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PubMed1999

Effects of cannabinoids in membrane bilayers containing cholesterol.

The thermotropic and dynamic properties of the biologically active Delta(8)-tetrahydrocannabinol (Delta(8)-THC) and its inactive congener O-methyl-Delta(8)-tetrahydrocannabinol (Me-Delta(8)-THC) in DPPC/cholesterol (CHOL) bilayers have been studied using a combination of DSC and solid-state NMR spectroscopy. The obtained results showed differential effects of the two cannabinoids under study. These are summarized as follows: (a) the presence of the active compound fluidizes more significantly the DPPC/CHOL bilayers than the inactive analog as it is revealed by DSC and NMR spectroscopy results; (b) cholesterol seems to play a significant role in the way cannabinoids act in membrane bilayers; (c) the observed additional peaks in (13)C/MAS-NMR spectra which were cannabinoid specific offer an evidence of their different dynamic properties in membranes. In particular, the aromatic part of the inactive cannabinoid appears more mobile than that of the active one. This finding is in agreement with previously obtained X-ray data which locate the inactive cannabinoid in the hydrophobic core of the bilayer while the active one in the polar region; and (d) the observed downfield shift of C-1 carbon in the preparation containing the active cannabinoid is a strong evidence that Delta(8)-THC resides nearby the polar region where also cholesterol is well known to locate itself. Such downfield shift is absent when Me-Delta(8)-THC is resided in the membrane bilayer. These differential effects of the two cannabinoids propose that the phospholipid/cholesterol core of the membrane may play an important role in the mode of cannabinoid action by regulating their thermotropic and dynamic properties.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1966

Structure and organization of the living mitotic spindle of Haemanthus endosperm.

New details of mitotic spindle structures in the endosperm of Haemanthus katherinae (Bak) have been demonstrated by differential interference microscopy. Spindle fibers are clearly seen in the living spindle extending from the kinetochores to the polar region. Individual spindle fibers consist of a bundle of smaller filaments which diverge slightly from the kinetochore and intermingle with filaments from other spindle fibers as they approach the polar region. The degree of intermingling increases during metaphase and anaphase. The chromosomes stop moving when the spindle fibers are still 5 to 10microns long; then the fibers disappear. These observations explain some aspects of spindle movements which were difficult to reconcile with earlier concepts of spindle organization.

Cell Division↗
PubMed1980

The blood supply of duck muscle spindles.

The blood supply of duck muscle spindles from the extensor pollicis and the extensor digitorum comminis wing muscles has been studied by light and electron microscopy. Capillaries usually accompany the nerve bundle that innervates the spindle, approaching at an oblique angle around the midequatorial region. Capillaries may run for some distance along the surface of the outer capsule, or penetrate partially into the outer capsule and lie between layers of the capsule cell processes. Some capillaries also penetrate the outer capsule, running into the periaxial space and continuing further towards the polar region. They have been shown to be in close contact with intrafusal muscle fibres, from the juxta-equatorial to the polar region, but have not been encountered among sensory terminals in the mid-equatorial region.

Animals↗
PubMed2006

Characterization of the HIV N-terminal fusion peptide-containing region in context of key gp41 fusion conformations.

Central to our understanding of human immunodeficiency virus-induced fusion is the high resolution structure of fragments of the gp41 fusion protein folded in a low energy core conformation. However, regions fundamental to fusion, like the fusion peptide (FP), have yet to be characterized in the context of the cognate protein regardless of its conformation. Based on conformation-specific monoclonal antibody recognition, we identified the polar region consecutive to the N36 fragment as a stabilizer of trimeric coiled-coil assembly, thereby enhancing inhibitory potency. This tertiary organization is retained in the context of the hydrophobic FP (N70 fragment). Our data indicate that the N70 fragment recapitulates the expected organization of this region in the viral fusion intermediate (N-terminal half of the pre-hairpin intermediate (N-PHI)), which happens to be the prime target for fusion inhibitors. Regarding the low energy conformation, we show for the first time core formation in the context of the FP (N70 core). The alpha-helical and coiled-coil stabilizing polar region confers substantial thermal stability to the core, whereas the hydrophobic FP does not add further stability. For the two key fusion conformations, N-PHI and N70 core, we find that the FP adopts a nonhelical structure and directs higher order assembly (assembly of coiled coils in N-PHI and assembly of bundles in the N70 core). This supra-molecular organization of coiled coils or folded cores is seen only in the context of the FP. This study is the first to characterize the FP region in the context of the folded core and provides a basic understanding of the role of the elusive FP for key gp41 fusion conformations.

Antibodies, Monoclonal↗
PubMed2006

Free energy landscape analysis of two-dimensional dipolar solvent model at temperatures below and above the rotational freezing point.

Ionic solvation in a polar solvent is modeled by a central charge surrounded by dipolar molecules posted on two-dimensional distorted lattice sites with simple rotational dynamics. Density of states is calculated by applying the Wang-Landau algorithm to both the energy and polarization states. The free energy landscapes of solvent molecules as a function of polarization are depicted to explore the competition between the thermal fluctuation and solvation energy. Without a central charge, for temperatures higher than the energy scale of the dipole-dipole interactions, the energy landscape for the small polarization region exhibits a parabolic shape as predicted by Marcus [Rev. Mod. Phys. 65, 599 (1993)] for electron transfer reaction, while there is an additional quartic contribution to the landscape for the large polarization region. When the temperature drops, the simulated free energy landscapes are no longer smooth due to the presence of multiple local minima arising from the frustrated interaction among the dipoles. The parabolic contribution becomes negligible and the energy landscape becomes quartic in shape. For a strong central charge, the energy landscape exhibits an asymmetric profile due to the contributions of linear and cubic terms that arise from the charge-dipole interactions.

Journal Article↗
PubMed1988

Molecular interactions between sphingomyelin and phosphatidylcholine in phospholipid vesicles.

The molecular interactions between DPPC and sphingomyelin have been examined using differential scanning calorimetry and Fourier transform infrared spectroscopy (FT-IR). It has been observed using DSC that the mixtures of DPPC and sphingomyelin show a single peak, which indicates that they are very miscible. It has been shown, by means of FT-IR, that the incorporation of sphingomyelin into DPPC does not modify the gauche/all-trans ratio at temperatures either above or below that of the phase transition. However, the librational motion of the fatty acyl chains was affected, although only above the phase transition. Mixing sphingomyelin and DPPC induced a conformational change in the polar region of DPPC, as deduced from the changes observed in the frequencies of the sn-1 and sn-2 C = O groups of DPPC with relation to pure DPPC and also in the polar region of sphingomyelin as deduced from the change in the frequency of the amide band. The phosphate group of sphingomyelin was observed to participate in hydrogen bonds between the molecules of sphingomyelin and, possibly, DPPC. Some possibilities of the interaction between the molecules of DPPC and sphingomyelin are discussed.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2003

The actin-based motility defect of a Shigella flexneri rmlD rough LPS mutant is not due to loss of IcsA polarity.

Shigella flexneri requires the outer membrane protein IcsA(VirG) and lipopolysaccharide (LPS) for efficient actin-based motility (ABM) within mammalian cells which is essential for virulence. Wild type strains of S. flexneri 2a such as 2457T have smooth LPS whose O antigen (Oag) chains have two modal lengths and IcsA predominantly located at one pole on their cell surface. In contrast, rough LPS mutants lack Oag chains, have IcsA on lateral and polar regions of the cell surface, and are defective for ABM. In this study we directly compared the phenotype of a S. flexneri producing non-IcsP/SopA cleavable IcsA (IcsA*) with that of a rough LPS mutant. IcsA* was located on lateral and polar regions of smooth LPS bacteria, and was fully functional in ABM assays (HeLa cell monolayer plaque and F-actin comet tail formation) which contrasts with the R-LPS phenotype. This indicates that loss of polar IcsA localisation in R-LPS mutants is unrelated to their ABM defect, and suggests that Oag may directly contribute to IcsA-mediated ABM.

Actins↗
PubMed1996

Selective inhibition of cyclic AMP-dependent protein kinase by amphiphilic triterpenoids and related compounds.

A set of plant- and animal-derived amphiphilic triterpenoids have been shown to be potent and selective inhibitors of the catalytic subunit of rat liver cyclic AMP-dependent protein kinase (cAK). Thus plant-derived 18 alpha- and 18 beta-glycyrrhetinic acid, ursolic acid, oleanolic acid and betulin and animal-derived lithocholic acid, 5-cholenic acid and lithocholic acid methyl ester are inhibitors of cAK with IC50 values (concentrations for 50% inhibition) in the range 4-20 microM. These compounds are ineffective or relatively ineffective as inhibitors of various other eukaryote signal-regulated protein kinases namely wheat embryo Ca(2+)-dependent protein kinase (CDPK), avian calmodulin-dependent myosin light chain kinase (MLCK) and rat brain Ca(2+)- and phospholipid-dependent protein kinase C (PKC). These naturally occurring triterpenoids have a common structural motif involving polar residues located at opposite ends of an otherwise non-polar triterpenoid nucleus. A variety of triterpenoids not possessing this structural motif are relatively inactive as inhibitors of cAK and of CDPK, PKC and MLCK. The terpenoid amphiphilic compound crocetin is also a potent and relatively selective inhibitor of cAK (IC50 value for cAK 3.0 microM). 12-Hydroxystearic acid and 10-hydroxydecanoic acid do not inhibit CDPK, PKC or MLCK but are selective inhibitors of cAK (IC50 values 127 and 138 microM, respectively), consistent with a simple model for amphiphile inhibition of cAK involving two polar groups separated by a non-polar region. However, laurylgallate and 15-pentadecanolide are also potent and selective inhibitors of cAK (IC50 values 1.5 and 20 microM, respectively) although the structures of both of these compounds involve a large non-polar portion associated with only one polar region. Crocetin and the plant-derived amphiphilic triterpenoids described here are the most potent non-aromatic plant-derived inhibitors of cAK yet found.

Amino Acid Sequence↗
PubMed1983

Differential localization of two myosins within nematode thick filaments.

The body wall muscle cells of the nematode, Caenorhabditis elegans, contain two unique types of myosin heavy chain, A and B. We have utilized an immunochemical approach to define the structural location of these two myosins within body wall muscle thick filaments. By immunofluorescence microscopy, myosin B antibodies label the thick filament-containing A-bands of body wall muscle with the exception of a thin gap at the center of each A-band, and myosin A antibodies react to form a medial fluorescent stripe within each A-band. The complexes of these monoclonal antibodies with isolated thick filaments were negatively stained and studied by electron microscopy. The myosin B antibody reacts with the polar regions of all filaments but does not react with a central 0.9 micron zone. The myosin A antibody reacts with a central 1.8 micron zone in all filaments but does not react with the polar regions.

Animals↗
PubMed1986

Positional signalling by Hensen's node when grafted to the chick limb bud.

Hensen's node from stage 4 to stage 10 shows polarizing activity when grafted to the anterior margin of the chick limb bud. It can specify additional digits though its action is somewhat attenuated when compared with the effect of a grafted polarizing region. At stage 10 the activity disappears from the node and is found both posterior to the node and in the future wing region of the flank. The ability of Hensen's node to generate a positional signal suggests that the signal in the limb and early embryo may be similar. The results support the view of the polarizing region as a discrete signalling region.

Animals↗
PubMed1992

Possible methane-induced polar warming in the early Eocene.

Reconstructions of early Eocene climate depict a world in which the polar environments support mammals and reptiles, deciduous forests, warm oceans and rare frost conditions. At the same time, tropical sea surface temperatures are interpreted to have been the same as or slightly cooler than present values. The question of how to warm polar regions of Earth without noticeably warming the tropics remains unresolved; increased amounts of greenhouse gases would be expected to warm all latitudes equally. Oceanic heat transport has been postulated as a mechanism for heating high latitudes, but it is difficult to explain the dynamics that would achieve this. Here we consider estimates of Eocene wetland areas and suggest that the flux of methane, an important greenhouse gas, may have been substantially greater during the Eocene than at present. Elevated methane concentrations would have enhanced early Eocene global warming, and also might specifically have prevented severe winter cooling of polar regions because of the potential of atmospheric methane to promote the formation of optically thick, polar stratospheric ice clouds.

Atmosphere↗
PubMed1997

Electroporation-induced formation of individual calcium entry sites in the cell body and processes of adherent cells.

Electroporation is a widely used method for introducing macromolecules into cells. We developed an electroporation device that requires only 1 microl of sample to load adherent cells in a 10-mm2 surface area while retaining greater than 90% cell survivability. To better understand this device, field-induced permeabilization of adherent rat basophilic leukemia and neocortical neuroblastoma cells was investigated by using fluorescent calcium and voltage indicators. Rectangular field pulses led to the formation of only a few calcium entry sites, preferentially in the hyperpolarized parts of the cell body and processes. Individual entry sites were formed at the same locations when field pulses were repeated. Before calcium entry, a partial breakdown of the membrane potential was observed in both polar regions. Based on our results, a model is proposed for the formation and closure of macromolecule entry sites in adherent cells. First, the rapid formation of a large number of small pores leads to a partial membrane potential breakdown in both polar regions of the cell. Second, over tens of milliseconds, a few entry sites for macromolecules are formed, preferentially in the hyperpolarized part of cell body and processes, at locations defined by the local membrane structure. These entry sites reseal on a time scale of 50 ms to several seconds, with residual small pores remaining open for several minutes.

Animals↗
PubMed2003

Patterning the limb before and after SHH signalling.

The vertebrate limb is one of the most relevant experimental models for analysing cell-cell signalling during patterning of embryonic fields and organogenesis. Recently, the combination of molecular and genetic studies with experimental manipulation of developing limb buds has significantly advanced our understanding of the complex molecular interactions co-ordinating limb bud outgrowth and patterning. Some of these studies have shown that there is a need to revise some of the textbook views of vertebrate limb development. In this review, we discuss how signalling by the polarizing region is established and how limb bud morphogenesis is controlled by both long-range and signal relay mechanisms. We also discuss recent results showing that differential mesenchymal responsiveness to SHH signalling is established prior to its expression by the polarizing region.

Animals↗
PubMed1976

The innervation of muscle spindles in the snake, Elaphe quadrivirgata.

The morphology and distribution of sensory endings in snake (Elaphe quadrivirgata) muscle spindles was studied in silver preparations. The sensory axon supplying long-capsule spindles often bifurcates before or after penetrating the capsule and runs for some distance along the intrafusal fibre. The sensory axon supplying short-capsule spindles penetrates the capsule at a sharp angle, and, without ramification, terminates abruptly on the intrafusal fibre. The sensory terminal is composed of terminal bulbs and small links. In long-capsule spindles the sensory ending is arranged longitudinally in one or two rows. The sensory ending in short-capsule spindles ramifies extensively, covering densely the intrafusal fibre. The area occupied by sensory bulbs was estimated to be slightly higher in short-capsule (15-17?) THAN IN LONG-CAPSULE SPINDLES (12-13). The functional significance of these findings is discussed. Motor innervation of muscle spindles in the snake Elaphe quadrivirgata was studied using AchE staining for light microscopy in conjection with electron microscopy. In the polar region of either type of spindle, the majority of the motor endings are of grape type. In long-capsule spindles plate endings may occur in the spindle pole as well as in the capsular region. Motor endings in the capsular region are mostly of plate type. Plate endings rarely occur in short-capsule spindles. Identified single motor endings were studied by electron microscopy. Intrafusal grape endings are characterized by a smooth post-synaptic membrane. Intrafusal plate endings in the polar region are characterized by junctional folds or gutterings; plate endings in the capsular region show less developed gutterings or indentations. The distribution of intrafusal motor endings was examined along the length of single intrafusal fibres. The long-capsule spindle often receives asymmetrical motor innervation around the capsular region, whereas motor endings in the short-capsule spindle distribute more symmetrically.

Acetylcholinesterase↗
PubMed1995

Ulysses above the sun's south pole: an introduction.

Ulysses has explored the field and particle environment of the sun's polar region. The solar wind speed was fast and nearly constant above -50 degrees latitude. Compositional differences were observed in slow (low-latitude) solar wind and in fast (high-latitude) solar wind. The radial magnetic field did not change with latitude, implying that polar cap magnetic fields are transported toward the equator. The intensity of galactic cosmic rays was nearly independent of latitude. Their access to the polar region is opposed by outward-traveling, large amplitude waves in the magnetic field.

Cosmic Radiation↗
PubMed1989

Organization of the F0 sector of Escherichia coli H+-ATPase: the polar loop region of subunit c extends from the cytoplasmic face of the membrane.

The membrane-spanning F0 sector of the Escherichia coli H+-transporting ATP synthase (EC 3.6.1.34) contains multiple copies of subunit c, a 79 amino acid residue protein that is thought to insert in the membrane like a hairpin with two membrane traversing alpha-helices. The center of the protein is much more polar than the putative transmembrane alpha-helices and has been postulated to play a crucial role in coupling H+ translocation through F0 to ATP synthesis in the membrane extrinsic, F1 sector of the complex. However, the direction of insertion of subunit c in the membrane has not been established. We show here that the "polar loop" lies on the F1 binding side of the membrane. A peptide corresponding to Lys34----Ile46 of the polar loop was synthesized. Antisera were generated to the Lys34----Ile46 cognate peptide, and the polyclonal antipeptide IgG was shown to bind to a crude F0 fraction by using enzyme-linked immunosorbent assays. The antipeptide serum did not bind tightly enough to F0 to disrupt function. However, a polyclonal antiserum made to purified, whole subunit c was shown to block the binding of F1 to the F0 exposed in F1-stripped membranes. Incubation of the antisubunit c serum with the peptide reduced the inhibitory effect of the antiserum on the binding of F1 to F0. The reversal of inhibition by the peptide was specific to the antisubunit c serum in that the peptide had no effect on inhibition of F1 binding to F0 by antiserum to subunit a of F0.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1998

Psychological adaptation to the polar environment.

Until the 1950's, psychological adaptation to the polar regions was expressed by survivors more in terms of their endurance and heroism after the event than by any studied preparation beforehand, a careful monitoring of performance in situ, the follow-up of subjects afterwards, and a scholarly comparison of data-based research finding. This paper covers some of the omissions, and presents the results of a few studies that should be of value to the variety of people who for quite different periods and purposes visit polar regions. Because of the author's own background in Antarctic research it draws upon material collected in Antarctica, but it also includes empirical studies made by behavioural scientists in the Arctic. Its aim is to encourage potential venturers to the polar areas to prepare themselves mentally as well as physically and logistically for the task ahead of them, whether it be for the long winter night at base or for the long summer day in the field. It concludes with a plea for all the venturers to accept an obligation to provide research data on themselves. The outcome, if done systematically, could be of value both for future participants and for behavioural scientists, because in the past human factors in exploration have been given little attention.

Adaptation, Psychological↗
PubMed1986

Synthesis and structure-activity relationship studies of a series of 5-aryl-4,6-dithianonanedioic acids and related compounds: a novel class of leukotriene antagonists.

A series of 5-alkynyl- and 5-aryl-4,6-dithianonanedioic acids and related compounds has been prepared for evaluation of leukotriene antagonist activity. The alkynyl compounds were prepared by thioacetal exchange from the corresponding acetylenic acetals. The aryl derivatives were synthesized from the appropriate benzaldehydes, most of which were prepared by one of three general routes: Meyers' oxazolin method, a palladium coupling procedure, and a hydroxybenzaldehyde alkylation. The analogues were examined in vitro for their ability to antagonize an LTD4-induced contraction of isolated guinea pig tracheal smooth muscle and to compete with [3H]LTD4 for receptor sites on guinea pig lung membrane. A number of structure-activity relationships have emerged from this study. There is an optimal chain length of 10-12 atoms (or its equivalent) in the lipid tail and two methylenes in the polar region. In the aromatic series, the ortho- and meta-substituted compounds have comparable activity, whereas the para derivatives are inactive. Substitution in the aromatic ring and lipid tail is generally well tolerated, with the terminal phenyl (6) and acetylene (33) analogues having especially good activity. Conformational restriction of either the polar region or lipid tail produced compounds devoid of activity. A number of selected analogues were also evaluated in vivo as antagonists of LTD4-induced bronchoconstriction in the guinea pig. The data established these compounds as a novel class of leukotriene antagonists with potential utility for the treatment of asthma and other immediate hypersensitivity diseases.

Alkynes↗