Studies on the mechanism of membrane fusion. Role of head-group composition in calcium- and magnesium-induced fusion of mixed phospholipid vesicles

We have investigated the contribution of various phospholipids to membrane fusion induced by divalent cations. Fusion was followed by means of a new fluorescence assay monitoring the mixing of internal aqueous contents of large (0.1 μm diameter) unilamellar liposomes. The rate and extent of fusion induced by Ca²⁺ in mixed phosphatidylserine/phosphatidylcholine vesicles were lower compared to those in pure phosphatidylserine vesicles. The presence of 50% phosphatidylcholine completely inhibited fusion, although the vesicles aggregated upon Ca²⁺ addition. When phosphatidylserine was mixed with phosphatidylethanolamine, however, rapid fusion could be induced by Ca²⁺ even in mixtures that contained only 25% phosphatidylserine. Phosphatidylethanolamine also facilitated fusion by Mg²⁺ which could not fuse pure phosphatidylserine vesicles. In phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine mixtures, in which the phosphatidylcholine content was kept at 25%, phosphatidylethanolamine could not substitute for phosphatidylserine, and the fusogenic capacity of Mg²⁺ was abolished by the presence of merely 10% phosphatidylcholine. The initial rate of release of vesicle contents was slower than the rate of fusion in all the mixtures used. The presence of phosphate effected a considerable decrease in the threshold concentration of Ca²⁺ and also enhanced     

Isolation of a phosphatidylserine transfer protein from yeast cytosol.

A phospholipid transfer protein with a broad substrate specificity was isolated from yeast cytosol. The rate of transfer catalyzed by this protein in vitro is highest for phosphatidylserine; phosphatidylethanolamine, cardiolipin, phosphatidic acid and ergosterol are transported at a lower rate. In contrast to the yeast phosphatidylinositol transfer protein (Daum, G. and Paltauf, F. (1984) Biochim. Biophys. Acta 794, 385-391) the phosphatidylserine transfer protein does not catalyze the translocation of phosphatidylinositol or phosphatidylcholine. Using chromatographic methods the phosphatidylserine transfer protein was enriched approximately 3000-fold over yeast cytosol. The protein is inactivated by heat, detergents and proteinases. Divalent cations strongly inhibit the transfer of phosphatidylserine in vitro, and EDTA at low concentrations has a stimulatory effect.     

Phospholipid hydration studied by deuteron magnetic resonace spectroscopy

Deuteron magnetic resonance spectra were obtained from ²H₂O in mixtures with egg lecithin, egg phosphatidylethanolamine, and ox brain sodium phosphatidylserine. The acid form of phosphatidylserine does not hydrate. Details of the different hydration “shells” were obtained by studying the spectral splittings as a function of ²H₂O concentration. Several different types of water are present, including bulk water (exchanging only slowly with water associated with the lipid), “trapped” water (not present with phosphatidylethanolamine), and up to three types of bound water. The spectral splittings characteristic of each water environment yielded information about the water binding energies and degrees of anisotropy of motion of the phospholipid polar groups; lecithin polar groups have least motional restriction and sodium phosphatidylserine most, while phosphatidylethanolamine binds water most tightly.Spectra of some lecithin and phosphatidylserine dispersions varied with time, due to a slow reorganization of randomly oriented multilamellar regions into longer, more ordered systems, with a length of about 1 μm. At ?20°C the timescales of the change were of the order of a week and a month for lecithin and phosphatidylserine respectively.Complex changes in the spectra were observed as the temperature was raised; these are interpreted in terms of changes in the motions of the phospholipid molecules.     

Evaluation of SLS: APG Mixed Surfactant Systems as Carrier for Solid Dispersion

The present investigation aims at studying the effect of mixed surfactant system of sodium lauryl sulphate (SLS) and alkyl polyglucosides (C₁₀APG, C₁₂APG and C_12/14APG) on dissolution rate enhancement of poorly water soluble drug. Aceclofenac—a non-steroidal anti-inflammatory agent was used as a model drug as it has limited water solubility. The influence of the surfactant concentration in various blends on dissolution rate of Solid Dispersion (SD), prepared using solution method with ethanol as the solvent was studied and the advantage of mixed surfactant systems over the individual surfactants was illustrated by differences in the in-vitro dissolution profiles of SD. Physico chemical evaluation (critical micellar concentration, zeta potential and β-parameter calculations) was carried out to study the mixed surfactant systems. Solid mixtures were characterized by Infrared spectroscopy (FT-IR); X-ray diffraction studies (XRD) and scanning electron microscopy (SEM). It was seen that the dissolution rate of aceclofenac from SD increased with the increase in the APG proportion relative to SLS with the optimum ratio of 0.2 SLS:0.8 APG showing the best effect in all cases. Results obtained from physico-chemical evaluation (the decrease in the value of critical micelle concentration and higher negative value of β-parameters) suggested the existence of synergism between surfactants blends. The observed results in the dissolution rate enhancement could be attributed to the drug—surfactant interactions as evident from FT-IR, SEM and XRD results.     

The interaction of hemoglobin with phosphatidylserine vesicles

The interaction of hemoglobin with phosphatidylserine vesicles at low ionic strength and pH conditions was studied. The fluorescence intensity of a lipid embedded probe was quenched by bound Hb but could not be reversed by an elevation of ionic strength and pH. The irreversibility of the fluorescence quenching is a time-dependent process associated with changes in the heme Soret and visible spectra. The rate of these changes was much faster for methemoglobin than for either cyanomethemoglobin or oxyhemoglobin. Elevation of ionic strength released out of the bound hemoglobin into the water phase most of the globin but only a small fraction of the heme. The data are interpreted as demonstrating the ability of phosphatidylserine vesicles to compete with globin for the heme group. When Hb binds to the liposome, heme is being transferred into the lipid phase and the rate-limiting step is the dissociation of the heme-globin complex. The fact that binding of heme to the lipid vesicles is very strong was demonstrated by the failure of hemin to interact with globin when the two were rapidly mixed in the presence of phosphatidylserine vesicles. A multi-step process is suggested to explain the results of Hb phosphatidylserine interaction.     

Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by inositol. Inositol is an inhibitor of phosphatidylserine synthase activity

The addition of inositol to the growth medium of Saccharomyces cerevisiae resulted in rapid changes in the rates of phospholipid biosynthesis. The partitioning of the phospholipid intermediate CDP-diacylglycerol was shifted to phosphatidylinositol at the expense of phosphatidylserine and its derivatives phosphatidylethanolamine and phosphatidylcholine. Serine at 133-fold greater concentrations than that of inositol shifted the partitioning of CDP-diacylglycerol to phosphatidylserine at the expense of phosphatidylinositol but to a much lesser degree. Kinetic experiments with pure phosphatidylserine synthase and phosphatidylinositol synthase indicated that the partitioning of CDP-diacylglycerol between phosphatidylserine and phosphatidylinositol was not governed by the affinities both enzymes have for their common substrate CDP-diacylglycerol. Instead, the main regulation of phosphatidylinositol and phosphatidylserine synthesis was through the exogenous supply of inositol. The Km of inositol (0.21 mM) for phosphatidylinositol synthase was 9-fold higher than cytosolic concentration of inositol (24 microM). The Km of serine (0.83 mM) for phosphatidylserine synthase was 3-fold below the cytosolic concentration of serine (2.6 mM). Therefore, inositol supplementation resulted in a dramatic increase in the rate of phosphatidylinositol synthesis, whereas serine supplementation resulted in little affect on the rate of phosphatidylserine synthesis. Inositol also contributed to the regulation of phosphatidylinositol and phosphatidylserine synthesis by having a direct affect on phosphatidylserine synthase activity. Kinetic experiments with pure phosphatidylserine synthase showed that inositol was a noncompetitive inhibitor of the enzyme with a Ki of 65 microM.     

Bioconversion of Phosphatidylserine by Phospholipase D from Streptomyces racemochromogenes in a Microaqueous Water-Immiscible Organic Solvent

Phospholipase D (PLD)-mediated transphosphatidylation of phosphatidylcholine (PC) in a biphasic system was limited by the hydrolysis reaction. A biphasic system can produce a large amount of water. To solve this problem, a microaqueous water-immiscible organic solvent was used for the first time in the bioconversion of phosphatidylserine (PS). The transphosphatidylation among 40 µmol PC, 800 µmol L-serine, and 0.17 U/mL PLD in 2.133 mL of butyl acetate with 6.25% water (V/V) was conducted at a trans-phosphatidylation rate of 88% (mol/mol), and no hydrolytic reaction was observed. Compared to commonly used biphasic systems, this system shows a similar transphosphatidylation rate, whereas the undesirable hydrolysis of phospholipids was completely suppressed.     

A new NMR method for directly monitoring and quantifying the dissolution kinetics of starch in DMSO

The kinetics of dissolution of starch is needed for (i) understanding digestive processes; (ii) providing data that could correlate with higher levels of starch structure; (iii) improving techniques for starch characterization in solution. A novel method is presented here to directly monitor these dissolution kinetics by time-resolved (1)H solution-state nuclear magnetic resonance (NMR); studies were carried out in deuterated dimethyl sulfoxide (DMSO-d(6)). By assuming pseudo-first-order kinetics with respect to starch concentration, the data for various starch samples yield values of the apparent rate coefficients for the rate of appearance of completely dissolved anhydroglucose units, results which have not been obtained hitherto. The presence of a limited amount of water in DMSO had a drastic effect on dissolution kinetics (slowing it down at high temperatures), indicating multiple pathways for the dissolution mechanism. Dynamic light scattering (DLS) appears to be more limited than the NMR method to monitor the kinetics of dissolution. The newly developed NMR method can be extended to other solvents and polysaccharides.     

Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by cyclic AMP-dependent protein kinase.

The addition of cyclic AMP (cAMP) to Saccharomyces cerevisiae cyr1 mutant cells resulted in an increase in the rate of phosphatidylinositol synthesis at the expense of phosphatidylserine synthesis. The decrease in phosphatidylserine synthesis correlated with the down regulation of phosphatidylserine synthase activity by cAMP-dependent protein kinase phosphorylation. The increase in phosphatidylinositol synthesis was not due to the regulation of phosphatidylinositol synthase by cAMP-dependent protein kinase.     

Disruption of phosphatidylserine translocation to the mitochondria in baby hamster kidney cells

Phosphatidylserine synthase is found predominantly in the microsomal fraction, and phosphatidylserine decarboxylase is found predominantly in the mitochondrial fraction of baby hamster kidney (BHK-21) cells. This segregation of enzymes of phosphatidylserine metabolism allows serine metabolism to phosphatidylserine and phosphatidylethanolamine to be used as an indicator of the intracellular movement of phosphatidylserine. After BHK-21 cells were pulse-labeled with [3H]serine, phosphatidylserine was efficiently labeled, and subsequently 40-50% of this radiolabeled lipid turned over to form phosphatidylethanolamine during a 7.5-h chase. Treatment of cells with NaN3 plus NaF or cycloheximide at the end of the pulse labeling period markedly inhibited the rate and extent of phosphatidylserine turnover during the chase period. The inhibition of phosphatidylserine turnover could not be attributed to inhibition of either phosphatidylserine decarboxylase or phosphatidylserine exchange protein activity. Subcellular fractionation of the BHK-21 cells demonstrated that cells poisoned with NaN3 plus NaF accumulated phosphatidylserine in the microsomal fraction relative to unpoisoned cells. The results indicate that metabolic energy is required for the transport of phosphatidylserine to the mitochondria.     

不同土地利用方式土下岩溶溶蚀速率及影响因素

以重庆中梁山为例,通过野外埋放标准溶蚀试片、土壤CO2收集装置和进行亮蓝染色示踪试验,测试不同土地利用方式下不同土层碳酸盐岩溶蚀速率、土壤CO2浓度、土壤溶解性有机碳(DOC)含量及土壤含水量、土壤pH值和孔L隙度等性质,探讨不同土地利用方式土下溶蚀速率差异及其影响因素.结果表明不同土地利用方式对土壤理化性质产生影响,形成特定的岩溶微环境,进而影响土下不同层次的岩溶作用:土壤CO2浓度是影响林地和草地旱季土下溶蚀速率的重要驱动力;土壤含水量和供水能力是影响旱季不同土地利用方式溶蚀速率的关键因素;菜地产生的酸性物质较多,土壤pH值最低,其平均溶蚀速率高于林地;土壤DOC随水下渗迁移性强,是林地土下50 cm处溶蚀速率高于土下20 cm处的原因之一.该文为西南岩溶区土下岩溶机理、岩溶碳汇提供理论依据和数据参考.     

Phosphatidylserine affects specificity of protein kinase C substrate phosphorylation and autophosphorylation

Protein kinase C substrate phosphorylation and autophosphorylation are differentially modulated by the phosphatidylserine concentration in model membranes. Both substrate phosphorylation and auto-phosphorylation display a cooperative dependence on phosphatidylserine in sonicated vesicles composed of diacylglycerol and either phosphatidylcholine or a mixture of cell lipids (cholesterol, sphingomyelin, phosphatidylethanolamine, and phosphatidylcholine). However, the concentration of phosphatidylserine required to support phosphorylation varies with individual substrates. In general, autophosphorylation is favored at intermediate phosphatidylserine concentrations, while substrate phosphorylation dominates at high phosphatidylserine concentrations. These different phosphatidylserine dependencies may reflect different affinities of particular substrates for negatively charged membranes. Increasing the negative surface charge of sonicated vesicles increases the rate of substrate phosphorylation. In contrast to the modulation exerted by phosphatidylserine, diacylglycerol activates protein kinase C equally toward substrate phosphorylation and autophosphorylation. These results indicate that both diacylglycerol and phosphatidylserine regulate protein kinase C activity in the membrane: diacylglycerol turns the enzyme on, while phosphatidylserine affects the specificity toward different substrates.     

Physicochemical Properties and Dissolution Studies of Dexamethasone Acetate-β-Cyclodextrin Inclusion Complexes Produced by Different Methods

Inclusion complexes between dexamethasone acetate (DMA), a poorly water soluble drug, and β-cyclodextrin (βCD) were obtained to improve the solubility and dissolution rate of this drug. Phase-solubility profile indicated that the solubility of DMA was significantly increased in the presence of βCD (33-fold) and was classified as A_L-type, indicating the 1:1 stoichiometric inclusion complexes. Solid complexes prepared by different methods (kneading, coevaporation, freeze drying) and physical mixture were characterized by differential scanning calorimetry, thermogravimetry, infrared absorption and optical microscopy. Preparation methods influenced the physicochemical properties of the products. The dissolution profiles of solid complexes were determined and compared with those DMA alone and their physical mixture, in three different mediums: simulated gastric fluid (pH 1.2), simulated intestinal fluid (pH 7.4) and distilled water. The dissolution studies showed that in all mediums DMA presented an incomplete dissolution even in four hours. In contrast, the complexes formed presented a higher dissolution rate in simulated gastric fluid (SGF pH 1.2), which indicate that these have different ionization characteristics. According to the results, the freeze–dried and kneaded products exhibited higher dissolution rates than the drug alone, in all the mediums.     

Impact of a water temperature shift on xenoma clearance and recovery time during a Loma salmonae (Microsporidia) infection in rainbow trout Oncorhynchus mykiss

Previous studies have modelled the relationship between water temperature and the rate of sporulation as defined by xenoma formation during microsporidial gill disease (MGD) in salmon caused by Loma salmonae. Although offering insight into the epidemiology of MGD, a key unexplored area is the role of temperature in the rate of xenoma dissolution including spore release into the environment, and this is crucial to our ability to model horizontal transmission of MGD within confined net-pen populations of farmed salmon. Results from a previous trial suggested that xenoma dissolution may be dramatically hastened as water temperature declines, thus introducing a critical anomaly into any predictive exercise. The data generated herein was evaluated using the statistics of survival analysis to re-establish the baseline relationship of xenoma formation and dissolution relative to water temperature and to compare these results with those of previous studies. We infected 30 individuals of Oncorhynchus mykiss (Walbaum) with macerated xenoma-laden gill material, and afterwards allocated them to tanks with water temperatures of 11, 15, or 19 degrees C and monitored them through a disease cycle. Xenoma onset and clearance times were similar to previous findings with both events being accelerated at higher water temperatures, thereby suggesting a similar temperature response in the current strain to those used in previous studies. Another group of 45 fish was infected with L. salmonae and held at 15 degrees C until xenomas formed, and were subsequently shifted to 11, 15, or 19 degrees C. The median xenoma dissolution time in these tanks was 49, 35 and 28 d, respectively, similar to rates observed when water temperature remained constant. Thus we rejected the hypothesis that a sudden change in water temperature triggers rapid or anomalous xenoma dissolution.     

New Nanometric Solid Dispersions of Glibenclamide in Neusilin<Superscript>®</Superscript> UFL2

To improve the poor water solubility and dissolution rate of the oral hypoglycemic drug glibenclamide, it was molecularly dispersed in Neusilin^® UFL2, an amorphous synthetic form of magnesium aluminometasilicate, at different proportions; the physicochemical and biopharmaceutical properties, as well as the stability of the four different batches recovered were characterised, and it was determined that complete dispersion of glibenclamide in the amorphous polymer was obtained at the drug to Neusilin ratio of 1 to 2.5. Completely amorphous dispersion was proven by Thermal Analysis and X-Ray Powder Diffractometry. Very small particles were obtained, ranging from approximately 200 to 400 nm. The amorphous batches were physically and chemically stable for the entire duration of experiments. The physicochemical properties of the four batches were compared to those of the starting materials and physical mixtures of Neusilin^® UFL2 and glibenclamide, the latter showing the typical behaviour of simple mixes, i.e., the additivity of properties of single components. The dissolution studies of the four solid dispersions revealed a very high dissolution rate of the completely amorphous batches (Batches 3 and 4), behaviour that was ascribed to their high Intrinsic dissolution rate due to the amorphous characteristics of the solid dispersions, to their very small particle size, and to the presence of polysorbate 80 that improved solid wettability. The technique under investigation thus proved effective for recovering stable amorphous dispersions of very small particle sizes.     

Determination of dissolution profiles for several commercially available therapeutic [131I]sodium iodide capsules

The dissolution profiles for [¹³¹I]sodium iodide therapeutic capsules from three commerical vendors were studied. The ¹³¹I release rate in water was rapid for the capsules with 100% release attained within 35 min. There was no significant difference in dissolution profiles for the capsules from the three vendors.     

Investigation into the role of phosphatidylserine in modifying the susceptibility of human lymphocytes to secretory phospholipase A2 using cells deficient in the expression of scramblase

Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A₂ but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitt's lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A₂. Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100 s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A₂. These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A₂, it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine.     

The Improvement of the Dissolution Rate of Ziprasidone Free Base from Solid Oral Formulations

This work aims at increasing solubility and dissolution rate of ziprasidone free base—Biopharmaceutics Classifaction System (BCS) class II compound. The authors describe a practical approach to amorphization and highlight problems that may occur during the development of formulations containing amorphous ziprasidone, which was obtained by grinding in high-energy planetary ball mills or cryogenic mills. The release of ziprasidone free base from the developed formulations was compared to the reference drug product containing crystalline ziprasidone hydrochloride—Zeldox® hard gelatin capsules. All preparations were investigated using compendial tests (USP apparatuses II and IV) as well as novel, biorelevant dissolution tests. The novel test methods simulate additional elements of mechanical and hydrodynamic stresses, which have an impact on solid oral dosage forms, especially during gastric emptying. This step may prove to be particularly important for many formulations of BCS class II drugs that are often characterized by narrow absorption window, such as ziprasidone. The dissolution rate of the developed ziprasidone free base preparations was found to be comparable or even higher than in the case of the reference formulation containing ziprasidone hydrochloride, whose water solubility is about 400 times higher than its free base.KEY WORDS: amorphization, dissolution stress test device, enhanced dissolution, solubility improvement, ziprasidone free base formulations     

Rates of dissolution and biodegradation of water-insoluble organic compounds.

We conducted a study of the relationship between the dissolution rates of organic compounds that are sparingly soluble in water and the biodegradation of these compounds by mixed cultures of bacteria. The rates of dissolution of naphthalene and 4-chlorobiphenyl were directly related to their surface areas. The bacteria caused a decline in the concentration of the soluble substrate. The rate of bacterial growth fell abruptly when 4-chlorobiphenyl or naphthalene was no longer detectable in solution. The population continued to increase in media with different surface areas of insoluble 4-chlorobiphenyl, but the final counts were higher in media in which the surface areas of the substrate were larger. The rates of dissolution of palmitic acid, octadecane, di(2-ethylhexyl) phthalate, and 1-naphthyl N-methylcarbamate were determined in the absence of microorganisms. A mixed culture of microorganisms mineralized palmitic acid, di(2-ethylhexyl) phthalate, and Sevin (1-naphthyl N-methylcarbamate) at a logarithmic rate, but octadecane mineralization was linear. The rates of mineralization at the end of the active phase of the biodegradation were lower than the rate of dissolution of palmitic acid but higher than the rate of dissolution of octadecane in the uninoculated medium. We suggest that spontaneous dissolution rates are only one of the factors that govern the rates of biodegradation.     

Rates of dissolution and biodegradation of water-insoluble organic compounds

We conducted a study of the relationship between the dissolution rates of organic compounds that are sparingly soluble in water and the biodegradation of these compounds by mixed cultures of bacteria. The rates of dissolution of naphthalene and 4-chlorobiphenyl were directly related to their surface areas. The bacteria caused a decline in the concentration of the soluble substrate. The rate of bacterial growth fell abruptly when 4-chlorobiphenyl or naphthalene was no longer detectable in solution. The population continued to increase in media with different surface areas of insoluble 4-chlorobiphenyl, but the final counts were higher in media in which the surface areas of the substrate were larger. The rates of dissolution of palmitic acid, octadecane, di(2-ethylhexyl) phthalate, and 1-naphthyl N-methylcarbamate were determined in the absence of microorganisms. A mixed culture of microorganisms mineralized palmitic acid, di(2-ethylhexyl) phthalate, and Sevin (1-naphthyl N-methylcarbamate) at a logarithmic rate, but octadecane mineralization was linear. The rates of mineralization at the end of the active phase of the biodegradation were lower than the rate of dissolution of palmitic acid but higher than the rate of dissolution of octadecane in the uninoculated medium. We suggest that spontaneous dissolution rates are only one of the factors that govern the rates of biodegradation.