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.

Missense mutations in desmin associated with familial cardiac and skeletal myopathy.

Desmin-related myopathy (OMIM 601419) is a familial disorder characterized by skeletal muscle weakness associated with cardiac conduction blocks, arrhythmias and restrictive heart failure, and by intracytoplasmic accumulation of desmin-reactive deposits in cardiac and skeletal muscle cells. The underlying molecular mechanisms are unknown. Involvement of the desmin gene (DES) has been excluded in three families diagnosed with desmin-related myopathy. We report two new families with desmin-related cardioskeletal myopathy associated with mutations in the highly conserved carboxy-terminal end of the desmin rod domain. A heterozygous A337P mutation was identified in a family with an adult-onset skeletal myopathy and mild cardiac involvement. Compound heterozygosity for two other mutations, A360P and N393I, was detected in a second family characterized by childhood-onset aggressive course of cardiac and skeletal myopathy.

Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis.

Niemann-Pick type C (NP-C) disease, a fatal neurovisceral disorder, is characterized by lysosomal accumulation of low density lipoprotein (LDL)-derived cholesterol. By positional cloning methods, a gene (NPC1) with insertion, deletion, and missense mutations has been identified in NP-C patients. Transfection of NP-C fibroblasts with wild-type NPC1 cDNA resulted in correction of their excessive lysosomal storage of LDL cholesterol, thereby defining the critical role of NPC1 in regulation of intracellular cholesterol trafficking. The 1278-amino acid NPC1 protein has sequence similarity to the morphogen receptor PATCHED and the putative sterol-sensing regions of SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase.

Immunodominance of a low-affinity major histocompatibility complex-binding myelin basic protein epitope (residues 111-129) in HLA-DR4 (B1*0401) subjects is associated with a restricted T cell receptor repertoire.

The pathogenesis of multiple sclerosis (MS) is currently ascribed in part to a T cell-mediated process targeting myelin components. The T cell response to one candidate autoantigen, myelin basic protein (MBP), in the context of HLA-DR15Dw2, has been previously studied in detail. However, the characteristics of cellular immunity in the context of other MS-associated HLA-DR haplotypes are scarcely known. MBP-specific T cell lines (TCL) were generated from HLA-DR4 (B1*0401)-positive MS subjects. Out of 275 MBP-specific TCL, 178 (64. 7%) specifically recognized region MBP(111-129), predominantly in the context of DRB1*0401. The major T cell epitope for MBP recognition corresponded to residues MBP(116-123). These TCL expressed disparate profiles of cytokine secretion and cytotoxicity. T cell receptor analysis, on the other hand, revealed a strikingly limited heterogeneity of rearrangements. In contrast to MBP(81-99), which binds with high affinity to HLA-DR15 and is recognized by a diverse T cell repertoire, MBP(111-129) binds weakly to DRB1*0401, suggesting that only high affinity T cell receptors might be able to efficiently engage such unstable MHC/peptide complexes, thus accounting for the T cell receptor restriction we observed. This study provides new insight about MBP recognition and proposes an alternative mechanism for immunodominance of self-antigen T cell epitopes in humans.

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.

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.

Lateral compressibility of lipid mono- and bilayers. Theory of membrane permeability.

The passive sodium permeability of pure lipid vesicles and dispersions has a large peak at the bilayer phase transition temperature. We discuss this anomaly in terms of density fluctuations, which can open up cavities in the headgroup region into which small ions can enter, and which may be large if bilayer conditions at the melting point are similar to those near the critical point which seems to exist in monolayers. We present two arguments, one thermodynamic and one microscopic, which suggest that the permeability is proportional to the lateral compressibility. We then calculate the lateral compressibility for two previously published theoretical models and compare the results with experiment.

Specific heats of lipid dispersions in single phase regions.

Differential scanning calorimetry has been used for the first time to measure the specific heat, Cp, as a function of temperature in the single phase regions above and below the main phase transition temperature, Tm, for dispersions of saturated phosphatidylcholines and phosphatidylethanolamines. Within error limits Cp, when expressed per gram, does not vary in any systematic way with chain length or headgroup. Its temperature dependence in both single phase regions qualitatively resembles that of n-alkanes. Contributions to Cp from intrachain vibrations and interchain van der Waals' interactions have been calculated and account for nearly all the measured Cp at temperatures above Tm. However, these contributions do not yield the observed temperature dependence below Tm. It is conjectured that such a temperature dependence arises from the unhindering of chain vibrations as the lipids undergo thermal expansion, and the result of a preliminary calculation which supports this conjecture is presented.

Structure of fully hydrated bilayer dispersions.

A systemic formalism is developed that shows how the results for absolute specific volumes of multilamellar lipid dispersions may be combined with results from diffraction studies to obtain quantitative characterizations of the average structure of fully hydrated lipid bilayers. Quantities obtained are the area per molecule, the thickness and volumes of the bilayer, the water layer, the hydrocarbon chain layer and the headgroup layer, and where appropriate, the tilt angle of the hydrocarbon chains. In the case of the C phase of DPPC this formalism leads to the detection of inconsistencies between three data. Results for the G phases of DPPC and DLPE are in reasonable agreement with, though more comprehensive than, previous work that used fewer data and equations. Various diffraction data for the F phase of DPPC are in disagreement and it is shown how this disagreement affects results for the bilayer structure. A recent method of McIntosh and Simon for obtaining fluid phase structure utilizing gel phase structure is slightly modified to obtain results for the F phase of DLPE. Methods of obtaining the average methylene and methyl volumes in the fluid phases are critically examined.

Anomalous phase behavior of long chain saturated lecithin bilayers.

X-ray scattering has been performed on fully hydrated unoriented multilamellar vesicles of lecithins with even chain lengths n from 16 to 24 as a function of temperature in chain ordered phases. The longer chain lengths, n > or = 20, show anomalous behavior compared to the shorter chain lengths, n < 20. This report concentrates on n = 24. Although the history and time dependence shows that equilibrium was not always achieved, it appears that there is a second gel-like phase G2 below 40 degrees C. The G2 phase has a small tilt angle and opposite hexagonal symmetry breaking from the usual G1 gel phase. Also, as T is raised above 45 degrees C, the wide-angle data suggest the appearance of a phase with hexagonal chain packing and small chain tilt angle.

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.

Kinetics of subgel formation in DPPC: X-ray diffraction proves nucleation-growth hypothesis.

Wide-angle and low-angle X-ray diffraction data were obtained during the time course of the gel to subgel phase transformation in fully hydrated DPPC. When the system was kept close to equilibrium by following a T-jump protocol, the X-ray data unequivocally demonstrate the coexistence of growing subgel and shrinking gel domains. When the system was supercooled and held further from equilibrium as in previous studies, the kinetic behavior was more complicated. These data prove that the basic mechanism for the gel to subgel phase transformation is one of nucleation of subgel domains followed by growth of the domains.

Thermodynamic studies of purple membrane.

Differential dilatometric and differential scanning calorimetric measurements have been made of purple membrane with an emphasis upon the temperature range 5 degrees C less than T less than 45 degrees C. The coefficient of thermal expansion alpha is about 7 X 10(-4)/Cdeg up to 30 degrees C and decreases at higher temperatures. The specific heat increases rapidly with temperature with absolute values in the range 0.30-0.45 cal/Cdeg per g. A nearly constant alpha juxtaposed with a rapidly increasing specific heat is similar to the properties of lipid bilayers in the gel phase and alkanes in the solid phase. This behavior is explained by the concept of hindered vibrations which would now appear to apply to at least one integral membrane protein. There may also be a small broad transition centered near 20-25 degrees C that would correspond to the melting of less than 25 degrees of freedom per bacteriorhodopsin molecule and associated lipids. Using our measured apparent specific volume the average thickness of purple membrane is calculated to be 43.5 A. The specific volume of interaction of lipids and proteins is estimated, using the amino acid sequence of bacteriorhodopsin and average amino acid volumes from structural studies of other proteins, to be about 11% of the specific volume of the purple membrane lipids or 4% of the volume of the bacteriorhodopsin protein. A positive volume of interaction is consistent with lipid-protein interactions being an important determinant of the thermodynamic properties of purple membrane.

New phases of DPPC/water mixtures.

Hydration of DPPC at low temperatures yielded two new phases, a non-lamellar C1 phase and a lamellar C2 phase, as well as the normal gel phase, depending upon the initial physical state of the dry lipid. From the results of wide-angle diffraction and calorimetry the C2 phase appears very similar to the normal C phase, but the D spacing is considerably larger, suggesting that the C2 phase is a C phase with untilted chains.

DMSO produces a new subgel phase in DPPC: DSC and X-ray diffraction study.

Equilibrium phases and the kinetics of subgel phase transformation of dipalmitoylphosphatidylcholine (DPPC) hydrated with mixtures of dimethylsulfoxide (DMSO)/water have been studied using differential scanning calorimetry (DSC) and X-ray diffraction (XRD). The rate of gel-to-subgel transformation is decreased with a small increase in X, the DMSO/water mole fraction, but then speeds up and becomes faster than in pure water by X = 0.16. The DSC scans show multiple subgel peaks, some of which can be attributed to impacted domain growth. For X greater than 0.10, XRD shows that there is a new, stable subgel phase, S, which also accounts for some of the multiplicity of DSC peaks. Our electron density profiles show that the thickness of the bilayer in the S phase is greater than in the usual C subgel phase. We suggest that the S subgel phase is characterized by different headgroup ordering and smaller chain tilt angle than in the C subgel phase. Electron density profiles show that increasing X decreases the water space between bilayers in all phases, subgel, gel and fluid (L alpha). For X = 0.20, a different gel phase is also observed that may be due to subtle changes in the orientation of chain tilt first observed in partially dehydrated DMPC. The dehydrating effect of DMSO explains the results of a previous study, confirmed in this study, that increasing the concentration of DMSO raises the main transition temperature and eliminates the ripple phase.

Specific volumes of lipids in fully hydrated bilayer dispersions.

The neutral buoyancy method of obtaining absolute specific volumes of lipid in multilamellar dispersions is critically investigated. Control experiments show that there is no preferential partitioning of 2H2O vs. H2O into the liposomes, and several thermodynamic properties of the samples, such as the enthalpy change and the volume change of the main transition, are changed very little with deuteration of the solvent. The assumption that the molecular volume of the solvent in the interlamellar space is essentially the same as in bulk solution is discussed; and it is shown to introduce rather small corrections. Previous procedures have been modified to avoid possible kinetic limitations in phases with low water permeability. It is concluded that the molecular volume of lipid in bilayers can be obtained to an accuracy better than 0.002 nm3 (2A3) which is less than 0.2% of typical molecular volumes of lipids.

Dilatometric studies of isobranched phosphatidylcholines.

Absolute apparent specific volumes have been obtained for phosphatidylcholine lipids with saturated, isobranched hydrocarbon chains with ni = 15 to 20 carbons, with an emphasis upon phase transition behavior, both equilibrium and kinetic. The temperature of the chain-melting transition extrapolates with increasing chain length to the melting temperature of polyethylene with a small odd/even alternation. There are also odd/even alternations in the volume of transition and in the hysteresis of the chain-melting transition, but with the odd and even reversed when compared with the larger odd/even alternation in the lower solid-solid transition that occurs in the longer chain ni lipids. A phenomenological picture is given for the coalescence of the two transitions for shorter ni lipids and this picture is used to sharpen the discussion of the kinetic mechanism of melting. A temperature-reversal experiment shows that the melting from the lowest temperature crystal or C phase to the fluid F phase does not proceed via the metastable gel G phase for 16i. The dilatometric results are combined with recent X-ray structural results for the C and G phases of 17i and 20i to deduce various structural information, including the hydration numbers and the volume of the headgroup, VH = 341 A3, which agrees very well with VH for straight-chain phosphatidylcholines. For the chain-melted F phase the assumption that the methylene volumes of the different ni lipids should be the same at the same temperature is used to obtain the volumes of the methylene and the methyl groups.

Structural organization, expression and chromosomal mapping of the mouse cystatin-C-encoding gene (Cst3).

Cystatin C (CstC) is a potent cysteine-proteinase inhibitor. The structure of the mouse CstC-encoding gene (Cst3) was examined by sequencing a 6.1-kb genomic DNA containing the entire gene, as well as 0.9 kb of 5' flanking and 1.7 kb of its 3' flanking region. The sequence revealed that the overall organization of the gene is very similar to those of the genes encoding human CstC and other type-2 Cst, with two introns at positions identical to those in the human gene. The promoter area does not contain typical TATA or CAAT boxes. Two copies of a Sp1-binding motif, GGGCGG, are present in the 5' flanking region within 300 bp upstream from the initiation codon. A hexa-nucleotide, TGTTCT, which is a core sequence of the androgen-responsive element (ARE), is found in the promoter region. This region also contains a 21-nucleotide sequence, 5'-AGACTAGCAGCTGACTGAAGC, which contains two potential binding sites for the transcription factor, AP-1. The mouse Cst3 mRNA was detected in all of thirteen tissues examined by Northern blot analysis. Cst3 was mapped in the mouse to a position on distal chromosome 2.

Cloning and expression analysis of a human cDNA homologous to Xenopus TFIIIA.

We report here the nucleotide sequence of a clone, C2H2-34.10, isolated from a human brain cDNA library using degenerate oligodeoxyribonucleotide hybridization. C2H2-34.10 has extensive homology to the Xenopus laevis 5S DNA/RNA-binding protein, TFIIIA. The deduced amino acid (aa) sequence of the human clone gives a protein of 363 aa with identity to TFIIIA from both X. laevis (57%) and Rana pipiens (59%). This human clone contains nine C2H2-type zinc fingers like frog TFIIIA. Northern blot analysis indicates that the C2H2-34.10 RNA is expressed in human ovary, as well as human neuronal cell lines.