Solution structure of amyloid beta-peptide(1-40) in a water-micelle environment. Is the membrane-spanning domain where we think it is?

The three-dimensional solution structure of the 40 residue amyloid beta-peptide, A beta(1-40), has been determined using NMR spectroscopy at pH 5.1, in aqueous sodium dodecyl sulfate (SDS) micelles, In this environment, which simulates to some extent a water-membrane medium, the peptide is unstructured between residues 1 and 14 which are mainly polar and likely solvated by water. However, the rest of the protein adopts an alpha-helical conformation between residues 15 and 36 with a kink or hinge at 25-27. This largely hydrophobic region is likely solvated by SDS. Based on the derived structures, evidence is provided in support of a possible new location for the transmembrane domain of A beta within the amyloid precursor protein (APP). Studies between pH 4.2 and 7.9 reveal a pH-dependent helix-coil conformational switch. At the lower pH values, where the carboxylate residues are protonated, the helix is uncharged, intact, and lipid-soluble. As the pH increases above 6.0, part of the helical region (15-24) becomes less structured, particularly near residues E22 and D23 where deprotonation appears to facilitate unwinding of the helix. This pH-dependent unfolding to a random coil conformation precedes any tendency of this peptide to aggregate to a beta-sheet as the pH increases. The structural biology described herein for A beta(1-40) suggests that (i) the C-terminal two-thirds of the peptide is an alpha-helix in membrane-like environments, (ii) deprotonation of two acidic amino acids in the helix promotes a helix-coil conformational transition that precedes aggregation, (iii) a mobile hinge exists in the helical region of A beta(1-40) and this may be relevant to its membrane-inserting properties and conformational rearrangements, and (iv) the location of the transmembrane domain of amyloid precursor proteins may be different from that accepted in the Literature. These results may provide new insight to the structural properties of amyloid beta-peptides of relevance to Alzheimer's disease.

Membrane-bounded nucleoids in microbial symbionts of marine sponges

In thin sections of resin-embedded samples of glutaraldehyde- and osmium tetroxide-fixed tissue from five genera of marine sponges, Stromatospongia, Astrosclera, Jaspis, Pseudoceratina and Axinyssa, cells of a bacteria-like symbiont microorganism which exhibit a membrane-bounded nuclear region encompassing the fibrillar nucleoid have been observed within the sponge mesohyl. The nuclear region in these cells is bounded by a single bilayer membrane, so that the cell cytoplasm is divided into two distinct regions. The cell wall consists of subunits analogous to those in walls of some Archaea. Cells of the sponge symbionts observed here are similar to those of the archaeal sponge symbiont Cenarchaeum symbiosum. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

In vitro human epidermal and polyethylene membrane penetration and retention of the sunscreen benzophenone-3 from a range of solvents

Purpose. To study epidermal and polyethylene membrane penetration and retention of the sunscreen benzophenone-3 (BP) from a range of single solvent vehicles and evaluate solvent effects on permeability parameters. Methods. The solubility of BP was measured in a number of solvents. Penetration of BP across human epidermis and high density polyethylene (HDPE) membranes was studied from 50% saturated solutions in each solvent. Results. Maximal BP fluxes from the solvents across the two membranes varied widely. Highest fluxes were observed from 90% ethanol (EtOH) for epidermis and from isopropyl myristate (IPM) and C12-15 benzoate alcohols (C12-15 BA) for HDPE membrane. Both the flux and estimated permeability coefficient and skin-vehicle partitioning of BP appeared to be related to the vehicle solubility parameter (delta(v)). The major effects of solvents on BP flux appear to be via changes in BP diffusivity through the membranes. Conclusions. Minimal penetration of sunscreens such as BP is best achieved by choosing vehicles with a delta(v) substantially different to the solubility parameter of the membrane.

Determination of chemical shielding tensor of an indole carbon and application of tryptophan orientation of a membrane peptide

Using tryptophan C-13-enriched at the C-4 (C epsilon(3)) of the indole, the orientation of the C epsilon(3) chemical shift tensor relative to the C epsilon(3)-H dipolar axis was determined from the C-13 chemical shift/C-13-H-1 dipolar 2D NMR powder pattern. The principal values obtained were 208, 137 and 15 ppm with sigma(33) perpendicular to the indole plane, and sigma(11) (least shielded direction) 5 degrees off the C epsilon(3)-H bond toward C xi(3). The side off the C epsilon(3)-H bond was determined by comparing the reduced chemical shift anisotropies obtained by solid-state NMR and from molecular dynamics calculations of [4-C-13] tryptophans in gramicidin A aligned in phospholipid membranes. (C) 1999 Elsevier Science B.V. All rights reserved.

The plasma membrane calcium pump, its role and regulation: New complexities and possibilities

Significant progress has been achieved in elucidating the role of the plasma membrane Ca2+-ATPase in cellular Ca2+ homeostasis and physiology since the enzyme was first purified and physiology since the enzyme was first purified and cloned a number of years ago. The simple notion that the PM Ca2+-ATPase controls resting levels of [Ca2+](CYT) has been challenged by the complexity arising from the finding of four major isoforms and splice variants of the Ca2+ pump, and the finding that these are differentially localized in various organs and subcellular regions. Furthermore, the isoforms exhibit differential sensitivities to Ca2+, calmodulin, ATP, and kinase-mediated phosphorylation. The latter pathways of regulation can give rise to activation or inhibition of the Ca2+ pump activity, depending on the kinase and the particular Ca2+ pump isoform. Significant progress is being made in elucidating subtle and more profound roles of the PM Ca2+-ATPase in the control of cellular function. Further understanding of these roles awaits new studies in both transfected cells and intact organelles, a process that will be greatly aided by the development of new and selective Ca2+ pump inhibitors. (C) 1999 Elsevier Science Inc.

Isolation and characterization of a Clostridium sp with cinnamoyl esterase activity and unusual cell envelope ultrastructure

Microorganisms that hydrolyse the ester linkages between phenolic acids and polysaccharides in plant cell walls are potential sources of enzymes for the degradation of lignocellulosic waste. An anaerobic, mesophilic, spore-forming, xylanolytic bacterium with high hydroxy cinnamic acid esterase activity was isolated from the gut of the grass-eating termite Tumilitermes pastinator. The bacterium was motile and rod-shaped, stained gram-positive, had an eight-layered cell envelope, and.formed endospores. Phylogenetic analysis based on 16S rRNA indicated that the bacterium is closely related to Clostridium xylanolyticum and is grouped with polysaccharolytic strains of clostridia. A wide range of carbohydrates were fermented, and growth was stimulated by either xylan or cellobiose as substrates. The bacterium hydrolysed and then hydrogenated the hydroxy cinnamic acids (ferulic and p-coumaric acids), which are esterified to arabinoxylan in plant cell walls. Three cytoplasmic enzymes with hydroxy cinnamic acid esterase activity were identified using non-denaturing gel electrophoresis. This bacterium possesses an unusual multilayered cell envelope in which both leaflets of the cytoplasmic membrane, the peptidoglycan layer and the S layer are clearly discernible. The fate of all these components was easily followed throughout the endospore formation process. The peptidoglycan component persisted during the entire morphogenesis. It was seen to enter the septum and to pass with the engulfing membranes to surround the prespore. It eventually expanded to form the cortex, verification for the peptidoglycan origin of the cortex. Sporogenic vesicles, which are derived from the cell wall peptidoglycan, were associated with the engulfment process. Spore coat fragments appeared early, in stage II, though spore coat formation was not complete until after cortex formation.

Thylakoid membrane architecture

Confocal scanning laser microscopic observations were made on live chloroplasts in intact cells and on mechanically isolated, intact chloroplasts. Chlorophyll fluorescence was imaged to observe thylakoid membrane architecture. C-3 plant species studied included Spinacia oleracea L., Spathiphyllum sp. Schott, cv. 'Mauna Loa', and Pisum sativum L. C-4 plants were also investigated: Saccharum officinarum L., Sorghum bicolor L. Moench, Zea mays L. and Panicum miliaceum L. Some Spinacia chloroplasts were treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to enhance or sodium dithionite (SD) to reduce the photosystem II fluorescence signal. Confocal microscopy images of C-3 chloroplasts differed from electron microscopy pictures because they showed discrete spots of bright fluorescence with black regions between them. There was no evidence of fluorescence from stroma thylakoids. The thylakoid membrane system at times appeared to be string-like, with brightly fluorescing grana lined up like beads. C-4 bundle sheath chloroplasts were imaged from three different types of C-4 plants. Saccharum and Sorghum bundle sheath chloroplasts showed homogeneous fluorescence and were much dimmer than mesophyll chloroplasts. Zea had rudimentary grana, and dim, homogeneous intergrana fluorescence was visualised. Panicum contained thylakoids similar in appearance and string-like arrangement to mesophyll chloroplasts. Isolated Pisum chloroplasts, treated with a drop of 5 mM MgCl2 showed a thylakoid membrane system which appeared to be unravelling. Spongy mesophyll chloroplasts of Spinacia treated with 5 mM sodium dithionite showed a granal thylakoid system with distinct regions of no fluorescence. A time-series experiment provided evidence of dynamic membrane rearrangements over a period of half an hour.

Evolutionary conservation of the membrane fusion machine

Recent structural studies of proteins mediating membrane fusion reveal intriguing similarities between diverse viral and mammalian systems. Particularly striking is the close similarity between the transmembrane envelope glycoproteins from the retrovirus HTLV-1 and the filovirus Ebola. These similarities suggest similar mechanisms of membrane fusion. The model that fits most currently available data suggests fusion activation in viral systems is driven by a symmetrical conformational change triggered by an activation event such as receptor binding or a pH change. The mammalian vesicle fusion mediated by the SNARE protein complex most likely occurs by a similar mechanism but without symmetry constraints.

Structure of the tegument and ectocommensal microorganisms of Gyliauchen nahaensis (Digenea : Gyliauchenidae), an inhabitant of herbivorous fish of the Great Barrier Reef, Australia

The ultrastructure of the tegument and tegument-associated microorganisms of the gyliauchenid digenean Gyliauchen nahaensis is described by transmission and scanning electron microscopy. The tegument is devoid of surface spines and is characterized by a moderately folded apical membrane, abundant vesicles, basal mitochondria, a folded basal plasma membrane, and a thick basal matrix. Microorganisms form a dense biofilm on the tegument of the posterodorsal surface and the excretory papilla. At least 7 microbial morphotypes were identified, including eubacteria, spirochaetes, and nanobacteria.

Vesicle-associated proteins and transmitter release from sympathetic ganglionic boutons

A method is reported for introducing peptides derived from SNARE proteins that control exocytosis of vesicles at boutons formed by sympathetic ganglion cells in tissue culture. These peptides were coupled to the DNA binding domain of the Drosophila transcription factor antennapedia, called penetratin, This facilitated the passage of peptides across the bouton membrane. FMI-43 was used to monitor the exocytosis of transmitter from depolarized boutons after their exposure to the penetratin-peptide sequences IETRHNEIIKLETSIRELHD of syntaxin and KGFLSSLFGGSSK of alpha -SNAP. both of which blocked secretion, whereas the peptide sequences SELDDRA-DALQAGASQFETSAAKLKRK of synaptobrevin did not. This report introduces a readily applicable method for determining the effect of different peptide sequences of vesicle-associated proteins on secretion at vertebrate boutons and presents an account of the effects of a selection of such peptides on exocytosis. NeuroReport 12:607-610 (C) 2001 Lippincott Williams & Wilkins.

Association of bovine papillomavirus type 1 with microtubules

Transport of BPV-1 virus from the cell membrane to the nucleus was studied in vitro in CV-1 cells. At reduced temperature (4 degreesC). BPV-I binding to CV-1 cells was unaffected but there was no transport of virions across the cytosol. Electron microscopy showed BPV-I virions in association with microtubules in the cytoplasm, a finding confirmed by co-immunoprecipitation of L1 protein and tubulin. Internalization of virus was unimpaired in cells treated with the microtubule-depolymerizing drug nocodazole but virions were retained in cytoplasmic vesicles and not transported to the nucleus. We conclude that a microtubule transport mechanism in CV-1 cells moves intact BPV-1 virions from the cell surface to the nuclear membrane. (C) 2001 Academic Press.

Interaction of RTD-1, a cyclic antimicrobial defensin from Rhesus macaque leukocytes, and its open chain analogue with membrane mimics

In contrast to other mammalian defensins, rhesus theta defensin-1 (RTD-1) is composed of just 18 amino acids with the backbone cyclized through peptide bonds. Antibacterial activities of both the native cyclic peptide and a linear form were examined, showing that the cyclic form was 3-fold more active than the open chain analogue, oRTD-1, although both peptides adopt very similar structures in water. It was suggested that the additional charges at the termini of oRTD-1 are the cause for its lower antimicrobial activity. Therefore, we studied the interaction of both peptides with membrane mimics composed of zwitterionic (PC) and negatively charged (PG) phospholipids, major lipid components of erythrocyte and bacterial cell membranes, respectively. Microcalorimetry showed that RTD-1 and oRTD-1 did not affect the phase behavior of PC liposomes, while in PG liposomes both peptides induced new phase transitions above the chain melting transition of the lipid. The shape and fraction differed between both peptides, depending also on their concentration, which will be discussed in terms of their antimicrobial activity.

Tomosyn interacts with the t-SNAREs Syntaxin4 and SNAP23 and plays a role in insulin-stimulated GLUT4 translocation

The Sec1p-like/Munc18 (SM) protein Munc18a binds to the neuronal t-SNARE Syntaxin1A and inhibits SNARE complex assembly. Tomosyn, a cytosolic Syntaxin1A-binding protein, is thought to regulate the interaction between Syntaxin1A and Munc18a, thus acting as a positive regulator of SNARE assembly. In the present study we have investigated the interaction between b-Tomosyn and the adipocyte SNARE complex involving Syntaxin4/SNAP23/VAMP-2 and the SM protein Munc18c, in vitro, and the potential involvement of Tomosyn in regulating the translocation of GLUT4 containing vesicles, in vivo. Tomosyn formed a high affinity ternary complex with Syntaxin4 and SNAP23 that was competitively inhibited by VAMP-2. Using a yeast two-hybrid assay we demonstrate that the VAMP-2-like domain in Tomosyn facilitates the interaction with Syntaxin4. Overexpression of Tomosyn in 3T3-L1 adipocytes inhibited the translocation of green fluorescent protein-GLUT4 to the plasma membrane. The SM protein Munc18c was shown to interact with the Syntaxin4 monomer, Syntaxin4 containing SNARE complexes, and the Syntaxin4/Tomosyn complex. These data suggest that Tomosyn and Munc18c operate at a similar stage of the Syntaxin4 SNARE assembly cycle, which likely primes Syntaxin4 for entry into the ternary SNARE complex.