Neutral glycolipids of human and bovine milk

The neutral glycolipids of milk, a small fraction of the total lipids, are of potential biological importance. The simultaneous quantitation of the simple (less than five sugars) glycosphingolipids of human milk samples was achieved by high-pressure liquid chromatography. The samples, representing various stages of lactation, parity of the nursing child, and age of the mother, contained similar glycolipid patterns, but with varying individual glycolipid concentrations. The cerebrosides are major glycosphingolipids of human milk: the non-hydroxylated fatty acid (NFA)-containing species are present at 1.8 μM, and the hydroxylated and/or short-chain fatty acid-containing species (HFA) are present at 1.7 μM; NFA lactosylceramide is present at 931 nM. The cerebrosides appear to be primarily galactosylceramides (galactocerebrosides); glucosylceramides (glucocerebrosides) are a minor component. Globotriaosylceramide (Gb₃) is found at 50 nM and 73 nM for the NFA and HFA species, respectively, while globoside (Gb₄) is found at 45 nM and 46 nM for the NFA and HFA species. Bovine milk glycosphingolipids differ from those of human milk, with bovine milk containing mainly NFA glucosylceramide (8 μM) and NFA lactosylceramide (17 μM); bovine milk contains little Gb₃ or Gb₄. Based on a paper presented at the Symposium on Milk Lipids held at the AOCS Annual Meeting, Baltimore, MD, April 1990.     

Systematic analysis of glycosphingolipids in the human gastrointestinal tract: Enrichment of sulfatides with hydroxylated longer-chain fatty acids in the gastric and duodenal mucosa

The composition of the glycosphingolipids of the human gastrointestinal tract was studied. The major neutral glycosphingolipids were ceramide monohexosides (e.g., GalCer, GlcCer), LacCer, Gb₃Cer, Gb₄Cer and more polar ones with more than four sugars, whereas neither Gg₃Cer nor Gg₄Cer were present. The acidic glycosphingolipids consisted of sulfatides and gangliosides such as G_M3, G_M1, G_D3 and G_D1a. Also a large amount of sulfatides was found in the gastric mucosa and duodenum. The concentrations of sulfatides in the fundic mucosa, antral mucosa and duodenum amounted to 416.0, 933.8 and 682.9 nmol/g of dry weight, respectively, exceeding those in the gastric mucosa and kidney of other mammals. The major molecular species of the sulfatides were identified as I³SO₃-GalCer with hydroxylated longer-chain fatty acids based on the analyses by gas-liquid chromatography and negative ion fast-atom bombardment mass spectrometry. In contrast, gangliosides in these regions showed a tendency to be lower than sulfatides, and the molar ratios of sulfatides to gangliosides were about 2.0, whereas those in other parts were less than 0.5. A high content of sulfatides in the gastric and duodenal mucosa, where mucosa is easily insulted by acid, pepsin and bile salts, may be closely related to their roles in mucosal protection. The nomenclature used for gangliosides and other glycosphingolipids follows the system of Svennerholm (Ref. 1) and the recommendation of the IUPAC-IUB Commission (Ref. 2), respectively.     

Synthesis and aggregation behavior of an amphiphilic cobalt (III) complex that coordinates higher diacylglyceryldiamine ligands

An amphiphilic cobalt complex that coordinates a higher diacylglycerylethylenediamino group was prepared. They extensively aggregated in aqueous solution upon ultrasonication. Electron microscopy indicated formation of stable bilayer structures, which further formed vesicles and fibrous aggregates. We observed an intermediate state that led to a network morphology in the fusion process of small unilamellar vesicles. Differential scanning calorimetry experiments establiched the presence of the phase transition from gel to liquid crystal for all probes in which a C₁₄ to C₁₈ acyl group was bound. The phase-transition temperature was elevated with increasing carbon numbers of the acyl group.     

Action of cobra venom phospholipase A2 on large unilamellar vesicles: Comparison with small unilamellar vesicles and multibilayers

Phospholipase A₂ (Naja naja naja) catalyzes the hydrolysis of dipalmitoyl phosphatidylcholine in small unilamellar vesicles (SUVs) with a faster initial rate than in large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). For the SUVs, the hydrolysis was initially faster for gel phase than liquid crystalline phase phospholipid. For both LUVs and MLVs, hydrolysis was low except in a small temperature range around the thermotropic phase transition of the phospholipid. In this temperature range, the reaction time course of phospholipase action on dipalmitoyl phosphatidylcholine in LUVs and MLVs included a lag period. With SUVs, a lag period also was observed above the phase transition temperature, but it was not observed below it.     

Observations of Membrane Domain Reorganization in Mechanically Compressed Artificial Cells

Giant unilamellar vesicles (GUVs) are considered to be the gold standard for assembling artificial cells from the bottom up. In this study, we investigated the behavior of such biomimetic vesicles as they were subjected to mechanical compression. A microfluidic device is presented that comprises a trap to capture GUVs and a microstamp that is deflected downwards to mechanically compress the trapped vesicle. After characterization of the device, we show that single-phase GUVs can be controllably compressed to a high degree of deformation (D=0.40) depending on the pressure applied to the microstamp. A permeation assay was implemented to show that vesicle bursting is prevented by water efflux. Next, we mechanically compressed GUVs with co-existing liquid-ordered and liquid-disordered membrane phases. Upon compression, we observed that the normally stable lipid domains reorganized themselves across the surface and fused into larger domains. This phenomenon, observed here in a model membrane system, not only gives us insights into how the multicomponent membranes of artificial cells behave, but might also have interesting consequences for the role of lipid rafts in biological cells that are subjected to compressive forces in a natural environment.     

Optimization of the Inverted Emulsion Method for High-Yield Production of Biomimetic Giant Unilamellar Vesicles

In the field of bottom-up synthetic biology, lipid vesicles provide an important role in the construction of artificial cells. Giant unilamellar vesicles (GUVs), due to their membrane's similarity to natural biomembranes, have been widely used as cellular mimics. So far, several methods exist for the production of GUVs with the possibility to encapsulate biological macromolecules. The inverted emulsion-based method is one such technique, which has great potential for rapid production of GUVs with high encapsulation efficiencies for large biomolecules. However, the lack of understanding of various parameters that affect production yields has resulted in sparse adaptation within the membrane and bottom-up synthetic biology research communities. Here, we optimize various parameters of the inverted emulsion-based method to maximize the production of GUVs. We demonstrate that the density difference between the emulsion droplets, oil phase, and the outer aqueous phase plays a crucial role in vesicle formation. We also investigated the impact that centrifugation speed/time, lipid concentration, pH, temperature, and emulsion droplet volume has on vesicle yield and size. Compared to conventional electroformation, our preparation method was not found to significantly alter the membrane mechanical properties. Finally, we optimize the parameters to minimize the time from workbench to microscope and in this way open up the possibility of time-sensitive experiments. In conclusion, our findings will promote the usage of the inverted emulsion method for basic membrane biophysics studies as well as the development of GUVs for use as future artificial cells.     

Dispersed phase characteristics in three phase (liquid-liquid-solid) fluidized beds

Two ideal droplet length (l,) distributions have been derived for two different droplet shapes. The dispersed phase holdup (?_d) increases with increasing dispersed phase velocity (U_d), but decreases with increasing continuous phase velocity, (U_c) in three-phase fluidized beds. In the droplet-coalescing flow regime, l_v and the droplet rising velocity (v_d) increase, but the spherical droplet fraction (k) decreases with increasing U_d and u_c. In the droplet-disintegrating flow regime, the effects of u_d and U_c on l_v and k are insignificant, but v_d increases with increasing U_c. Maximum values of l_v, occur in the bed containing 1.7 mm diameter particles and l_v has an uniform length of around 2.0 mm in beds with particle size larger than 3.0 mm.     

Trimeric Supra-Amphiphile with Diverse Lamellar Self-Assemblies

A new kind of trimeric supra-amphiphile [M-4-M-4-M][DBS]₃ that exhibits diverse self-assembly behaviours was fabricated through ion exchange between linear tricationic imidazolium chloride ([M-4-M-4-M]Cl₃) and sodium dodecyl benzene sulfonate (SDBS). Micelles are spontaneously transformed into unilamellar vesicles at a quite low concentration (1.5 mM), and then evolved to multilamellar vesicles and planar bilayers successively with a further increase in the concentration of [M-4-M-4-M][DBS]₃ in the absence of any additives. The presence of π-π stacking interactions has been proved to promote the lamellar arrangement of the supra-amphiphile. With increasing temperatures, [M-4-M-4-M][DBS]₃ exhibits interesting “clouding” and phase separation in the L_a phase, behaving like common nonionic surfactants. Chain-like aggregates are formed in the bottom phase, which are probably induced by the hierarchical assembly of vesicles. This work provides useful insights into the role of weak interactions in the fabrication and self-assembly of oligomeric supra-amphiphiles, and paves a way to enrich the structures and functions of self-assemblies.     

A Trifunctional Linker for Palmitoylation and Peptide and Protein Localization in Biological Membranes

Attachment of lipophilic groups is an important post-translational modification of proteins, which involves the coupling of one or more anchors such as fatty acids, isoprenoids, phospholipids, or glycosylphosphatidyl inositols. To study its impact on the membrane partitioning of hydrophobic peptides or proteins, we designed a tyrosine-based trifunctional linker. The linker allows the facile incorporation of two different functionalities at a cysteine residue in a single step. We determined the effect of the lipid modification on the membrane partitioning of the synthetic α-helical model peptide WALP with or without here and in all cases below; palmitoyl groups in giant unilamellar vesicles that contain a liquid-ordered (L_o) and liquid-disordered (L_d) phase. Introduction of two palmitoyl groups did not alter the localization of the membrane peptides, nor did the membrane thickness or lipid composition. In all cases, the peptide was retained in the L_d phase. These data demonstrate that the L_o domain in model membranes is highly unfavorable for a single membrane-spanning peptide.     

Reconstitution and anchoring of cytoskeleton inside giant unilamellar vesicles

Among the requirements for all life forms is the ability to self-replicate. In eukaryotic cellular systems, this division is achieved through cytokinesis, and is facilitated by the (re)arrangement and interaction of cytoskeletal proteins with lipids and other proteins localized to the plasma membrane. A fascinating challenge of modern synthetic biology is the bottom-up reconstitution of such processes for the generation of an artificial cell. One crucial step towards this goal is the functional reconstitution of the protein-anchoring machinery to facilitate cytokinesis into lipid vesicles. True to the ideal of a minimal cell-like system, we here describe the formation of an actin-based cytoskeleton within giant unilamellar vesicles (GUVs) made from porcine brain lipid extracts. We demonstrate that the actin filaments are localised and anchored to the interior walls of the GUVs through the spectrin/ankyrin proteins, and produce tightly packed actin bundles. These studies allow for the examination of cytoskeletal rearrangements within a cell-like model membrane system and represent important first steps in reconstituting the minimal machinery required for the division of an artificial cell. In addition, the study of such minimal systems can shed light on protein functions that are commonly unobservable or hidden within the overwhelming complexity of cells.     

Translational Diffusion and Interaction of a Photoreceptor and Its Cognate Transducer Observed in Giant Unilamellar Vesicles by Using Dual‐Focus FCS

In order to monitor membrane–protein binding in lipid bilayers at physiological protein concentrations, we employed the recently developed dual‐focus fluorescence correlation spectroscopy (2fFCS) technique. In a case study on a photoreceptor consisting of seven transmembrane helices and its cognate transducer (two transmembrane helices), the lateral diffusion for these integral membrane proteins was analyzed in giant unilamellar vesicles (GUVs). The two‐dimensional diffusion coefficients of both separately diffusing proteins differ significantly, with D=2.2×10^?8 cm² s^?1 for the photoreceptor and with D=4.1×10^?8 cm² s^?1 for the transducer. In GUVs with both membrane proteins present together, we observed significantly smaller diffusion coefficients for labelled transducer molecules; this indicates the presence of larger diffusing units and therefore intermolecular protein binding. Based on the phenomenological dependence of diffusion coefficients on the molecule's cylindrical radius, we are able to estimate the degree of membrane protein binding on a quantitative level.     

Mass spectra of prostaglandins: II. Trimethylsilyl and alkyloxime-trimethylsilyl derivatives of prostaglandins B1 and B2

The mass spectra of the trimethylsilyl ester trimethylsilyl ether derivatives of prostaglandins B₁ and B₂, and of theirO-ethyl andO-methyl oximes are reported and discussed. The high resolution spectra of these compounds are also considered. These spectra are compared with those of the correspondingd ₉-trimethylsilyl derivatives and of the selectively labeled trimethylsilyl esterd ₉-trimethylsilyl ethers. The base peaks of the spectra of the trimethylsilyl derivatives are formed by sequential loss of C₅H₁₁ (by cleavage of C-15/6) and of CO (from the ring). The spectra of the oxime-trimethylsilyl derivatives are dominated by ions resulting from loss of the alkoxy group of the oxime moiety. For Part I, see Reference 1.     

Effect of hydration on morphology of thin phosphonate block copolymer electrolyte membranes studied by electron tomography

The morphological changes of phosphonate polypeptoid electrolyte membranes, poly-N-(2-ethyl)hexylglycine-block-poly-N-phosphonomethylglycine (pNeh_m-b-pNpm_n), in hydrated and dry states were characterized by cryogenic transmission electron microscopy (cryo-TEM) and cryogenic electron tomography (cryo-ET). The analysis of 3D tomograms revealed that the pNeh₉-b-pNpm₉ thin films absorbed a large amount of water, resulting in the formation of membranes that were nearly flat and giant multicompartment vesicles dispersed in the water phase. A simple lamellar phase appeared when the films were dried. In contrast, pNeh₁₈-b-pNpm₁₈ thin films absorbed little water and formed small highly curved unilamellar and multilamellar vesicles. Water was located mainly outside the closely-packed vesicles. When water was removed by drying, the walls of adjacent vesicles collapsed to form honeycomb-like capsules. The changes in domain size reflected changes in chain conformations. The pNpm₉ blocks were saturated by water and fully extended, while pNpm₁₈ blocks were neither saturated by water nor fully extended. In addition, the thicknesses of hydrophobic blocks in the hydrated films of both pNeh₉-b-pNpm₉ and pNeh₁₈-b-pNpm₁₈ were smaller than those in the dry films, reflecting an increase of the average distance between the neighboring junctions of polypeptoid molecules.     

Destabilizing effects of fructose-1,6-bisphosphate on membrane bilayers

Fructose-1,6-bisphosphate (FBP) is a high-energy glycolytic intermediate that decreases the effects of ischemia; it has been used successfully in organ perfusion and preservation. How the cells utilize external FBP to increase energy production and the mechanism by which the molecule crosses the membrane bilayer are unclear. This study examined the effects of FBP on membrane bilayer permeability, membrane fluidity, phospholipid packing, and membrane potential to determine how FBP crosses the membrane bilayer. Large unilamellar vesicles composed of egg phosphatidylcholine (Egg PC) were made and incubated with 50 mM FBP spiked with ¹⁴C-FBP at 30°C. Uptake of FBP was significant (P<0.05) and dependent on the lipid concentration, suggesting that FBP affects membrane, bilayer permeability. With added calcium (10 mM), FBP uptake by lipid vesicles decreased significantly (P<0.05). Addition of either 5 or 50 mM FBP led to a significant increase (P<0.05) in Egg PC carboxyfluorescein leakage. We hypothesized that the membrane-permeabilizing effects of FBP may be due to a destabilization of the membrane bilayer. Small unilamellar vesicles composed of dipalmitoyl pC (DPPC) were made containing either diphenyl-1,3,5-hexatriene (DPH) or trimethylammmonia-DPH (TMA-DPH) and the effects of FBP on the fluorescence anisotropy (FA) of the fluorescent labels examined. FBP caused a significant decrease in the FA of DPH in the liquid crystalline state of DPPC (P<0.05), had no effect on FA of TMA-DPH in the liquid crystalline state of DPPC, but increased the FA of TMA-DPH in the gel state of DPPC. From phase transition measurements with DPPC/DPH or TMA-DPH, we calculated the slope of the phase transition as an indicator of the cooperativity of the DPPC molecules. FBP significantly decreased the slope, suggesting a decrease in fatty acyl chain interaction (P<0.05). The addition of 50 mM FBP caused a significant decrease (P<0.05) in the liquid crystalline/gel state fluorescence ratio of merocyanine 540, indicating increased head-group packing. To determine what effects these changes would have on cellular membranes, we labeled human endothelial cells with the membrane potential probe 3,3′-dipropylthiacarbocyanine iodide (DiSC₃) and then added FBP. FBP caused a significant, dose-dependent decrease in DiSC₃ fluorescence, indicating membrane depolarization. We suggest that FBP destabilizes membrane bilayers by decreasing fatty acyl chain interaction, leading to significant increases in membrane permeability that allow FBP to diffuse into the cell where it can be used as a glycolytic intermediate.     

Macroscopic estimation method of the mixedness of Kenics type static mixer

We investigated the mixing process of the Kenics type static mixer, in which the mixing is enhanced by both advective mixing and mixing due to molecular diffusion in order to propose an estimation method of the mixedness. The results show that the element divides, folds and stretches the mixing fluids and forms a lamellar structure with striation width l _a . The value of l _a decreases with an increase in the number of elements by l _a -(2 ⁿ⁻¹)⁻¹, which represents the characteristics length of advective mixing. The characteristic length of mixing due to molecular diffusion l _d can be estimated by solving the one-dimensional unsteady species conservation equation analytically. The macroscopic estimation and prediction method of mixedness is proposed as function of l _a and l _d . When l _a >l _d , the advective mixing is dominant, while, when l _a <l _d , the mixing due to molecular diffusion plays an important role.     

Instrumented impact testing of glass reinforced polypropylene

The impact response of a glass fiber reinforced polypropylene was studied in a 3-point drop-weight impact test between ?15 and 85°C and at a constant impact velocity of 2.2 m/s (5 mph). The response is a combination of tension and shear and can be expressed in terms of an apparent modulus, E_A: 1 . Where E₁₁ is the tensile modulus, G₁₂: shear modulus, d: specimen thickness, and l: specimen length. For a 40 weight-percent glass reinforced polypropylene, E₁₁ was found to have a room temperature value of 5.8 GPa, and shear modulus of 0.43 GPa. Both decreased with temperature increase, with the shear modulus showing greater sensitivity to a temperature change. The fracture initiation and propagation energies were relatively independent of temperature. The fracture initiation energy per unit deformed volume was of the order of 1 MJ/m³. The total fracture energy was found to be sensitive to l/d: about 7 MJ/m³ at l/d of 5.3 and about 1.7 MJ/ m³ at l/d of 16.     

Tryptophanase-Catalyzed l-Tryptophan Synthesis from d-Serine in the Presence of Diammonium Hydrogen Phosphate

Tryptophanase, an enzyme with extreme absolute stereospecificity for optically active stereoisomers, catalyzes the synthesis of l-tryptophan from l-serine and indole through a β-substitution mechanism of the ping-pong type, and has no activity on d-serine. We previously reported that tryptophanase changed its stereospecificity to degrade d-tryptophan in highly concentrated diammonium hydrogen phosphate, (NH₄)₂HPO₄ solution. The present study provided the same stereospecific change seen in the d-tryptophan degradation reaction also occurs in tryptophan synthesis from d-serine. Tryptophanase became active to d-serine to synthesize l-tryptophan in the presence of diammonium hydrogen phosphate. This reaction has never been reported before. d-serine seems to undergo β-replacement via an enzyme-bonded α-aminoacylate intermediate to yield l-tryptophan.     

Site‐Specific Antibody Labeling by Covalent Photoconjugation of Z Domains Functionalized for Alkyne–Azide Cycloaddition Reactions

Antibodies are extensively used in research, diagnostics, and therapy, and for many applications the antibodies need to be labeled. Labeling is typically performed by using amine‐reactive probes that target surface‐exposed lysine residues, resulting in heterogeneously labeled antibodies. An alternative labeling strategy is based on the immunoglobulin G (IgG)‐binding protein domain Z, which binds to the Fc region of IgG. Introducing the photoactivable amino acid benzoylphenylalanine (BPA) into the Z domain makes it possible for a covalent bond to be be formed between the Z domain and the antibody on UV irradiation, to produce a site‐specifically labeled product. Z₃₂BPA was synthesized by solid‐phase peptide synthesis and further functionalized to give alkyne‐Z₃₂BPA and azide‐Z₃₂BPA for Cu^I‐catalyzed cycloaddition, as well as DBCO‐Z₃₂BPA for Cu‐free strain‐promoted cycloaddition. The Z₃₂BPA variants were conjugated to the human IgG1 antibody trastuzumab and site‐specifically labeled with biotin or fluorescein. The fluorescently labeled trastuzumab showed specific staining of the membranes of HER2‐expressing cells in immunofluorescence microscopy.     

Plate wettability and droplet diameter in a pulsed perforated-plate extraction column

The liquid-liquid-plate contact angles of several plate materials were measured using a “preferential wetting method” proposed by the authors. The adhesional work (I_SL), calculated on the basis of contact angles was utilized to evaluate plate wettability. The experimental results indicate that different plate materials affect differently plate wettability. The plate wettability of stainless steel in an aqueous phase was observed to be different from that in an organic phase. The droplet diameter in the mixer-settler region of operation of a pulsed perforated-plate extraction column using several different plate materials, was measured. An empirical correlation of mean droplet diameter: d_p = 0.222(a_f/l_SL^1/3)^?0.13 is proposed except for alumina plates with dispersed aqueous phase operation.