Enzymatic preparation of food-grade l-α-glycerylphosphorylcholine from soy phosphatidylcholine or fractionated soy lecithin

l -α-Glycerylphosphorylcholine (l -α-GPC) is a biosynthetic precursor for the neurotransmitter acetylcholine in humans, making it a useful as a cognitive enhancer for treating patients with stroke and dementia, including Alzheimer's disease. The aim of this study was to prepare l -α-GPC via Novozym 435 (an immobilized Candida antarctica lipase B)-catalyzed hydrolysis of soy phosphatidylcholine or a fractionated soy lecithin, from which triacylglycerols were completely removed, followed by food-grade solvent extraction of l -α-GPC from the reaction products. The reaction was performed in n-hexane–water biphasic media in a stirred-batch reactor. Phosphatidylcholine was completely hydrolyzed to l -α-GPC under optimal conditions: temperature, 55°C; water content, 100 wt% of the substrate weight; enzyme loading, 10 wt% of the substrate weight; and reaction time of 6 hr (for soy phosphatidylcholine) or 8 hr (for fractionated soy lecithin). Water-soluble fractions of the reaction products containing 98.6 area% l -α-GPC (from soy phosphatidylcholine) or 52.4 area% glycerophosphodiesters, including l -α-GPC (from fractionated soy lecithin), were obtained after phase separation of the media. The resulting products would be suitable for use as food-grade cognitive enhancers because of the use of enzymatic reaction and food-grade solvent extraction.     

Incorporation of newly formed lecithin into peripheral nerve myelin

Radioactive choline was used to study the metabolism and movement of choline-containing phospholipids in peripheral nerve myelin of adult mice. Incorporation at various times after intraperitoneal injection was measured in serial segments of sciatic nerve as well as in myelin isolated from those segments. At no time (1 h to 35 days) could a proximal-distal difference in the extent of labeling be demonstrated. This finding suggests that incorporation of precursor choline phospholipids into nerve membranes is a local event, with little contribution from the neuronal perikaryon via axoplasmic transport. Autoradiographic investigations were undertaken to elucidate the pattern of movement of radioactive choline-labeled phospholipids, predominantly lecithin, into the myelin sheaths of the sciatic nerve. A sequence of autoradiographs was prepared from animals sacrificed between 20 min and 35 days after a microinjection of precursor directly into the nerve. Analysis of these autoradiograms revealed that labeling is initially concentrated in the Schwann cell cytoplasm. Later, the label moves first into the outer regions of the myelin sheaths and is eventually distributed evenly throughout the inner and outer layers of the sheath. At no time is there a build-up of label in the axon. The rate of uptake of precursor and subsequent redistribution of lecithin into the myelin were also examined in frog sciatic nerve (18 degrees C). Both uptake and redistribution processes were considerably slower in the cold-blooded animal.     

Microparticles from condensed DNA formed in the process of polymerase chain reaction

DNA microparticle formation in the course of a polymerase chain reaction (PCR) is reported. PCR with gene-specific and partially complementary primers and yeast genomic DNA as a template was shown to yield spherical DNA-composed microparticles as well as their aggregates and conglomerates, along with routine linear DNA. Microparticles were formed at late PCR stages and could be easily identified by the reaction with fluorescently labeled oligonucleotide primers or by staining of the PCR mixture with fluorescent dyes (acridine orange, propidium iodide or DAPI). According to the data of epifluorescent and electron microscopy, the microparticle size varied from 500 nm to 3–4 μm and the particles were multimeric star-shaped spheres or aggregates formed by several fused microspheres. Some properties of the microspheres were studied. It was found that the Mg⁺² cations comprising the PCR buffer played a key role in the formation of microparticles and the stabilization of their structures.     

Substrate specificity of plasma lysolecithin acyltransferase and the molecular species of lecithin formed by the reaction

Human plasma lecithin-cholesterol acyltransferase also converts lysolecithin to lecithin in the presence of low density lipoproteins. To understand the physiological importance of this lysolecithin acyltransferase reaction, we investigated the molecular species of lysolecithin available for acylation in normal plasma and the lecithins which are formed by the acylation of each of these lysolecithins. Palmitate- and stearate-containing lysolecithins were formed by the lecithin-cholesterol acyltransferase reaction, whereas oleate- and linoleate-containing lysolecithins were formed by the action of post-heparin lipase(s). All the natural lysolecithins were esterified at comparable rates by the isolated enzyme. Lyso platelet-activating factor was esterified about 70% as efficiently as the lysolecithins, while lysophosphatidylethanolamine was esterified at about 30% the rate observed with lysolecithin. The 2-acyl isomers of lysolecithin were acylated to the same extent as the 1-acyl isomers, although considerable isomerization of the former took place during the incubation. There were no net changes in the concentrations of lecithin and lysolecithin after 6 h of incubation with the enzyme, although over 10% of the labeled lysolecithin was converted to lecithin, indicating that the endogenous lecithin serves as the acyl donor in the reaction. When the molecular species of lecithin formed were analyzed by high performance liquid chromatography, the same pattern of fatty acid incorporation was observed with all the lysolecithins used. The bulk of the radioactivity was incorporated into molecular species formed by the acylation with linoleic, oleic, and palmitic acids, in decreasing order. However, in each case, the lecithins formed by acylation with palmitic acid had the highest specific radioactivity, followed by those acylated with linoleic and oleic acids. From these results it is postulated that the enzyme alters the molecular species composition of lecithin in plasma without increasing the net amount of total lecithins.     

Determination of the IgE-binding activity of soy lecithin and refined and non-refined soybean oils

In the present study refined and non-refined soybean oils as well as soy lecithins were investigated for residual allergenicity and compared with extracts from native soybeans. By means of immunoblotting and EAST inhibition experiments no IgE-binding activity was detectable in refined soybean oils, which is probably due to thermal treatment during the refining. The investigated non-refined oils and soy lecithins showed a residual IgE-binding activity. In addition in the lecithin extracts a new IgE-binding structure with a molecular mass of approximately 16 kDa was detectable.     

Optimization of enzymatic hydrolysis of triglycerides in soy deodorized distillate with supercritical carbon dioxide

Enzymatic hydrolysis of triglycerides of soy deodorized distillate (DOD), using immobilized Candida rugosa lipase under supercritical carbon dioxide (SC-CO₂) medium, was carried out. Optimization of the reaction parameters using response surface methodology based on Box-Behnken model at three levels of pressure (120–180 bar), temperature (40–60 °C) and moisture content (40–80% of triglyceride content) for maximum hydrolysis of triglycerides was arrived by multilinear regression of the experimental results. The optimum conditions for maximum degree of triglyceride hydrolysis (94%) were found to be: pressure of 180 bar, temperature of 43 °C and moisture content of 40% to the triglyceride content. Maximum degree of hydrolysis was achieved with short incubation time of 1.5 h under SC-CO₂. Whereas conventional method of hydrolysis in hexane under similar reaction conditions of temperature, moisture and enzyme concentration, needs 5 h to achieve 88% of triglyceride hydrolysis.     

The adsorption of adrenocorticotropin-(1-24)-tetracosapeptide to lecithin bilayer membranes formed from liposomes

The interaction of the peptide hormone adrenocorticotropin (ACTH_1-24) with solvent-free planar lipid bilayers has been studied by use of the capacitance minimization method. The membranes were formed from artificial vesicles according to the method described by Schindler. In contrast to analogous studies with hexane-containing membranes, experiments with these vesicle-derived bilayers were completely reproducible and gave no indication that ACTH_1-24 spans such hexane-free bilayers.     

Modulation of phytohemagglutinin-mediated lymphocyte stimulation by egg lecithin

Egg lecithin at 200 μg/ml or greater was found to abrogate blastogenic responses of human leukocyte cultures stimulated with phytohemagglutinin (PHA), concanavalin A (ConA), purified protein derivatives of tuberculin (PPD), candidin or streptokinase and streptodornase (SKSD) while leukocyte aggregation mediated by PHA, however, occurred irrespective of the presence of the lipid. Inhibition of PHA-mediated responses occurred when the lecithin was added simultaneously with the mitogen but the extent of inhibition decreased with delay in the addition of the lipid. When added 48 h after their exposure to PHA, the presence of lecithin did not change the pattern of [³H]TdR incorporation. Prior sonic treatment of an aqueous suspension of lecithin was found to render the lipid more effective in arresting lymphocyte responses than the unsonicated control. In entity, these results indicated that the lipid did not affect the viability of the leukocyte cultures nor did it seem that the lipid may interfere with the binding of the mitogen per se. It was concluded therefore, that inhibition by lecithin may most likely occur early at a time following binding of the mitogen but before the cells are committed to cellular division and it seems that the plasma membrane might be a likely site of action of the lipid.     

Perturbation of lecithin bilayer structure by globoside.

The ultrastructure of aggregates formed by mixtures of pig erythrocyte lecithin, cholesterol and globoside in aqueous systems was studied by electron microscopy and X-ray diffraction. Globoside and lecithin in up to equimolar amounts formed a lamellar mesophase, although the structure of the lamellae was perturbed. Mixtures containing excess globoside formed complex tubular or reticular aggregates. Cholesterol appeared to promote mixing of lecithin and globoside. The flexibility gradient of the hydrocarbon (hc) region of the lipid bilayers was studied using electron spin resonance (esr) spectroscopy of various nitroxide-labelled stearic acid probes. Globoside in equimolar amounts greatly perturbed the order parameters of lecithin bilayers, reducing the fluidity of the hc region and flattening the flexibility gradient near the polar (p) surface. The effect of globoside on lecithin-cholesterol bilayers was not so pronounced, since the latter was already more ordered than lecithin bilayers. A phase transition of pure globoside at 55 degrees C, involving 'melting' of the hc chains was also detected using X-ray and esr spectroscopic techniques. The interbilayer spacing, dw, of equimolar lecithin-globoside lamellar phase increased by 42% from that of lecithin bilayers, indicating that the glycolipid p group may increase the net repulsive force between bilayers, as was previously predicted theoretically.     

Small Staphylococcus aureus plasmids are transduced as linear multimers that are formed and resolved by replicative processes

The molecular processes involved in the transduction of small staphylococcal plasmids by a generalized transducing phage, phi 11, have been analysed. The plasmids are transduced in the form of linear concatemers containing only plasmid DNA; plasmid-initiated replication is required for their generation but additive interplasmid recombination is not. Concatemers are probably generated by the interaction of one or more phage functions with replicating plasmid DNA. Insertion of any restriction fragment of the phage into the plasmid causes an approximately 10(5)-fold increase in transduction frequency, regardless of the size or genetic content of the fragment. The resulting transducing particles (Hft particles) contain mostly pure linear concatemers composed of tandem repeats of the plasmid::phage chimera, and their production requires active plasmid-initiated replication. The high frequency of transduction is a consequence of homologous recombination between the linear chimeric and phage concatemers, which has the effect of introducing an efficient pac site into the former. Following introduction into lysogenic recipient bacteria, the transducing DNA is first converted to the supercoiled form, then processed to monomers by a mechanism that requires the active participation of the plasmid replication system.