M51K, Y123M, G261D), nonsense point mutations (E251X) and small insertions or deletions creating a premature translational termination signal (P6X, D93X, W162X, K240X, H302X, I303X, L403X, S345X, G375X, F396X). Residual alpha-galactosidase A activity was significantly lower in patients with neuropathic pain (p=0.01) and in patients with mutations leading to a nonconservative amino acid change (p=0.04). Our findings emphasize the wide variety of genetic mechanisms leading to Fabry disease. A significant genotype-phenotype relationship was found.

The thermotropic phase behavior of cationic lipids: calorimetric, infrared spectroscopic and X-ray diffraction studies of lipid bilayer membranes composed of 1,2-di-O-myristoyl-3-N,N,N-trimethylaminopropane (DM-TAP).

The thermotropic phase behavior of lipid bilayer model membranes composed of the cationic lipid 1,2-di-O-myristoyl-3-N,N,N-trimethylaminopropane (DM-TAP) was examined by differential scanning calorimetry, infrared spectroscopy and X-ray diffraction. Aqueous dispersions of this lipid exhibit a highly energetic endothermic transition at 38.4 degrees C upon heating and two exothermic transitions between 20 and 30 degrees C upon cooling. These transitions are accompanied by enthalpy changes that are considerably greater than normally observed with typical gel/liquid--crystalline phase transitions and have been assigned to interconversions between lamellar crystalline and lamellar liquid--crystalline forms of this lipid. Both infrared spectroscopy and X-ray diffraction indicate that the lamellar crystalline phase is a highly ordered, substantially dehydrated structure in which the hydrocarbon chains are essentially immobilized in a distorted orthorhombic subcell. Upon heating to temperatures near 38.4 degrees C, this structure converts to a liquid-crystalline phase in which there is excessive swelling of the aqueous interlamellar spaces owing to charge repulsion between, and undulations of, the positively charged lipid surfaces. The polar/apolar interfaces of liquid--crystalline DM-TAP bilayers are not as well hydrated as those formed by other classes of phospho- and glycolipids. Such differences are attributed to the relatively small size of the polar headgroup and its limited capacity for interaction with moieties in the bilayer polar/apolar interface.

Method for obtaining structure and interactions from oriented lipid bilayers.

Precise calculations are made of the scattering intensity I(q) from an oriented stack of lipid bilayers using a realistic model of fluctuations. The quantities of interest include the bilayer bending modulus Kc, the interbilayer interaction modulus B, and bilayer structure through the form factor F(qz). It is shown how Kc and B may be obtained from data at large q(z) where fluctuations dominate. Good estimates of F(qz) can be made over wide ranges of q(z) by using I(q) in q regions away from the peaks and for q(r) not equal0 where details of the scattering domains play little role. Rough estimates of domain sizes can also be made from smaller q(z) data. Results are presented for data taken on fully hydrated, oriented DOPC bilayers in the L(alpha) phase. These results illustrate the advantages of oriented samples compared to powder samples.

Anomalous swelling in phospholipid bilayers is not coupled to the formation of a ripple phase.

Aligned stacks of monomethyl and dimethyl dimyristoyl phosphatidylethanolamine (DMPE) lipid bilayers, like the much studied dimyristoyl PC (DMPC) bilayers, swell anomalously in a critical fashion as the temperature is decreased within the fluid phase towards the main transition temperature, T(M). Unlike DMPC bilayers, both monomethyl and dimethyl DMPE undergo transitions into a gel phase rather than a rippled phase below T(M). Although it is not fully understood why there is anomalous swelling, our present results should facilitate theory by showing that the formation of the phase below T(M) is not related to critical phenomena above T(M).

Rapid identification of local T cell expansion in inflammatory organ diseases by flow cytometric T cell receptor Vbeta analysis.

Oligoclonal expansion of antigen-specific T cells occurs frequently during inflammatory diseases. These cells may persist for a long time at high frequency in the body and be enriched in the affected tissues. As a screening test for expanded cell T cell populations at sites of inflammation, we developed an optimized methodology for flow-cytometry-based quantification of T cell receptor Vbeta (TCRBV) expression. We first validated the specificity of a TCRBV-specific monoclonal antibody set by direct comparison with PCR-based analysis of mono- and polyclonal T cell samples. This monoclonal antibody (mAb) panel recognized approximately two thirds of the T cell receptor alpha/beta repertoire in a group of 64 healthy donors and allowed defining TCR usage in the CD4+ and CD8+ subsets. The reliable detection of expanded Vbeta gene families in T cell populations was confirmed in experiments on superantigen-stimulated T cells. Through differential TCR analysis on T cell subpopulations in cerebrospinal fluid and blood in patients with acute encephalitis, we were able to identify locally expanded CD8+ T cells. The power of this approach affords not only high-throughput comparative TCR analysis for immunological studies in vitro, but also rapid ex vivo identification of cell populations enriched in organ compartments during inflammatory diseases.

Structure of lipid bilayers.

The quantitative experimental uncertainty in the structure of fully hydrated, biologically relevant, fluid (L(alpha)) phase lipid bilayers has been too large to provide a firm base for applications or for comparison with simulations. Many structural methods are reviewed including modern liquid crystallography of lipid bilayers that deals with the fully developed undulation fluctuations that occur in the L(alpha) phase. These fluctuations degrade the higher order diffraction data in a way that, if unrecognized, leads to erroneous conclusions regarding bilayer structure. Diffraction measurements at high instrumental resolution provide a measure of these fluctuations. In addition to providing better structural determination, this opens a new window on interactions between bilayers, so the experimental determination of interbilayer interaction parameters is reviewed briefly. We introduce a new structural correction based on fluctuations that has not been included in any previous studies. Updated measurements, such as for the area compressibility modulus, are used to provide adjustments to many of the literature values of structural quantities. Since the gel (L(beta)') phase is valuable as a stepping stone for obtaining fluid phase results, a brief review is given of the lower temperature phases. The uncertainty in structural results for lipid bilayers is being reduced and best current values are provided for bilayers of five lipids.

Mucolipidosis type IV is caused by mutations in a gene encoding a novel transient receptor potential channel.

Mucolipidosis type IV (MLIV) is a developmental neurodegenerative disorder characterized by severe neurologic and ophthalmologic abnormalities. The MLIV gene, ML4 (MCOLN1), has recently been localized to chromosome 19p13.2-13.3 by genetic linkage. Here we report the cloning of a novel transient receptor potential cation channel gene and show that this gene is mutated in patients with the disorder. ML4 encodes a protein, which we propose to call mucolipin, which has six predicted transmembrane domains and is a member of the polycystin II subfamily of the Drosophila transient receptor potential gene family. The role of a potential receptor-stimulated cation channel defect in the pathogenesis of mucolipidosis IV is discussed.

Clarification of the ripple phase of lecithin bilayers using fully hydrated, aligned samples.

Aligned samples of lipid bilayers have been fully hydrated from water vapor in a different type of x-ray chamber. Our use of aligned samples resolves issues concerning the ripple phase that were ambiguous from previous powder studies. In particular, our x-ray diffraction data conclusively demonstrate that, on cooling from the L alpha to the P beta' phase, both chiral and racemic samples of dipalmitoyl phosphatidylcholine (DPPC) exhibit phase coexistence of long and short ripples with a ripple wavelength ratio lambda L/lambda S approximately 1.8. Moreover, the long ripple always forms an orthorhombic unit cell (gamma L = 90 degrees), strongly supporting the possibility that these ripples are symmetric. In contrast, gamma S for short ripples was consistently different from 90 degrees, implying asymmetric ripples. We continue to find no evidence that chirality affects the structure of rippled bilayers. The relative thermodynamic stability of the two types of ripples was investigated and a qualitative free energy diagram is given in which the long ripple phase is metastable. Finally, we suggest a kinetic mechanism, involving loss of water, that promotes formation of the metastable long ripple phase for special thermal protocols.

Lipid bilayer structure.

Fluctuations, inherent in flexible and biologically relevant lipid bilayers, make quantitative structure determination challenging. Shortcomings in older methods of structure determination have been realized and new methodologies have been introduced that take fluctuations into account. The large uncertainty in literature values of lipid bilayer structural parameters is being reduced.

Characterization and sequencing of prototypic human T-lymphotropic virus type 1 (HTLV-1) from an HTLV-1/2 seroindeterminate patient.

Serological screening for human T-lymphotropic virus type 1 (HTLV-1) parallels the standard screening process for human immunodeficiency virus (HIV), in which samples found positive by enzyme-linked immunosorbent assay (ELISA) are confirmed with a modified Western blot procedure. There are a significant number of cases in which HTLV-1/2 ELISA-positive specimens demonstrate an incomplete banding pattern on this Western blot. Individuals providing these atypical antibody responses are categorized as seroindeterminate for HTLV-1/2. Although HTLV-1 genomic sequences are readily detectable in the peripheral blood lymphocytes (PBL) of seropositive individuals, previous studies have repeatedly demonstrated that PBL from the vast majority of HTLV-1/2 seroindeterminate individuals are PCR negative for HTLV-1. As a result, identification of the agent responsible for this indeterminate reactivity has been of interest. We have generated an HTLV-1-positive B-cell line (SI-1 B) from one of these seroindeterminate individuals. Previous screening for HTLV-1 in PBL from this patient had been routinely negative by primary PCR; however, HTLV-1 tax had been periodically detected by nested PCR. DNA sequence data generated with genomic DNA from the SI-1 B cell line and HTLV-1-specific primers demonstrated the presence of a full-length viral genome with >97% homology to the Cosmopolitan form of HTLV-1. A 12-bp deletion was identified in the 3'-gag/5'-prot region, which would predict translation of altered or nonfunctional proteins from these genes. We propose that this HTLV-1/2-seroindeterminate patient is infected with a prototypic form of HTLV-1 at an extremely low viral load and that this finding may explain HTLV-1/2 seroindeterminate reactivity in at least a subset of these individuals.

Intraligand charge transfer in the Pd(II) oxinate complex Pd(qol)2. Site-selective emission, excitation, and optically detected magnetic resonance.

The first spectroscopic investigation of Pd(qol)2 (qol- = 8-quinolinolato-N,O = oxinate) dissolved in an n-octane matrix (Shpol'skii matrix) is reported. Application of several spectroscopic methods at liquid helium temperatures (typically, T = 1.2 K), such as site-selective and highly resolved luminescence and excitation spectroscopy, time-resolved emission spectroscopy, optically detected magnetic resonance, microwave recovery, phosphorescence microwave double-resonance, and magnetic fields, allows us to characterize the lowest excited electronic states in detail. In accord with previous assignments for the related Pt(qol)2 it is shown that these lowest states represent-intraligand charge-transfer states, namely, 1ILCT and 3ILCT. The electronic origin of the 1ILCT state lies at 20,617 cm(-1) (site A). It exhibits a nearly homogeneous line width with a half-width of about 80 cm(-1) (fwhm), which corresponds to a lifetime of tau(1ILCT) approximately equals 2 x 10(-13) s. This value is even shorter than that found for Pt(qol)2, presumably due to intersystem crossings and relaxations to dd* states. The electronic origin of the 3ILCT state lies at 16 090 cm(-1) (site A), and its zero-field splittings (zfs) into three sublevels are 2E = 2356 MHz (0.0785 cm(-1)) and D - E = 5241 MHz (0.175 cm(-1)). The emission decay times of the three sublevels are determined as tauI = 90 +/- 30 ms, tau(II) = 180 +/- 10 mus, and tau(II) = 80 +/- 10 mus. (Slightly different values are found for a second site B at 16,167 cm(-1).) From the small values of zfs and the long emission decay times it is concluded that metal-d or MLCT admixtures to 3ILCT are very small. This result clearly reflects the ligand-centered character of the transition. The assignment as an ILCT transition is supported by the occurrence of relatively strong vibrational satellites of Pd-N and Pd-O character in highly resolved emission spectra. Although the transition is ascribed to a charge-transfer process, the geometry changes between the ground state and 3ILCT are very small. The results found for Pd(qol)2 are compared to those of companion studies of Pt(qol)2 and Pt(qtl)2 (qtl- = 8-quinolinethiolato-N,S).

Re-analysis of magic angle spinning nuclear magnetic resonance determination of interlamellar waters in lipid bilayer dispersions.

A recent method to obtain the number of water molecules of hydration of multilamellar lipid vesicles using magic angle spinning nuclear magnetic resonance has been re-examined. The previous interpretation divided the water into bulk and interlamellar water and ignored water in defects (lakes) that are intrinsic to multilamellar lipid vesicles; the result was inconsistent with x-ray results for the lipid DOPC. The new interpretation takes advantage of the reduction of lake water with increased spinning and it uses osmotic pressure measurements to determine the loss of interlamellar water. The new result for DOPC from magic angle spinning is consistent with x-ray results.

The genomic organization and polymorphism analysis of the human Niemann-Pick C1 gene.

Niemann-Pick C (NP-C) is a fatal autosomal recessive storage disorder characterized by progressive neurodegeneration and variable hepatosplenomegaly. At the cellular level, cells derived from an affected individual accumulate unesterified cholesterol in lysosomes when cultured with low-density lipoprotein. The NP-C gene was identified at 18q11. The transcript is 4.9 kb encoding a 1278-amino-acid protein. We have defined the genomic structure of NPC1 along with the 5' flanking sequence. The NPC1 gene spans greater than 47 kb and contains 25 exons. Exons range in size from 74 to 788 bp with introns ranging in size from 0.097 to 7 kb. All intron/exon boundaries follow the GT/AG rule. The 5' flanking sequence has a CpG island containing multiple Sp1 sites indicative of a promoter region. The CpG island is located in the 5' flanking sequence, exon 1 and the 5' end of intron 1. We have also identified multiple single nucleotide polymorphisms in the coding and intronic sequences.

Novel exon 3B proteolipid protein gene mutation causing late-onset spastic paraplegia type 2 with variable penetrance in female family members.

Spastic paraplegia type 2 (SPG2) is allelic to Pelizaeus-Merzbacher disease (PMD), with both conditions resulting from mutations in the proteolipid protein gene (PLP). We report an SPG2 family in which 3 male members and a heterozygous female member were affected with spastic paraplegia characterized by relatively late onset and mild clinical manifestations. A unique H147Y mutation in exon 3B of the PLP altering the proteolipid protein (PLP) but not the alternatively spliced DM20 isoform was identified as the cause of this distinct disease phenotype. Cellular pathology studies of SPG2 mutations offer an explanation for the paradoxical finding that mutations associated with the mildest phenotype in male family members also affect female carriers.

Analysis of simulated NMR order parameters for lipid bilayer structure determination.

The conventional formula for relating CD2 average order parameters to average methylenic travel is flawed when compared to molecular dynamics simulations of dipalmitoylphosphatidylcholine. Inspired by the simulated probability distribution functions, a new formula is derived that satisfactorily relates these quantities. This formula is used to obtain the average chain length , and the result agrees with the direct simulation result for . The simulation also yields a hydrocarbon thickness 2. The result = is consistent with a model of chain packing with both early chain termination and partial interdigitation of chains from opposing monolayers. The actual simulated area per lipid is easily obtained from the order parameters. However, when this method is applied to NMR order parameter data from dimyristoylphosphatidylcholine, the resulting is 10% larger than the currently accepted value.

Absence of a vestigial vapor pressure paradox.

The enigmatic but much accepted vapor pressure paradox for oriented lipid bilayer samples was recently justified theoretically. Subsequently, recent experiments have shown that there is no vapor pressure paradox. The first result of this paper is to consider another degree of freedom that reverses the previous theoretical conclusion, so that theory and experiment are now in agreement that there is no vapor pressure paradox. However, this analysis also suggests the possibility of a vestigial vapor pressure paradox that would rationalize why the vapor pressure paradox was historically so persistent and that would have led to an improved protocol for obtaining bilayer structure. This vestigial vapor pressure paradox would involve a phase transition as a function of applied osmotic pressure. We test this possibility experimentally using combined neutron and x-ray scattering data. The conclusion from these experiments is that there is not even a vestigial vapor pressure paradox. However, this negative result validates an improved method for calibrating osmotic pressure in x-ray studies of oriented samples.

Polymorphism in myristoylpalmitoylphosphatidylcholine.

This study focuses on the mixed-chain lipid myristoylpalmitoylphosphatidylcholine (MPPC) near full hydration. The lipid, synthesized according to the procedure of (Mason et al., 1981a, has a low degree of acyl chain migration. When MPPC is temperature-jumped (T-jumped) from the L alpha phase (T = 38 degrees C) to T = 20 degrees C or below, a subgel phase forms; this formation takes less than 1 h at a temperature below T = 12 degrees C. The subgel remains stable up to T = 29 degrees C. When MPPC is T-jumped from the L alpha phase to T = 24 degrees C or above, a ripple phase forms with coexisting ripple wavelengths of 240 A and 130 A. In contrast, when MPPC is melted from the subgel phase, the ripple phase is characterized by bilayers having a single ripple wavelength of 130 A. In agreement with earlier studies (Stumpel et al., 1983; Serrallach et al., 1984. Structure and thermotropic properties of mixed-chain phosphatidylcholine bilayer membranes. Biochemistry 23:713-720.), no stable gel phase was observed. Instead, an ill-defined low-angle X-ray pattern is initially observed, which gradually transforms into the subgel phase below 20 degrees C, or into the ripple phase above 24 degrees C. In the wide-angle X-ray diffraction, a single peak is observed, similar to the ripple phase wide-angle pattern, that either persists above 24 degrees C or transforms into a multi-peaked subgel wide-angle pattern below 20 degrees C. The absence of a gel phase can be understood phenomenologically as the relative dominance of the subgel phase in mixed-chain PCs compared to same-chain PCs. The subgel structure and molecular interactions responsible for this comparative behavior are interesting open issues.