The diameters of membrane vesicles fit in geometric series

Vesicles formed in vitro by fragmentation of biomembranes are restricted to certain dimensions; the diameters are represented by two geometric series. The diameters of membrane vesicles found in intact cells, including viral envelopes, are terms of the same two series. A tentative mathematical model is proposed, to explain this phenomenon by fusion of equally sized vesicles.     

Erratum

Vesicular fragments of biomembranes either induced or present as vesicles inside cells, have diameters which fall into two geometric series. Cells with intact cytoskeletons can take up a range of sizes but at mitosis, the cytoskeleton disappears, the cells round up and the plasma membrane can be considered as a vesicle in a stable phase which according to the theory should fall within one of the two possible geometric series. Thus, the size of the mother cell at mitosis and hence of the daughter cells are determined by these geometric series. The diameters of lymphoid cells fall into series which are consistent with the theory.     

Comparison between the size of granular vesicles in intact cells and vesicles Obtained by fragmentation of biomembranes

Summary Preparations of biomembranes, consisting of membrane vesicles, were analyzed with the analytical ultracentrifuge. Under certain conditions depending on the speed of rotation and the temperature, a sedimentation profile was observed that was highly characteristic for membranous material. From the sedimentation coefficients obtained, we calculated particle weights for the various well-defined membrane components. In certain types of preparations the particle weights of two adjacent components differed on average by a factor of 2. When vesicles obtained by fragmentation of biomembranes were compared with the granular vesicles present in intact cells, the accordance in diameters was striking. This may indicate that the size of vesicles is determined by purely physical factors.     

Dynamic light scattering from polydisperse suspensions of thin ellipsoidal shells of revolution with submicron diameters.

Osmotic swelling of membrane vesicles has been studied in combination with dynamic light scattering, to obtain information about the elastic properties of biomembranes. In such studies, there arise some technical problems specific to dynamic light scattering, which include the effects on the light-scattering results of the size distribution and nonsphericity of the vesicles with submicron sizes. Even for highly monodisperse suspensions of spherical vesicles (sigma/dn = [(the mean of d2)/d2n-1]1/2 = 0.1; dn being the number-average diameter of vesicles), the average diameter d obtained from dynamic light scattering is shown to be strongly dependent on dnK, where K is the length of the scattering vector. This is solely due to the shell structure of the vesicles. For ellipsoidal vesicles, another complication appears which is due to the rotational motion of ellipsoids.     

Hexagonal columnar liquid crystal in the cells secreting spider silk

The liquid crystallinity of spider dragline silk dope is thought to be important for both the spinning process and the extreme mechanical properties of the final thread. Although the formation of the liquid crystalline units is poorly understood, it has been suggested that spider silk proteins are secreted in a random coil and then aggregate end-to-end into rod-shaped units to form supramolecular liquid crystals. However, evidence presented here from transmission electron microscopy indicates that coat protein of the dragline silk of a Nephila spider is stored as hexagonal columnar liquid crystals within the intracellular secretory vesicles. This implies that this component is already folded into short rods within the gland cells and forms molecular rather than supramolecular liquid crystals.     

A membrane filtering method for the purification of giant unilamellar vesicles

The use of giant unilamellar vesicles (GUVs) for investigating the properties of biomembranes is advantageous compared to the use of small-sized vesicles such as large unilamellar vesicles (LUVs). Experimental methods using GUVs, such as the single GUV method, would benefit if there was a methodology for obtaining a large population of similar-sized GUVs composed of oil-free membranes. We here describe a new membrane filtering method for purifying GUVs prepared by the natural swelling method and demonstrate that, following purification of GUVs composed of dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) membranes suspended in a buffer, similar-sized GUVs with diameters of 10–30 μm are obtained. Moreover, this method enabled GUVs to be separated from water-soluble fluorescent probes and LUVs. These results suggest that the membrane filtering method can be applied to GUVs prepared by other methods to purify larger-sized GUVs from smaller GUVs, LUVs, and various water-soluble substances such as proteins and fluorescent probes. This method can also be used for concentration of dilute GUV suspensions.     

Cryoelectron microscopy of a nucleating model bile in vitreous ice: formation of primordial vesicles

Because gallstones form so frequently in human bile, pathophysiologically relevant supersaturated model biles are commonly employed to study cholesterol crystal formation. We used cryo-transmission electron microscopy, complemented by polarizing light microscopy, to investigate early stages of cholesterol nucleation in model bile. In the system studied, the proposed microscopic sequence involves the evolution of small unilamellar to multilamellar vesicles to lamellar liquid crystals and finally to cholesterol crystals. Small aliquots of a concentrated (total lipid concentration = 29.2 g/dl) model bile containing 8.5% cholesterol, 22.9% egg yolk lecithin, and 68.6% taurocholate (all mole %) were vitrified at 2 min to 20 days after fourfold dilution to induce supersaturation. Mixed micelles together with a category of vesicles denoted primordial, small unilamellar vesicles of two distinct morphologies (sphere/ellipsoid and cylinder/arachoid), large unilamellar vesicles, multilamellar vesicles, and cholesterol monohydrate crystals were imaged. No evidence of aggregation/fusion of small unilamellar vesicles to form multilamellar vesicles was detected. Low numbers of multilamellar vesicles were present, some of which were sufficiently large to be identified as liquid crystals by polarizing light microscopy. Dimensions, surface areas, and volumes of spherical/ellipsoidal and cylindrical/arachoidal vesicles were quantified. Early stages in the separation of vesicles from micelles, referred to as primordial vesicles, were imaged 23-31 min after dilution. Observed structures such as enlarged micelles in primordial vesicle interiors, segments of bilayer, and faceted edges at primordial vesicle peripheries are probably early stages of small unilamellar vesicle assembly. A decrease in the mean surface area of spherical/ellipsoidal vesicles was correlated with the increased production of cholesterol crystals at 10-20 days after supersaturation by dilution, supporting the role of small unilamellar vesicles as key players in cholesterol nucleation and as cholesterol donors to crystals. This is the first visualization of an intermediate structure that has been temporally linked to the development of small unilamellar vesicles in the separation of vesicles from micelles in a model bile and suggests a time-resolved system for further investigation.     

Giant proteoliposomes prepared by freezing-thawing without use of detergent: reconstitution of biomembranes usually inaccessible to patch-clamp pipette microelectrode

When sarcoplasmic reticulum membrane vesicles or synaptosomes were mixed with sonicated phospholipid vesicles and subjected to freezing-thawing, giant vesicles of up to 50 microns in diameter were formed. When the biomembrane vesicles were labeled with a covalently binding fluorescent dye, the resultant giant vesicles were fluorescent, thereby suggesting that the freezing-thawing process induces fusion of phospholipid and biomembrane vesicles. When membranes of the giant proteoliposomes thus prepared were studied using the patch-clamp technique, potassium channels of the biomembranes were detectable. The present method of the giant proteoliposome preparation is simple and rapid, and provides a system suitable for the study of ion channels of various biomembranes usually inaccessible to a patch-pipette microelectrode.     

药敏药片对临床235株细菌实验观察报告

药敏药片经临床对金黄色葡萄球菌、大肠埃希氏菌、铜绿假单胞菌等２３５株考核．表明药片工艺研究先进,药片与培养基结合牢固,无断裂、崩解,不渗出颗粒,抑菌圈呈同心园扩散．边缘清楚。药物含量均匀,释放度好。药片抑菌差仅１～３ｍｍ;而纸片抑菌差为２～１２ｍｍ。药片变黑系数ＣＶ为２．７１～４．２１;而纸片ＣＶ为３．８２～１４．３６。表明纸片片间差大,药片精密度明显好于纸片。     

Membrane properties of branched polyprenyl phosphates, postulated as primitive membrane constituents

We have postulated earlier that the highly branched isoprenoid alkanes, which are distributed widely in many sediments, may have been derived from the corresponding branched polyprenyl phosphates, potentially present in biomembranes in primitive organisms. These polyprenyl-branched polyprenyl phosphates might be derived by a simple alkylation from non-substituted polyprenyl phosphates, which we postulate to be the precursors of all membrane terpenoids. We have now synthesized a series of 6-(poly)prenyl-substituted polyprenyl phosphates and studied the formation of vesicles from these phosphates, as a function of the substituted-chain length, the position of the double bond, and pH. Nine of the branched polyprenyl phosphates containing 20-30 C-atoms do form vesicles at a 'physiological' pH; the lipophilicity/hydrophilicity ratio is as expected an important factor. We have also studied the water permeability through membranes of these branched polyprenyl phosphate vesicles by our stopped-flow/light-scattering method. These highly branched polyprenyl phosphates can more effectively reduce the water permeability than non-substituted polyprenyl phosphates: the vesicles formed by the former are more stable against mechanical stress. This reinforces our hypothesis about the origin of the sedimentary polyprenyl-substituted polyprene hydrocarbons.     

The length of DNA determines the degree of regularity of crystals of the cro-repressor complex

The DNA-cro-repressor complex crystals have been obtained, five DNA fragments of the same nucleotide sequence and different length being used. The rotation function for crystals of complexes with hexamer (pGpT)3 . (pApC)3 and with octamer (pGpT)3 . (pApC)3 have been calculated. The order of cro-DNA complex crystals is shown to vary with DNA length, the crystal of the complex with octamer being the most perfect among all investigated complexes.     

Organization and dynamics of pyrene and pyrene lipids in intact lipid bilayers. Photo-induced charge transfer processes.

The dynamics of fluorescence quenching and the organization of a series of pyrene derivatives anchored in various depths in bilayers of phosphatidylcholine small unilamellar vesicles was studied and compared with their behavior in homogeneous solvent systems. The studies include characterization of the environmental polarity of the pyrene fluorophore based on its vibronic peaks, as well as the interaction with three collisional quenchers: the two membrane-soluble quenchers, diethylaniline and bromobenzene, and the water soluble quencher potassium iodide. The system of diethylaniline-pyrene derivatives in the membrane of phosphatidylcholine vesicles was characterized in detail. The diethylaniline partition coefficient between the lipid bilayers and the buffer is approximately 5,800. Up to a diethylaniline/phospholipid mole ratio of 1:3 the perturbation to membrane structure is minimal so that all photophysical studies were performed below this mole ratio. The quenching reaction, in all cases, was shown to take place in the lipid bilayer interior and the relative quenching efficiencies of the various probe molecules was used to provide information on the distribution of both fluorescent probes and quencher molecules in the lipid bilayer. The quenching efficiency by diethylaniline in the lipid bilayer was found to be essentially independent on the length of the methylene chain of the pyrene moiety. These findings suggest that the quenching process, being a diffusion controlled reaction, is determined by the mobility of the diethylaniline quencher (with an effective diffusion coefficient D approximately 10(-7) cm2 s-1) which appears to be homogeneously distributed throughout the lipid bilayer. The pulsed laser photolysis products of the charge-transfer quenching reaction were examined. No exciplex (excited-complex) formation was observed and the yield of the separated radical ions was shown to be tenfold smaller than in homogenous polar solutions. The decay of the radical ions is considerably faster than the corresponding process in homogenous solutions. Relatively high intersystem crossing yields are observed. The results are explained on the basis of the intrinsic properties of a lipid bilayer, primarily, its rigid spatial organization. It is suggested that such properties favor ion-pair formation over exciplex generation. They also enhance primary geminate recombination of initially formed (solvent-shared) ion pairs. Triplet states are generated via secondary geminate recombination of ion pairs in the membrane interior. The results bear on the general mechanism of electron transfer processes in biomembranes.     

Extracellular matrix vesicle distribution in primary mineralization two weeks after injury to rat tibial bone (ultrastructural tissue morphometry)

Primary mineralization on the 14th day of bone healing served as a model to study the distribution of extracellular matrix vesicles by means of transmission electron microscopy combined with computerized morphometry. Vesicles were traced on electron micrographs and classified according to diameter, distance from the calcified front, and type. The different types were determined as follows: electron-lucent vesicles ("empty"), vesicles with amorphous contents ("amorphic"), vesicles containing crystalline depositions ("crystal"), and vesicles with crystals and ruptured membranes ("rupture"). The majority of the vesicles measured between 0.02 and 0.07 micron and were located at a distance of less than 3 micron from the calcified front. They were distributed according to "empty", "amorphic", "crystal" and "rupture" type in concentrations of 10%, 31%, 51% and 8%, respectively. The diameters of the "rupture" vesicles were significantly larger than those of the "empty" and "amorphic" types. The sequence of their location, starting at the calcified front, ran as follows: "rupture", "crystal", "amorphic" and "empty", with the "rupture" type proximate to the front. According to the working hypothesis on calcification via extracellular matrix vesicles, it is thought that the "empty" vesicles are secreted by the cell and that subsequently amorphous Ca and Pi accumulate intravesicularly to form a hydroxyapatite crystal which, in turn, brings about rupture of the vesicle's membrane. The results of the present study support this theory and, additionally, show that the maturation process is accompanied by an increase of the vesicular diameter and by its approximation to the calcifying front.     

The preendosomal compartment comprises distinct coated and noncoated endocytic vesicle populations

The transfer of molecules from the cell surface to the early endosomes is mediated by preendosomal vesicles. These vesicles, which have pinched off completely from the plasma membrane but not yet fused with endosomes, form the earliest compartment along the endocytic route. Using a new assay to distinguish between free and cell surface connected vesicle profiles, we have characterized the preedosomal compartment ultrastructurally. Our basic experimental setup was labeling of the entire cell surface at 4 degrees C with Con A-gold, warming of the cells to 37 degrees C to allow endocytosis, followed by replacing incubation medium with fixative, all within either 30 or 60 s. Then the fixed cells were incubated with anti-Con A-HRP to distinguish truly free (gold labeled) endocytic vesicles from surface-connected structures. Finally, analysis of thin (20-30 nm) serial sections and quantification of vesicle diameters were carried out. Based on this approach it is shown that the preendosomal compartment comprises both clathrin-coated and non-coated endocytic vesicles with approximately the same frequency but with distinct diameter distributions, the average noncoated vesicle being smaller (95 nm) than the average coated one (110 nm). In parallel experiments, using an anti-transferrin receptor gold-conjugate as a specific marker for clathrin-dependent endocytosis it is also shown that uncoating of coated vesicles plays only a minor role for the total frequency of noncoated vesicles. Furthermore, after perturbation of clathrin-dependent endocytosis by potassium depletion where uptake of transferrin is blocked, noncoated endocytic vesicles with Con A-gold, but not coated vesicles, exist already after 30 and 60 s. Finally, it is shown that the existence of small, free vesicles in the short-time experiments cannot be ascribed to recycling from the early endosomes.     

小约化体积膜泡形状的研究

目的:研究ADE模型下小约化体积膜泡的形状。方法:应用数值计算方法中的打靶法,运用Mathematica 7.0软件进行编程。结果:在ADE模型下,计算得到了一系列小约化体积的膜泡的形状,解决了已往小约化体积区域内不存在稳定膜泡的问题。结论:研究表明,在ADE模型下通过适当的边界条件把黏附双层区域的接触势能考虑进去,数值计算结果与实验上的非常相似。     

Vesicle formation by amphiphilic 10-alkyl-3-methyl isoalloxazine in aqueous medium.

The synthetic 10-alkyl isoalloxazines have been found to form vesicles in aqueous and binary solvent systems and confirmed by UV-visible, fluorescence,transmission electron microscopy and quasi elastic light scattering experiments. The mean external diameters of vesicles have been calculated for isoalloxazine with different carbon atom chain at position 10 by transmission electron microscopy and quasi elastic laser light scattering. The gel to liquid phase transition of liposomes measured by differential scanning calorimetry shows reproducible endothermic peak which lies well in the range of typical aqueous vesicles.     

Formation of asymmetric vesicles via phospholipase D-mediated transphosphatidylation

Most biomembranes have an asymmetric structure with regard to phospholipid distribution between the inner and outer leaflets of the lipid bilayers. Control of the asymmetric distribution plays a pivotal role in several cellular functions such as intracellular membrane fusion and cell division. The mechanism by which membrane asymmetry and its alteration function in these transformation processes is not yet clear. To understand the significance of membrane asymmetry on trafficking and metabolism of intracellular vesicular components, a system that experimentally reproduces the asymmetric nature of biomembranes is essential. Here, we succeeded in obtaining asymmetric vesicles by means of transphosphatidylation reactions with phospholipase D (PLD), which acts exclusively on phosphatidylcholine (PC) present in the outer leaflet of vesicles. By treating PC vesicles with PLD in the presence of 1.7 M serine and 0.3 M ethanolamine, we obtained asymmetric vesicles that are topologically similar to intracellular vesicles containing phosphatidylserine and phosphatidylethanolamine in the cytosolic leaflet. PLD and other unwanted compounds could be removed by trypsin digestion followed by dialysis. Our established technique has a great advantage over conventional methods in that asymmetric vesicles can be provided at high yield and high efficiency, which is requisite for most physicochemical assays.     

Proton translocation by ATPase and bacteriorhodopsin.

Stable membrane proteins and lipids are convenient to study biomembranes. Two stable proton translocating proteins were purified and reconstituted into vesicles capable of proton translocation. One was a thermostable ATPase (TF0-F1) of thermophilic bacterium PS3 and the other was rhodopsin of Halobacterium halobium. TF0-F1 was composed of a proton pump moiety (TF1) and a proton channel moiety (TF0). TF1 was the first membrane ATPase which was crystallized and reconstituted from its five polypeptides. Like TF0 and TF1, the rhodopsin in purple membrane was highly stable against dissociating agents, acids and alkali. Phospholipids of these biomembranes were also stable and contained no unsaturated fatty acyl groups. The molecular species of the phospholipids of PS3 were determined by mass chromatography. Measurements were made of the difference in electrochemical potential of protons (deltamicronH+) across the membrane of the reconstituted vesicles. The deltamicronH+ attained was 312 mV in TF0-F1 vesciles and was 230 mV in the rhodopsin vesicles. To conclude that electron transport components are not necessary for ATP synthesis in energy yielding biomembranes, two experiments were performed: The ATP synthesis was observed i) on acid-base treatment of TF0-F1 vesicles, and ii) on illumination of the rhodopsin-TF0-F1 vesicles.     

How iron is transported into magnetosomes

Magnetotactic bacteria are microaerophilic organisms found in sediments or stratified water columns at the oxic-anoxic transition zone or the anoxic regions below. They use magnetite-filled membrane vesicles, magnetosomes, to passively align with, and actively swim along, the geomagnetic field lines in a magneto-aerotactic search for the ideal concentration of molecular oxygen. Such an efficient chemotaxis needs magnetosomes that contain nearly perfect magnetite crystals. These magnetosomes originate as invaginations of the inner membrane and the empty vesicles are aligned in a chain by an actin-like protein. Subsequently, the vesicles are filled with iron, which then is converted to magnetite crystals. Until now it was unclear how such a process might be accomplished. In this issue, Uebe et al., 2011 unveil a part of this complicated bio-mineralization process. In Magnetospirillum gryphiswaldense, MamM and MamB, two members of the cation diffusion facilitator (CDF) transport protein family, are required for magnetite formation. MamM increases the stability of MamB by forming a heterodimer. The MamBM heterodimer strongly influences the biomineralization process by controlling the size and the shape of the crystals, and even the nature of the formed iron mineral. Thus, these two CDF proteins not only transport iron, but they also control the magnetite biomineralization.     

Optical characterization of liposomes by right angle light scattering and turbidity measurement

Liposomes have frequently been used as models of biomembranes or vehicles for drug delivery. However, the systematic characterization of lipid vesicles by right angle light scattering and turbidity has not been carried out despite the usefulness of such studies for size estimation. In this study, liposomes of various sizes were prepared by sonication and extrusion. The mean cumulant radii of the vesicles were determined by dynamic light scattering. The lamellarities were estimated based on fluorescence quenching of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)dipalmitoyl-L-alpha-phosph ati dylethanolamine by sodium dithionite. Right angle light scattering intensity and optical density at 436 nm per unit lipid concentration were measured as a function of vesicle radius. With a vesicle radius < or =100 nm, the optical parameters could be well explained by the Rayleigh-Gans-Debye theory in which the liposomes were modeled as homogeneous spheres with mean refractive indices determined by the volume fractions of lipids in vesicles.