Use of deuterated phospholipids in Raman spectroscopic studies of membrane structure. I. Multilayers of dimyristoyl phosphatidylcholine (and its -d54 derivative) with distearoyl phosphatidylcholine

The temperature dependence of the Raman spectrum has been studied for binary phospholipid mixtures of dimyristoyl phosphatidylcholine (and its chain deuterated -d54 derivative) with distearoyl phosphatidylcholine. Two distinct melting regions are observed for the 1 : 1 mole ratio mixture. The use of deuterated phospholipid permits the identification of the lower (approximately 22 degrees C) transition with primarily the melting of the shorter chain component, and the higher (approximately 47 degrees C) transition primarily with the melting of the longer chains. The C-H stretching vibrations of the distearoyl component respond to the melting of the dimyristoyl component, an apparent consequence of alterations in the lateral interactions of the distearoyl chains. These changes in the C-H spectral region suggest that phase separation does not occur in the gel state for this system. The results are in reasonable accord with recent calorimetric studies (Mabrey, S. and Sturtevant, J.M. (1976) Proc. Natl. Acad. Sci. U.S. 73, 3862-3866). The feasibility of using deuterated phospholipids to monitor the conformation of each component in a binary phospholipid mixture is demonstrated.     

On the mechanism for formation of RNA . DNA complexes from lymphocytes.

Early intermediates in DNA synthesis by human lymphocytes were studied for the possible association of RNA with nascent DNA. Nucleic acid extracts from cells pulse-labeled with [3H] uridine contain RNA that is associated with DNA in Cs2SO4 equilibrium density gradients. The amount of RNA bound to DNA was greatly reduced by repeated denaturation and equilibrium centrifugation. An apparently similar complex between RNA and DNA was formed in reconstruction experiments in which purified [3H] uridine-labeled RNA was mixed with purified DNA. The association between RNA and DNA could be eliminated in the reconstruction experiments and greatly reduced in extracts from pulse-labeled cells by denaturation and equilibrium centrifugation in the presence of formaldehyde. These studies demonstrate that noncovalent bonding between RNA and DNA can account for most, and possibly all, of the RNA with density close to DNA in Cs2SO4 gradients of nascent DNA preparations. In addition, the results indicate that ribonucleotide, demonstrated by other methods to be covalently bound to nascent DNA, must constitute less than 1/5 of the total nucleotide in the molecule.     

Novel Rickettsiella Bacterium in the Leafhopper Orosius albicinctus (Hemiptera: Cicadellidae)

Bacteria in the genus Rickettsiella (Coxiellaceae), which are mainly known as arthropod pathogens, are emerging as excellent models to study transitions between mutualism and pathogenicity. The current report characterizes a novel Rickettsiella found in the leafhopper Orosius albicinctus (Hemiptera: Cicadellidae), a major vector of phytoplasma diseases in Europe and Asia. Denaturing gradient gel electrophoresis (DGGE) and pyrosequencing were used to survey the main symbionts of O. albicinctus, revealing the obligate symbionts Sulcia and Nasuia, and the facultative symbionts Arsenophonus and Wolbachia, in addition to Rickettsiella. The leafhopper Rickettsiella is allied with bacteria found in ticks. Screening O. albicinctus from the field showed that Rickettsiella is highly prevalent, with over 60% of individuals infected. A stable Rickettsiella infection was maintained in a leafhopper laboratory colony for at least 10 generations, and fluorescence microscopy localized bacteria to accessory glands of the female reproductive tract, suggesting that the bacterium is vertically transmitted. Future studies will be needed to examine how Rickettsiella affects host fitess and its ability to vector phytopathogens.     

A polyalanine-based peptide cannot form a stable transmembrane alpha-helix in fully hydrated phospholipid bilayers

The conformation and amide proton exchangeability of the peptide acetyl-K(2)-A(24)-K(2)-amide (A(24)) and its interaction with phosphatidylcholine bilayers were examined by a variety of physical techniques. When dissolved in or cast from methanol as a dried film, A(24) is predominantly alpha-helical. In aqueous media, however, A(24) exists primarily as a mixture of helical (though not necessarily alpha-helical) and random coiled structures, both of which allow rapid H-D exchange of all amide protons. When incorporated into phospholipids in the absence of water, A(24) also exists primarily as a transmembrane alpha-helix. However, upon hydration of that system, rapid exchange of all amide protons also occurs along with a marked change in the amide I absorption band of the peptide. Also, when dispersed with phosphatidylcholine in aqueous media, the conformation and thermal stability of A(24) are not significantly altered by the presence of the phospholipid or by its gel/liquid-crystalline phase transition. Differential scanning calorimetric and electron spin resonance spectroscopic studies indicate that A(24) has relatively minor effects on the thermodynamic properties of the lipid hydrocarbon chain-melting phase transition, that it does not abolish the lipid pretransition, and that its presence has no significant effect on the orientational order or rates of motion of the phospholipid hydrocarbon chains. We therefore conclude that A(24) has sufficient alpha-helical propensity, but insufficient hydrophobicity, to maintain a stable transmembrane association with phospholipid bilayers in the presence of water. Instead, it exists primarily as a dynamic mixture of helices and other conformers and resides mostly in the aqueous phase where it interacts weakly with the bilayer surface or with the polar/apolar interfacial region of phosphatidylcholine bilayers. Thus, polyalanine-based peptides are not good models for the transmembrane alpha-helical segments of natural membrane proteins.     

Characterization of a new member of the fatty acid-binding protein family that binds all-trans-retinol

Cellular retinol-binding protein, type I (CRBP-I) and type II (CRBP-II) are the only members of the fatty acid-binding protein (FABP) family that process intracellular retinol. Heart and skeletal muscle take up postprandial retinol but express little or no CRBP-I or CRBP-II. We have identified an intracellular retinol-binding protein in these tissues. The 134-amino acid protein is encoded by a cDNA that is expressed primarily in heart, muscle and adipose tissue. It shares 57 and 56% sequence identity with CRBP-I and CRBP-II, respectively, but less than 40% with other members of the FABP family. In situ hybridization demonstrates that the protein is expressed at least as early as day 10 in developing heart and muscle tissue of the embryonic mouse. Fluorescence titrations of purified recombinant protein with retinol isomers indicates binding to all-trans-, 13-cis-, and 9-cis-retinol, with respective K(d) values of 109, 83, and 130 nm. Retinoic acids (all-trans-, 13-cis-, and 9-cis-), retinals (all-trans-, 13-cis-, and 9-cis-), fatty acids (laurate, myristate, palmitate, oleate, linoleate, arachidonate, and docosahexanoate), or fatty alcohols (palmityl, petrosenlinyl, and ricinolenyl) fail to bind. The distinct tissue expression pattern and binding specificity suggest that we have identified a novel FABP family member, cellular retinol-binding protein, type III.     

Visualizing sexual dimorphism in the brain

Sexually dimorphic behaviors are likely to involve neural pathways that express the androgen receptor (AR). We have genetically modified the AR locus to visualize dimorphisms in neuronal populations that express AR. Analysis of AR-positive neurons reveals both known dimorphisms in the preoptic area of the hypothalamus and the bed nucleus of the stria terminalis as well as novel dimorphic islands in the basal forebrain with a clarity unencumbered by the vast population of AR-negative neurons. This genetic approach allows the visualization of dimorphic subpopulations of AR-positive neurons along with their projections and may ultimately permit an association between neural circuits and specific dimorphic behaviors.     

Pitfalls of nonstandardized photography in facial plastic surgery patients

The authors tested the hypothesis that certain maneuvers (neck flexion/extension and head protrusion/retrusion) alter the appearance of the submental area, jawline, and melolabial groove. They used a questionnaire survey of 20 na?ve judges who assessed a standardized photograph album of three subjects. The subjects' faces (frontal and lateral views) were photographed in neutral, neck flexion/extension, and head protrusion/retrusion positions. High Kendall coefficients of correlation were observed in 10 of 12 questions evaluating an improvement in jawline definition with neck extension or head protrusion, as well as in 11 of 12 questions assessing decreased submental soft tissue. All questions relating to the melolabial groove had a correlation coefficient of less than 0.70. Small changes in patient positioning during photodocumentation for facial plastic surgical procedures can cause dramatic changes in the appearance of certain parameters. Standardizing patient positioning for preoperative and postoperative photographs is imperative.     

Hyperforin inhibits vesicular uptake of monoamines by dissipating pH gradient across synaptic vesicle membrane

Extracts of Hypericum perforatum (St. John's wort) have antidepressant properties in depressed patients and exert antidepressant-like action in laboratory animals. The phloroglucinol derivative hyperforin has become a topic of interest, as this Hypericum component is a potent inhibitor of monoamines reuptake. The molecular mechanism by which hyperforin inhibits monoamines uptake is yet unclear. In the present study we try to clarify the mechanism by which hyperforin inhibits the synaptic vesicle transport of monoamines. The pH gradient across the synaptic vesicle membrane, induced by vacuolar type H(+)-ATPase, is the major driving force for vesicular monoamines uptake and storage. We suggest that hyperforin, like the protonophore FCCP, dissipates an existing Delta pH generated by an efflux of inwardly pumped protons. Proton transport was measured by acridine orange fluorescence quenching. Adding Mg-ATP to a medium containing 130 mM KCl and synaptic vesicles caused an immediate decrease in fluorescence of acridine orange and the addition of 1 microM FCCP abolished this effect. H(+)-ATPase dependent proton pumping was inhibited by hyperforin in a dose dependent manner (IC(50) = 1.9 x 10(-7) M). Hyperforin acted similarly to the protonophore FCCP, abolishing the ATP induced fluorescence quenching (IC(50) = 4.3 x 10(-7) M). Hyperforin and FCCP had similar potencies for inhibiting rat brain synaptosomal uptake of [3H]monoamines as well as vesicular monoamine uptake. The efflux of [3H]5HT from synaptic vesicles was sensitive to both drugs, thus 50% of preloaded [3H]5HT was released in the presence of 2.1 x 10(-7) M FCCP and 4 x 10(-7) M hyperforin. The effect of hyperforin on the pH gradient in synaptic vesicle membrane may explain its inhibitory effect on monoamines uptake, but could only partially explain its antidepressant properties.     

An infrared reflection-absorption spectroscopy study of the secondary structure in (KL4)4K,a therapeutic agent for respiratory distress syndrome,in aqueous monolayers with phospholipids

KLLLLKLLLLKLLLLKLLLLK (KL(4)) has been suggested to mimic some aspects of the pulmonary surfactant protein SP-B and has been tested clinically as a therapeutic agent for respiratory distress syndrome in premature infants [Cochrane, C. G., and Revak, S. D. (1991) Science 254, 566-568]. It is of obvious interest to understand the mechanism of KL(4) function as a guide for design of improved therapeutic agents. Attenuated total reflection (ATR) IR measurements have indicated that KL(4) is predominantly alpha-helical with a transmembrane orientation in lipid multilayers (1), a geometry quite different from the originally proposed peripheral membrane lipid interaction. However, the lipid multilayer model required for ATR may not be the best experimental paradigm to mimic the in vivo function of KL(4). In the current experiments, IR reflection-absorption spectroscopy (IRRAS) was used to evaluate peptide secondary structure in monolayers at the air/water interface, the physical state that best approximates the alveolar lining. In contrast to the ATR-IR results, KL(4) (2.5-5 mol %) films with either DPPC or DPPC/DPPG (7/3 mol ratio) adopted an antiparallel beta-sheet structure at all surface pressures studied > or =5 mN/m, including pressures physiologically relevant for lung function (40-72 mN/m). In contrast, in DPPG/KL(4) films, the dominant conformation was the alpha-helix over the entire pressure range, a possible consequence of enhanced electrostatic interactions. IRRAS has thus provided unique molecular structure information and insight into KL(4)/lipid interaction in a physiologically relevant state. A structural model is proposed for the response of the peptide to surface pressure changes.