Gun Hedström

Effects of sphingomyelin degradation on cell cholesterol oxidizability and steady-state distribution between the cell surface and the cell interior

This study addresses questions related to (i) the distribution of cholesterol between the cell surface and intracellular membranes in cultured fibroblasts and (ii) the effects of plasma membrane sphingomyelin on this distribution. Cholesterol oxidase (Streptomyces sp.) converts cell cholesterol to cholestenone and was used in this study to probe the cellular distribution of cholesterol. The availability of cell cholesterol for oxidation by cholesterol oxidase was markedly influenced by the presence of sphingomyelin. In native, glutaraldehyde-fixed fibroblasts only about 20% of the cell cholesterol was oxidized under our experimental conditions. However, degradation of cell sphingomyelin with sphingomyelinase (Staphylococcus aureus) markedly enhanced the oxidation of cell surface cholesterol in glutaraldehyde-fixed fibroblasts. About 90% of the total unesterified cholesterol could be oxidized to cholestenone in confluent, sphingomyelin-depleted fibroblasts. These results suggest that about 90% of the unesterified cholesterol was at the cell surface in these cells. It was also observed that degradation of cell sphingomyelin exerted a dramatic effect on the distribution of cell cholesterol between the cell surface and intracellular membranes. Within 90 min after hydrolysis of cell sphingomyelin, about 30% of the total cell-associated unesterified cholesterol was transported from a cholesterol oxidase-susceptible pool to an oxidase-resistant pool. Together with the redistribution of cell cholesterol after sphingomyelin degradation, a marked enhancement of the endogenous cholesterol esterification reaction was observed. We conclude that the degradation of plasma membrane sphingomyelin resulted in a new apparent steady-state distribution of cellular cholesterol, with less cholesterol in the plasma membrane and more in intracellular membranes. It therefore appears that sphingomyelin is a major determinant of the distribution of cholesterol in intact cells.

Monolayer interaction of cholesterol with phosphatidylcholines: effects of phospholipid acyl chain length

Abstract The degree of association of cholesterol with phosphatidylcholines having symmetric acyl chains from 10 to 20 carbons in length have been examined in monolayers at the air/water interface using cholesterol oxidase as a probe. Mixed monolayers having three different cholesterol/phospholipid (C/PL) molar ratios (0.9:1.0, 1.0:1.0, and 1.5:1.0 C/PL) were prepared. In these monolayers (at a lateral surface pressure of 20 mN/m), cholesterol was most readily available for oxidation in monolayers having phosphatidylcholines with short (di-10-PC and di-12-PC) or long (di-18-PC and di-20-PC) acyl chains, whereas the oxidation susceptibility was lower in monolayers having phosphatidylcholines with intermediate length acyl chains (di-14-PC, di-15-PC, di-16-PC and di-17-PC). Mixed monolayers having a C/PL of 0.9:1.0 were prepared to include 0.5 mol% NBD-cholesterol, and the monolayer surface texture at 20 mN/m was examined using epifluorescence microscopy. It was clearly revealed that monolayers containing di-10-PC and di-12-PC were laterally heterogeneous (containing both liquid-expanded and liquid-condensed lateral domains). With intermediate chain phosphatidylcholines (14–17 carbons), there was no surface texture (dominantly dark monolayer areas), whereas occasional bright NBD-cholesterol-rich inclusions again began to appear in di-18-PC and di-20-PC monolayers. The increased oxidation susceptibility of cholesterol in di-18-PC and di-20-PC could possibly result from a partial lateral phase separation of cholesterol-rich phases in these monolayers, since cholesterol can be expected to be less readily soluble in a long chain than in intermediate chain phosphatidylcholine matrix, and since cholesterol-rich phases are expected to be oxidized more readily than cholesterol-poor phases. We conclude that the susceptibility of cholesterol to oxidation by cholesterol oxidase was most pronounced in monolayers containing phosphatidylcholines with acyl chain lengths that did not match the length of the sterol molecule.

Rational evaluation of the utilization of PEG-PEI copolymers for the facilitation of silica nanoparticulate systems in biomedical applications

HYPOTHESIS Polymer constructs are often applied in nanoparticulate systems to expand their applicability. One such common macromolecular modifier is poly(ethylene imine) - poly(ethylene glycol) copolymers. Despite their quite widespread use, and considering that interaction and stabilization mechanisms when combining a polyelectrolyte with a non-charged polymer are not trivial to pinpoint, these systems are generally poorly characterized in literature. Here, we attempt to provide a solid rationale to utilize PEG-PEI copolymers as surface modifiers and stabilizers/dispersion agents in solid colloidal systems with focus on biomedical applicability. EXPERIMENTAL mPEG grafted PEI copolymers with two different grafting densities and 100 nm sized non-porous silica nanoparticles (SiNP) were synthesized. Detailed physico-chemical characterization of all prepared materials was conducted with spectroscopic methods, while the interaction mechanisms between the produced copolymers and SiNP were investigated by calorimetry. The influence of increased PEG grafting ratio on the attained colloidal stability of copolymer functionalized SiNP was studied by multiple light scattering, and its further implications on the biobehavior of SiNP were evaluated. FINDINGS The interaction mechanism between SiNP and copolymers was concluded to be mainly directed by electrostatics, whereas an influence of PEG grafting density on the adsorption process was also observed. The implications of the surface modifications on the in vitro biobehavior of SiNP were investigated by combining the knowledge obtained by the detailed characterizations with microscopy evaluation under in vitro conditions.

Lipase-catalyzed enantioselective esterifications using different microemulsion-based gels

Chiral esters with high optical purity have been synthesized at 298.2 K from racemic 2-octanol and alkanoic acids using the commerical lipases fromChromobacterium viscosum (CV) orCandida sp. (SP 525) immobilized in microemulsion-based gelatin gels. The microemulsions consisted of water and alkanes stabilized by the anionic surfactant sodium 1,4-bis(2-ethylhexyl) sulfosuccinate (AOT) and the naturally occurring zwitterionic surfactant soybean lecithin, respectively. The enzymes were solubilized both in water-in-oil (W/O) microemulsions and in microemulsions with a bicontinuous structure. Different microstructures of the gels were chosen since the enzyme may undergo conformational changes in different environments resulting in different catalytic efficiencies toward competing substrates. Therefore, it is of great fundamental interest to know the phase behaviour and the microstructures of the used microemulsion systems. Phase diagrams were determined at 298.2 K for the systems water-hexane-AOT and ethanol/water (1∶1)-hexadecane-soybean lecithin. The former system exhibited a large one-phase W/O microemulsion region, while in the latter a small one-phase region with bicontinuous structure was present. The kinetic enantiomeric ratios (E-values), as determined from enantiomeric excess (e.e.) values at a conversion below 0.5, were higher both in the W/O microemulsion as well as in the bicontinuous microemulsion using the SP 525 lipase, than using the CV lipase. On the other hand, the conversions were higher using gels based on W/O microemulsions (AOT stabilized) than using gels based on microemulsions with a bicontinuous structure (lecithin stabilized).

Intracellular transport of cholesterol in type C Niemann-Pick fibroblasts

The purpose of this study was to determine the capacity of Niemann-Pick type C (NPC) fibroblasts to transport cholesterol from the cell surface to intracellular membranes. This is relevant in light of the observations that NPC cells display a sluggish metabolism of LDL-derived cholesterol, a phenomenon which could be explained by a defective intracellular transport of cholesterol. Treatment of NPC cells for 4 h with 0.1 mg/ml of LDL failed to increase the incorporation of [14C]oleic acid into cholesterol [14C]oleate, an observation consistent with previous reports on this cell type (Pentchev et al. (1985) Proc. Natl. Acad. Sci. USA 82, 8247). Normal fibroblasts, however, displayed the classical upregulation (6-fold over control) of the endogenous esterification reaction in response to LDL exposure. Incubation of normal or NPC fibroblasts with sphingomyelinase (100 mU/ml; Staphylococcus aureus) led to a rapid and marked increase (9- and 10-fold for normal and NPC fibroblasts, respectively, after 4 h) in the esterification of plasma-membrane-derived [3H]cholesterol suggesting that sphingomyelin degradation forced a net transfer of cholesterol from the cell surface to the endoplasmic reticulum. The similar response in normal and mutant fibroblasts to the degradation of sphingomyelin suggests that plasma membrane cholesterol can be transported into the substrate pool of ACAT to about the same extent in these two cell types. Degradation of cell sphingomyelin in NPC fibroblasts also resulted in the movement of 20-25% of the cellular cholesterol from a cholesterol oxidase susceptible pool into oxidase-resistant pools, implying that a substantial amount of plasma membrane cholesterol was internalized after sphingomyelin degradation. This cholesterol internalization was not accompanied by an increased rate of membrane internalization, as measured by [3H]sucrose uptake. Although NPC cells showed a relative accumulation of unesterified cholesterol and a sluggish esterification of LDL-derived cholesterol when exposed to LDL, these cells responded like normal fibroblasts with regard to their capacity to transport cholesterol from the cell surface into intracellular sites in response to sphingomyelin degradation. It therefore appears that NPC cells, in contrast to the impaired intracellular movement of lipoprotein-derived cholesterol, do not display a general impairment of cholesterol transport between the cell surface and the intracellular regulatory pool of cholesterol.

Acid–base interaction of probes at silica surface. Microcalorimetry and adsorption

The interaction of four benzyl derivatives with silica surface sites in anhydrous cyclohexane is investigated. The strength and extent of adsorption is determined from the Langmuir adsorption isotherm. The enthalpy of immersion is determined microcalorimetrically, which is recalculated to the enthalpy of adsorption. The aim is to distinguish between the Lewis-type (electron exchange) and Brønsted-type (proton exchange) of interaction by selecting methanol-acetone and acetic acid-methylamine, respectively as acid-base substituent in benzene. Moreover, the experiments were performed in anhydrous cyclohexane in order to screen out hydrocarbon interactions from the energy balance. The monomolecular adsorption is also distinguished from the bulk wetting of solids. The results are evaluated in terms of possible proton exchange in anhydrous media.

LIPASE-CATALYSED STEREOSELECTIVE ESTERIFICATIONS USING GELATIN-BASED HYDROGELS

A hydrogel stable in an organic solvent has been developed. This pseudo-solid aqueous gel (PAG) consists of only native gelatin and water, and has been used for immobilization of enzymes. A relatively high amount of gelatin is required in order to obtain stable gels. PAGs containing the enzyme Candida antarctica lipase (SP 525) were successfully used in catalysing the esterification of R/S-(±)-2-octanol and hexanoic acid in hexane. The conversions as well as the enantiomeric excess values of the product, R-(−)-2-octyl hexanoate, were high and comparable to those obtained with microemulsion-based gels. The PAGs containing immobilized lipase gave reproducible results and may be re-used several times. The gels are easy to prepare and use, non-toxic and biocompatible. The PAGs retain their integrity in organic solvents and may be used in preparative-scale synthesis of organic compounds.

Enzymatic esterifications and transesterifications in AOT-based gels with different composition

Abstract The enzyme-catalysed esterification between racemic 2-octanol and hexanoic acid using lipase immobilized in two different microemulsion-based gels has been investigated. The gels differ in the composition of the microemulsion used for gelation. Furthermore, transesterifications between vinyl butyrate and racemic 2-octanol or between vinyl butyrate and racemic menthol have been studied. The phase boundary of the L2 phase, consisting of water droplets in oil, of the system water-AOT-decane has been outlined. Two microemulsions, one in the low-conducting region and one in the high-conducting region, were chosen for gelation and enzyme immobilization. The reactions were performed at 298.2 K by immersing the sectioned gel in hexane, containing the substrates. The conversion and selectivity of the esterification between 2-octanol and hexanoic acid were independent of the gel composition, but the initial reaction rate differed for the gels used. The transesterification between vinyl butyrate and 2-octanol was very fast, resulting in a maximum conversion after only 10 min. A subsequent hydrolysis, of the formed (R)-(−)-2-octyl butyrate, took place due to nonequilibrium conditions. A final conversion of (R)-(−)-2-octyl butyrate, of 0.45, was reached after 2 h. The enantiomeric purity of all the products was very high in all the studies.

ENZYME CATALYSIS IN DIFFERENT GELATIN GELS

Abstract Enzymes in microemulsions, liposomes, aqueous monomer surfactant solutions or pure water have been entrapped in gelatin-based gels. These gels have been used as minireactors for stereoselective resolution of racemic 2-octanol in esterifications with alkanoic acids or in transesterifications with vinyl butyrate. The microstructures were stabilized by the anionic surfactant sodium 1,4-bis(2-ethylhexyl) sulfosuccinate (AOT) or the zwitterionic surfactant soybean lecithin. The enzymes used were commercial lipases from Chromobacterium viscosum or Candida antarctica (SP 525). The incubation temperature was 298.2 K. The enantiomeric excess (e.e.) values were high in all systems studied, but the reaction rates depended on the gel used. In AOT-stabilized and in ethanol-free lecithin-stabilized gels the conversion was close to 0.45. However, in the ethanol-containing lecithin gels, the conversion was lower and decreased with increased ethanol content, due to competing reactions. In the hydrogels, the conve...