Spontaneous Incorporation Research Articles

Prebiotic cytosine synthesis: a critical analysis and implications for the origin of life.

A number of theories propose that RNA, or an RNA-like substance, played a role in the origin of life. Usually, such hypotheses presume that the Watson-Crick bases were readily available on prebiotic Earth, for spontaneous incorporation into a replicator. Cytosine, however, has not been reported in analyses of meteorites nor is it among the products of electric spark discharge experiments. The reported prebiotic syntheses of cytosine involve the reaction of cyanoacetylene (or its hydrolysis product, cyanoacetaldehyde), with cyanate, cyanogen, or urea. These substances undergo side reactions with common nucleophiles that appear to proceed more rapidly than cytosine formation. To favor cytosine formation, reactant concentrations are required that are implausible in a natural setting. Furthermore, cytosine is consumed by deamination (the half-life for deamination at 25 degrees C is approximately 340 yr) and other reactions. No reactions have been described thus far that would produce cytosine, even in a specialized local setting, at a rate sufficient to compensate for its decomposition. On the basis of this evidence, it appears quite unlikely that cytosine played a role in the origin of life. Theories that involve replicators that function without the Watson-Crick pairs, or no replicator at all, remain as viable alternatives.

Sliding wear behaviour of aluminium-based metal matrix composites produced by a novel liquid route

TiC-reinforced MMCs have been produced in a range of aluminium alloys using a novel casting technique which results in spontaneous incorporation of the particles into the melt and thus strong bonding between the particles and the matrix. The sliding wear behaviour of the extruded composites has been studied as a function of load and particle volume fraction and has been compared with a commercially available SiC-reinforced composite. In all cases, alloy reinforcement resulted in a reduction in wear rate and an increase in the load at which the transition from low rate wear to high rate wear occurred. In the low rate wear regime, the wear coefficients of all the alloys in both the reinforced and unreinforced states were similar, and since the TiC-reinforced A356 alloy was the hardest (due in part to the grain refining action of TiC), it exhibited the lowest wear rate (lower than that of the SiC-reinforced composite). Wear of the steel counterface depended on the mechanism of wear of the composite. An increase in load generally resulted in an increase in wear rate of both the composite pin and counterface, and the reasons for this are presented. Increasing the volume fraction of particles in a composite reduces its wear rate but generally increases the wear rate of the counterface. It is suggested that when both counterface and composite wear are considered, an optimum volume fraction of particles exists at which wear is lowest.

Temperature dependence of a-C:H film deposition in a CH4 radio frequency plasma

The hydrogenated hard carbon films were deposited at elevated temperature in planar rf CH4 plasmas. The deposition rate and mechanical properties of the carbon films deposited at substrate temperature between room temperature and 400 °C were examined. The behavior of the film deposition rate indicated the negative temperature slope, and activation energies of around 0.04–0.05 eV at gas pressures of 40–100 mTorr were obtained from the Arrhenius plots of the deposition rates. Both the temperature behavior and the value of the activation energy suggest that the spontaneous incorporation of physisorbed CH3 precursor into the growing film is negligible, and the chemical reaction between the CH3 molecules and hydrocarbon ions bombarding the film surface is most probable in the present deposition process. The dense carbon films having film densities up to 2.7 g/cm3 were obtained at 40 mTorr.

Direct Observation of Mobile Protons in SiO2 Thin Films: Potential Application in a Novel Memory Device

AbstractIn this work we show that annealing of silicon/silicon‐dioxide/silicon structures in forming gas (N2:H2; 95:5) above 500°C leads to spontaneous incorporation of mobile H+ ions in the buried SiO2 layer. We demonstrate that, unlike the alkali ions feared as killer contaminants in the early days, the space charge distribution of these mobile protons within the buried oxide layer can be very well controlled and easily rearranged with relatively high speed at room temperature. The hysteresis in the flat band voltage shift provides a unique vehicle to study proton kinetics in silicon dioxide thin films. It is further shown how this effect has great potential as the basis for a reliable nonvolatile FET memory device that is expected to be competitive with state‐of‐the‐art Si‐based memory technologies. The power of this novel device is its simplicity; it requires few processing steps, all of which are standard in Si integrated‐circuit fabrication.

Actinobacillus actinomycetemcomitans leukotoxin forms large conductance, voltage-gate ion channels when incorporated into planar lipid bilayers

Actinobacillus actinomycetemcomitans leukotoxin is a member of the bacterial RTX (repeats in toxin) toxin family, produced by a diverse group of Gram-negative pathogens. Members of this group of toxins, although similar in sequence, differ in target cell specificity with Actinobacillus actinomycetemcomitans leukotoxin demonstrating a unique species-and cell-type specificity. Purified A. actinomycetemcomitans leukotoxin added to pre-formed POPE/POPS lipid bilayers showed no spontaneous incorporation (to concentrations of 250 ng/ml). Reproducible channel activity was seen when the bilayer was reformed from lipid monolayers in the presence of toxin (50 ng/ml) in one of the aqueous chambers. Control experiments with heat-inactivated toxin did not display channel activity under the same experimental conditions. The channel behavior showed a complex pattern of multiple conductance levels of 118 262 and 406 pS in solutions containing 0.140 M NaCL The first two states showed voltage-dependent channel gating with approximately equal but opposite apparent gating charges of 1.4 electrons. A model accounting for the multiple conducting states and gating properties is presented.

New Structural Type in Manganese Carboxylate Chemistry via Coupled Oxidation/Oxide Incorporation: Potential Insights into Photosynthetic Water Oxidation

There continues to be great interest in the mechanism by which the tetranuclear Mn aggregate at the photosynthetic water oxidation center (WOC) of green plants and cyanobacteria binds H{sub 2}O molecules and oxidatively couples them to O{sub 2}. The WOC cycles through a number of oxidation levels (S{sub n} states; n = 0-4), with the highest (S{sub 4}) relaxing spontaneously to the lowest (S{sub 0}) with O{sub 2} evolution. Although a variety of spectroscopic and physicochemical studies have been performed, the precise structure of the WOC Mn{sub 4} aggregate at any S{sub n} state remains unclear. It is seen that the one-electron oxidation of a [Mn{sub 4}O{sub 2}]{sup 8+} complex leads to spontaneous incorporation of a third O{sup 2-} to give a novel [Mn{sub 4}O{sub 3}]{sup 7+} core, a type of process speculatively suggested in the past as possibly involved in the WOC catalytic cycle. Its demonstration now provides precedence for this possibility. 9 refs., 3 figs.

Spontaneous transfer of ganglioside GM1 from its micelles to lipid vesicles of differing size.

The spontaneous incorporation of II3-N-acetylneuraminosylgangliotetraosylceramide (GM1) from its micelles into phospholipid bilayer vesicles has been investigated to determine whether curvature-induced changes in membrane lipid packing influence ganglioside uptake. Use of conventional liquid chromatography in conjunction with technically-improved molecular sieve gels permits ganglioside micelles to be separated from phospholipid vesicles of different average size including vesicles with diameters smaller than 40 nm and, thus, allows detailed study of native ganglioside GM1 incorporation into model membranes under conditions where complicating processes like fusion are readily detected if present. At 45 degrees C, the spontaneous transfer rate of GM1 from its micelles to small unilamellar vesicles (SUVs) comprised of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) is at least 3-fold faster than that to similar composition large unilamellar vesicles (LUVs) prepared by octyl glucoside dialysis. Careful analysis of ganglioside GM1 distribution among vesicle populations of differing average size reveals that GM1 preferentially incorporates into the smaller vesicles of certain populations. This behavior is observed in SUVs as well as in LUV-SUV mixtures and actually serves as a sensitive indicator for the presence of trace quantities of SUVs in various LUV preparations. Analysis of the results shows that both differences in the diffusional collision frequency between GM1 monomers and either SUVs or LUVs and curvature-induced changes in the interfacial lipid packing in either SUVs or LUVs can dramatically influence spontaneous ganglioside uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Spontaneous incorporation of the glycosyl-phosphatidylinositol-linked protein Thy-1 into cell membranes.

Thy-1 is a membrane protein that is attached to the plasma membrane by a glycosyl-phosphatidylinositol anchor. Purified rat brain Thy-1 could be reincorporated into the plasma membrane of murine Thy-1- cells directly from aqueous suspension and without the use of detergents. A peripheral staining pattern similar to that observed for endogenous Thy-1 was achieved. Treatment with phosphatidylinositol-specific phospholipase C removed nearly all antibody staining due to either endogenous or inserted Thy-1. Fluorescence recovery after photobleaching (FRAP) was used to compare the lateral mobility of endogenous and inserted Thy-1. Both forms exhibited large lateral diffusion coefficients, but with a substantial immobile fraction (approximately 50%) indicating that the immobile fraction was not due either to chemical differences between inserted and native Thy-1 or to some surface Thy-1 molecules having a protein anchor. However, the inserted Thy-1 failed to activate mouse T lymphocytes upon crosslinking as assayed by [3H]thymidine uptake. Since Thy-1 could be directly labeled with rhodamine, the effect of the size of the labeling ligand on the mobility obtained by the FRAP technique could be explored. Rhodamine-conjugated MRC-OX7 monoclonal antibody or its fragments [R-F(ab)2 or R-Fab] were compared with rhodamine as labels for Thy-1. The measured diffusion coefficients were 1.6 x 10(-9), 2.0 x 10(-9), and 3.2 x 10(-9) cm2/sec for Thy-1 labeled with R-F(ab)2, R-Fab, and rhodamine, respectively; mobile fractions were all in the 40-50% range. Thus, the size of the ligand affects the lateral mobility of this labeled membrane protein to a measurable extent.

Spontaneous incorporation of the glycosyl-phosphatidylinositol-linked protein Thy-1 into cell membranes.

Spontaneous incorporation of the glycosyl-phosphatidylinositol-linked protein Thy-1 into cell membranes.

Comparative studies on in vitro reactivity of fresh and cryopreserved pig lymphocytes

The effect of cryopreservation on in vitro reactivity of pig lymphocytes was studied. Peripheral blood mononuclear cells (PBMC) were frozen by controlled-rate freezing and stored in liquid nitrogen (LN 2) between 4 and 36 days. Following thawing 74.7 ± 2.6% of cells were recovered of which 94.5 ± 0.9% were viable as determined by trypan blue exclusion. Functional parameters measured included the concentration of free intracellular Ca 2+ ([Ca 2+] i) in resting and mitogen-stimulated PBMC, mitogen and alloantigen-induced blastogenesis, as well as cell-mediated cytotoxicity. Irrespective of storage time and cell donor, [Ca 2+] i in frozen-thawed PBMC (67.7 ± 4.3 n M) was significantly lower ( P < 0.001) when compared to fresh cells (96.2 ± 4.5 n M). In addition, cryopreserved PBMC only weakly responded with an increase of [Ca 2+] i after stimulation by various concentrations of phytohemagglutinin (PHA). Following activation by PHA (2 μg/ml) for 4 days fresh lymphocytes (84,047 ± 5475 cpm) incorporated significantly more ( P < 0.005) [ 3H]thymidine than frozen PBMC (66,001 ± 4117 cpm). A similar difference in proliferation rates ( P < 0.05) between fresh (10,046 ± 1915 cpm) and frozen-thawed PBMC (5852 ± 1304 cpm) was observed in one-way mixed lymphocyte cultures (MLC), while the spontaneous incorporation of radiolabel was unchanged in frozen stored cells. By using MLC-derived cytotoxic effector cells (E) and [ 3H]thymidine-labeled concanavalin A blasts as targets (T), cryopreserved PBMC displayed a severe deficiency of cytotoxic effector functions at all tested E:T ratios. These results indicate that pig PBMC are very sensitive to LN 2 storage although some immunological functions are more affected by cryopreservation than others.

Use of Size Fractionation of in Vitro‐Activated Human B Lymphocytes for Studies of Cell Cycle‐Dependent Growth Regulation

Cell cycle progression of in vitro-stimulated human B lymphocytes occurs asynchronously. In order to allow detailed studies of growth control in G1, B cells were stimulated with anti-mu and low molecular weight B-cell growth factor (LMW BCGF) for 50 h and subsequently separated into nine fractions of cells by means of centrifugal elutriation. As judged by volume profiles, activation antigen expression and DNA content, the cells in fractions 1-4 were in early to mid-G1, while fractions 5-7 mainly contained cells in late G1, and fractions 8-9 contained cells mainly in S and G2. Cells in fractions 5-7 had passed the commitment point, as demonstrated by a high spontaneous incorporation of [3H]thymidine when recultured in medium alone. Moreover, S-phase entry of these cells was largely unaffected by exogenous growth-promoting or growth-inhibitory signals. Cells in early (fractions 1-2) and intermediate fractions (fractions 3-4) showed a negligible spontaneous [3H]thymidine incorporation, but a significant proportion of these cells progressed to S phase upon restimulation. Moreover, while IL-4 or the anti-CD40 MoAb G28-5 potently stimulated cells in early and intermediate fractions, the responsiveness of LMW BCGF alone was obtained just prior to the commitment point.

Spontaneous incorporation of bacteriorhodopsin into large preformed vesicles

Bacteriorhodopsin, either as purple membrane sheets or as detergent-solubilized protein, was found to incorporate spontaneously into both large unilamellar vesicles (LUVs) and sized multilamellar vesicles (MLVs) under either gel-phase or liquid-phase conditions. These results were obtained with LUVs of various lipid compositions, including dimyristoylphosphatidylcholine (DMPC), DMPC and cholesterol, dioleoylphosphatidylcholine (DOPC), and DOPC and cholesterol. The lipid to protein (L/P) ratio of all proteoliposomes arising from these preformed vesicles continually increased in the presence of protein-free vesicles. Under fluid-phase conditions, the mixing of LUVs of DMPC with proteoliposomes showed vesicle growth independent of lipid concentration, suggesting that growth was due to lipid transfer. However, under gel-phase conditions a more rapid, concentration-dependent increase in the L/P ratio of the proteoliposome was observed, suggesting that growth occurred by a mechanism other than lipid transfer from vesicles to proteoliposomes. The use of the proteoliposome as an artificial lipid-protein membrane model is discussed.

Incorporation of amphotericin B into large unilamellar vesicles composed of phosphatidylcholine and phosphatidylglycerol

The spontaneous incorporation of the polyene antibiotic amphotericin B from a micellar solution into phospholipid vesicles was examined as a function of the lipid composition of the vesicles and their physical state. Virtually no insertion of the antibiotic into egg phosphatidylcholine vesicles was observed even when cholesterol was also present in the bilayer. In contrast, rapid incorporation occurred into systems containing an anionic phospholipid such as phosphatidylglycerol or phosphatidylserine with the fastest rates observed for lipids containing the saturated dimyristoyl fatty acyl species. Insertion of amphotericin B into vesicles composed of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol (7:3 mole ratio) was rapid either above, below or within the gel-to-liquid-crystalline phase transition temperature (23°C). The ability of amphotericin B to intercalate into lipid vesicles is discussed in relation to their relative bilayer stabilities.

Reconstitution of membrane proteins. Spontaneous incorporation of integral membrane proteins into preformed bilayers of pure phospholipid.

The spontaneous reconstitution of lipid-protein complexes was examined by mixing bacteriorhodopsin or UDP-glucuronosyltransferase with preformed, unilamellar bilayers of pure dimyristoylphosphatidylcholine. Spontaneous insertion of these proteins into vesicles of dimyristoylphosphatidylcholine was facilitated by resonicating the vesicles at 4 degrees C. The property of resonicated vesicles that led to spontaneous reconstitution could be annealed by melting the bilayers, which slowed down reconstitution. The overall process of reconstitution consisted, however, of two steps. There was an initial insertion of proteins into a small portion of vesicles followed by subsequent fusion between protein-free vesicles and vesicles containing lipid-protein complexes. The first step appeared to proceed rapidly in all vesicles in a gel phase, whether or not they were resonicated or whether or not resonicated vesicles were annealed. The rate of the second step was sensitive to these treatments. The membrane proteins also inserted into preformed vesicles in a liquid crystalline phase, but this step was slower than for vesicles in a gel phase. Fusion between protein-free and protein-containing vesicles in a liquid crystalline phase was extremely slow. The data show that the spontaneous insertion of pure membrane proteins into preformed vesicles can be a facile event and that the overall reconstitution of membrane proteins into preformed unilamellar vesicles may be simpler to achieve than has been appreciated.

124‐kDa PHYTOCHROME IN MODEL MEMBRANE SYSTEMS: ASSOCIATION STUDIES AND COVALENT BINDING TO PREFORMED LIPOSOMES

Abstract— 124‐kDa Phytochrome from oat has been covalently bound to the surface of preformed unilamellar liposomes doped with functionalized lipids. The extent of phytochrome binding varied from 100% (to soybean lecithin and dioleoyl phosphatidylcholine liposomes) to (90 ± 10)% (to dimyristoyl phosphatidylcholine liposomes) and (50 ± 10)% to dipalmitoyl phosphatidylcholine liposomes). The photochromic properties of phytochrome were fully retained in the liposome‐bound systems. Attempts to bring about spontaneous incorporation and binding of the phytochrome to neutral and positively charged liposomes failed. These results were independent of liposome size, the presence of cholesterol, and whether phytochrome was added prior to or after the liposome formation.

B cell hyperactivity and its relation to distinct clinical features and the degree of disease activity in patients with systemic lupus erythematosus.

Peripheral blood B cells that were actively proliferating, those actively secreting immunoglobulin, and those expressing an early activation marker, Ba antigens, on the surfaces were quantitated in 25 patients with systemic lupus erythematosus (SLE). B cell hyperactivity was found in almost all of the SLE patients, as demonstrated by any one of these measures of B cell activity. Moreover, we observed a strong positive correlation between the degree of disease activity and the amount of spontaneous incorporation of 3H-thymidine by B cells; the magnitude of the increase in frequency of spontaneous Ig-secreting cells in peripheral blood correlated strikingly with certain clinical features in these patients. Our findings suggest that there is heterogeneity of B cell hyperactivity in individual patients with SLE and, thus, that clinical subsets of SLE can be identified on the basis of B cell hyperactivity.

Transport study on [formula omitted] superlattices selectively doped by secondary implantation of Sb

Band offsets in strained layer Si Si 1− x Ge x heterostructures can be tailored by appropriate strain adjustment. The full potential for electronic device fabrication, however, becomes accessible only by methods which allow doping of these structures in a precise way. A new approach to assess doping profiles on an atomic scale is discussed which is applied as well to low temperature Sb spontaneous incorporation as to secondary implantation of Sb into Si 0.5Ge 0.5 layers.

Temperature-dependent osmotic permeability in glycoprotein containing liposomes.

The osmotic water outflow of large multilamellar liposomes containing alpha 1-acid glycoprotein was measured at a temperature near the lipid's phase transition temperature. The liposomes were formed from a mixture of DPPC, cholesterol and glycoprotein in molar ratios 100:20:1, by continuous sucrose density gradient centrifugation. These liposomes captured 35% of the radiolabeled glycoprotein. The temperature-dependent experiments showed that near phase transition temperature the initial rate of water outflow increased drastically in comparison with glycoprotein free liposomes incubated in buffer containing glycoprotein. We suggested that eventual a channel mechanism may be involved due to spontaneous incorporation of glycoprotein into the bilayer.

Reconstitution of membrane proteins: sequential incorporation of integral membrane proteins into preformed lipid bilayers.

Several integral membrane proteins can be inserted sequentially into preformed unilamellar vesicles (ULV's) composed of dimyristoylphosphatidylcholine (DMPC) and cholesterol in a gel phase. Thus, proteoliposomes of DMPC, cholesterol, and bacteriorhodopsin from Halobacterium halobium rapidly incorporate UDPglucuronosyltransferase (EC 2.4.1.17) from pig liver microsomes, cytochrome oxidase from beef heart mitochondria, and additional bacteriorhodopsin, added sequentially. This process of spontaneous incorporation can be regulated to produce complex artificial membranes that contain phospholipids and proteins at ratios (mol/mol) equivalent to what is found in biological membranes. The ability of the lipid-protein bilayers to incorporate additional integral membrane proteins is not affected by annealing of the proteoliposomes at 37 degrees C nor by the order of addition of the proteins. Bacteriorhodopsin-containing vesicles formed by the sequential addition of integral membrane proteins demonstrate light-driven proton pumping. Therefore, they have retained a vesicular structure. Vesicles containing one or two different proteins will fuse with each other at 21 degrees C or with ULV's devoid of proteins. Incorporation of bacteriorhodopsin or UDPglucuronosyltransferase into proteoliposomes containing DMPC, with or without cholesterol as impurity, also occurs above the phase transition for DMPC. The presence of a protein in a liquid-crystalline bilayer provides the necessary condition for promoting the spontaneous incorporation of other membrane proteins into preformed bilayers.

Phosphorylation and degradation of exogenous phosphatidylinositol incorporated into Friend erythroleukemic cells.

Phosphatidyl[2-3H]inositol (PtdIns) obtained from rat skeletal muscle and yeast was introduced into Friend erythroleukemic cells by use of the PtdInstransfer protein or by spontaneous route. The mammalian PtdIns incorporated by the transfer protein appeared metabolically inert while the spontaneously incorporated PtdIns was both phosphorylated to PtdIns-4-phosphate (i.e. 30% of the total PtdIns incorporated) and converted into lyso-PtdIns (i.e. 20% of the total PtdIns incorporated); formation of PtdIns, 4,5-bisphosphate was minimal. The extensive metabolism of the spontaneously incorporated PtdIns strongly suggests that this PtdIns does not rapidly equilibrate with the endogenous PtdIns pools. A similar spontaneous incorporation of yeast PtdIns was accompanied by a negligible degree of phosphorylation and hydrolysis. Evidence is provided that this difference in metabolism reflects the absence of arachidonate in the yeast PtdIns.