Prion protein expression in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system

Syrian hamster prion protein (PrPC) and a truncated Syrian hamster prion protein lacking the glycosylphosphatidylinositol (GPI) anchor C- terminal signal sequence (GPI-) were expressed in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system. The CHO cell clones expressing the GPI- PrP secreted the majority of the protein into the media, whereas most of the PrP produced by clones expressing the full-length protein with the GPI anchor was located on the cell surface, as demonstrated by its release upon treatment with phosphatidylinositol-specific phospholipase C (PIPLC). A cell clone that expressed the highest levels of full length PrP was subcloned to obtain clone 30C3-1. PrP from clone 30C3-1 was shown to be sensitive to proteolysis by proteinase K and to react with monoclonal and polyclonal antibodies that recognize native PrPC. The recombinant PrP migrated as a diffuse band of 19-40 kDa but removal of the N-linked oligosaccharides with peptide N-glycosidase F (PNGase F) revealed three protein species of 19, 17 and 15 kDa. The 19 kDa band corresponding to deglycosylated full-length PrP was quantified and found to be expressed at a level approximately 14-fold higher than that of PrPC found in Syrian hamster brain.      

Nonspecific lipid transfer proteins as probes of membrane structure and function

A protein that accelerates transfer of phospholipids of varying head group and fatty acid composition has been purified from bovine liver. As previously found for other phospholipid transfer proteins, “nonspecific lipid transfer protein” stimulates a kinetically biphasic transfer of radioactively labeled phospholipid from small unilamellar vesicles to unlabeled multilamellar vesicles. The kinetics are consistent with rapid transfer of phospholipid from the outer monalyer and slow transfer of that localized in the inner monolayer (half-times greater than 3 days for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol). Protein catalyzed transfer is inhibited by high ionic strength and has an activation energy of 35 kJ/mol. The broad lipid specificity and ease of large-scale purification make these proteins candidates for membrane phospholipid compositional modification. The compositions of rat liver mitochondrial and microsomal membranes and Morris hepatoma 7288c mitochondrial membranes were altered by incubation with lipid vesicles and nonspecific lipid transfer protein. Incubation with phosphatidylcholine vesicles led to increased levels of phosphatidylcholine and decreased levels of other transferrable lipids (phosphatidylethanolamine, phosphatidylinositol, and cholesterol) unless the latter were included in the vesicles. When vesicles containing dipalmitoylphosphatidylcholine were incubated with microsomal membranes, a large increase in disaturated phosphatidylcholine was also observed. These changes in composition were correlated with activities of membrane enzymes. It appears that microsomal glucose-6-phosphatase is inhibited by increased phosphatidylcholine saturation. Moreover, this enzyme is also inhibited by decreases in the phosphatidylethanolamine/phosphatidylcholine ratio whereas NADPH cytochrome c reductase is not. Likewise, decreased cholesterol to phospholipid ratios did not greatly affect the abnormally low levels of hepatoma succinate cytochrome c reductase activity. This paper was presented at the 73rd AOCS annual meeting, Toronto, Canada, May 1982.     

Role of prion protein aggregation in neurotoxicity

In several neurodegenerative diseases, such as Parkinson, Alzheimer's, Huntington, and prion diseases, the deposition of aggregated misfolded proteins is believed to be responsible for the neurotoxicity that characterizes these diseases. Prion protein (PrP), the protein responsible of prion diseases, has been deeply studied for the peculiar feature of its misfolded oligomers that are able to propagate within affected brains, inducing the conversion of the natively folded PrP into the pathological conformation. In this review, we summarize the available experimental evidence concerning the relationship between aggregation status of misfolded PrP and neuronal death in the course of prion diseases. In particular, we describe the main findings resulting from the use of different synthetic (mainly PrP106-126) and recombinant PrP-derived peptides, as far as mechanisms of aggregation and amyloid formation, and how these different spatial conformations can affect neuronal death. In particular, most data support the involvement of non-fibrillar oligomers rather than actual amyloid fibers as the determinant of neuronal death.     

Heparin inhibits membrane interactions and lipid-induced fibrillation of a prion amyloidogenic determinant

Glycosaminoglycans (GAGs), particularly heparin, are known to reduce the toxicities of various amyloidogenic proteins. The molecular factors underlying the antitoxic effects of GAGs, however, are still not fully understood. Because interactions of amyloidogenic proteins and their aggregates with membranes are believed to play major roles in affecting amyloid pathogenesis, our objective in this study was to elucidate the effect of heparin on membrane interactions of the 21-residue amyloidogenic determinant of the prion protein [PrP(106-126)]. Indeed, the experimental results indicate that heparin significantly interferes in membrane interactions of the prion peptide. Specifically, we show that there is direct competition for binding of PrP(106-126) between heparin on the one hand and negatively charged phospholipids on the other hand. The data reveal that heparin, even in very low molar concentrations, exhibited high affinity towards PrP(106-126) and consequently suppressed interactions of the peptide with lipid vesicles. Interestingly, whereas heparin significantly inhibited lipid-induced PrP(106-126) fibrillation, it still promoted fibril formation in aqueous solutions independently of the lipid vesicles present. Our results strongly suggest that the primary effects of GAGs in attenuating amyloid toxicities are due to blocking of membrane interactions of the amyloidogenic proteins rather than modulation of their fibrillation properties.     

Novel dominant-negative prion protein mutants identified from a randomized library

Prion diseases are untreatable neurodegenerative disorders characterized by accumulation of PrP(Sc), an aggregated isoform of the cellular prion protein (PrP(C)). We generated a library of PrP variants with random mutations in the helix-3 domain and screened for dominant-negative mutants (DNMs) that would inhibit replication of prions (the Rocky Mountain Laboratory strain) in infected N2a cells. Two of the identified PrP mutants, Q167R and Q218K, were already known to counteract prion replication, thereby validating the effectiveness of this approach. In addition, novel DNMs were found efficiently to antagonize PrP(Sc) propagation in cells. In contrast to Q167R and Q218K, the newly identified DNMs S221P and Y217C resided on the cell surface at a substantially lower level, suggesting that robust cell surface display of DNM might not be a general prerequisite for efficient prion antagonism. The newly identified DNMs point to useful target-selective therapeutic tools for the treatment of prion diseases.     

Changes in muscle lipid composition and resistance adaptation to temperature in the freshwater crayfish,Austropotamobius pallipes

The phospholipid and fatty acid composition of muscle lipid extracts from crayfish acclimated to 4 C and 25 C (18 hr-light photoperiod) were analyzed. The phospholipid content and class distribution, and cholesterol content were unaffected by the acclimation treatment. Unsaturation of muscle phosphoglycerides was higher in cold acclimated crayfish. Serine/inositol phosphoglycerides from cold-acclimated animals showed somewhat higher proportions of mono-and polyunsaturated fatty acids, whereas choline and ethanolamine phosphoglycerides were less affected. This was correlated with a decreased resistance of cold-acclimated crayfish to lethal high temperature. Acclimation at 4 C under an 8 hr-light photoperiod caused an increased fatty acid unsaturation of the total phospholipid fraction compared to the 4 C, 18 hr-light photoperiod acclimated animals. The resistance of 4 C acclimated crayfish to lethal high temperature, however, was unaffected by daylength treatment. The resistance of freshwater crayfish to lethal high temperature is not simply related to the degree of saturation of the muscle phospholipids. It is suggested that a breakdown in the integrity of a bulk-lipid bilayer is not involved in the process of heat death; rather, that a membrane-bound protein factor, whose thermal sensitivity is modified by changes in its phospholipid environment during temperature adaptation but not during photoperiod adaptation, is the primary site of heat injury.     

Distribution of cholesterol among its carriers in the bile of male and female hamsters

The distribution of cholesterol among its carriers was studied in the bile of male and female hamsters. Sasco hamsters (Sasco Inc., Omaha, NE) were fed a semipurified diet with 0.0% cholesterol and 4% butterfat (group 1, males; group 4, females); a semipurified diet with 0.3% cholesterol and 1.2% plamitic acid (group 2, males; group 5, females); and a semipirified diet with 0.3% cholesterol and 4% safflower oil (group 3, males; group 6, females). At the end of six weeks, gallstones were found only in male hamsters receiving both cholesterol and dietary fat (fatty acid) (incidence of cholesterol stones: 90% in group 2; 22% in group 3). The biliary cholesterol carriers were separated and isolated from the bile of the hamsters by gel filtration chromatography, using the method of Pattinson [Pattinson, N.R., Willis, K.E., and Frampton, C.M. (1991)J. Lipid Res. 32, 205–214]. In those male hamsters that formed cholesterol gallstones, significant amounts of cholesterol were present in the void volume which contained large cholesterol phospholipid vesicles (void volume vesicles) (23% in group 2 and 15% in group 3). Smaller cholesterol/phospholipid vesicles were eluted next (fractions 30–45) and contained 15% of biliary cholesterol in group 2 and 21% in group 3. The remainder of the cholesterol was associated with mixed cholesterol/phospholipid/bile salt micelles. The cholesterol/phospholipid ratio was larger in both the void volume vesicles and small vesicles (2.40 and 1.48 in group 2; 2.56 and 1.33 in group 3, respectively) compared to the micelles (about 0.3 in groups 2 and 3). In contrast, the bile of the female hasmters contained few vesicles (3% small vesciles in group 5) and the cholesterol/phospholipid ratio of these vesicles was lower (0.94). Hamsters fed cholesterol-free diets (groups 1 and 4) had no biliary cholesterol/phospholipid vesilces; and cholesterol was present in micelles. The results suggest that both the gender and the diet of the hamsters affected the distribution of biliary cholesterol between vesicles and micelles. The development of cholelithiasis in this animal model appears to depend on the rapid nucleation of cholesterol-rich phospholipid vesicles in bile.     

The Effect of Octapeptide Repeats on Prion Folding and Misfolding

Misfolding of prion protein (PrP) into amyloid aggregates is the central feature of prion diseases. PrP has an amyloidogenic C-terminal domain with three α-helices and a flexible tail in the N-terminal domain in which multiple octapeptide repeats are present in most mammals. The role of the octapeptides in prion diseases has previously been underestimated because the octapeptides are not located in the amyloidogenic domain. Correlation between the number of octapeptide repeats and age of onset suggests the critical role of octapeptide repeats in prion diseases. In this study, we have investigated four PrP variants without any octapeptides and with 1, 5 and 8 octapeptide repeats. From the comparison of the protein structure and the thermal stability of these proteins, as well as the characterization of amyloids converted from these PrP variants, we found that octapeptide repeats affect both folding and misfolding of PrP creating amyloid fibrils with distinct structures. Deletion of octapeptides forms fewer twisted fibrils and weakens the cytotoxicity. Insertion of octapeptides enhances the formation of typical silk-like fibrils but it does not increase the cytotoxicity. There might be some threshold effect and increasing the number of peptides beyond a certain limit has no further effect on the cell viability, though the reasons are unclear at this stage. Overall, the results of this study elucidate the molecular mechanism of octapeptides at the onset of prion diseases.     

Combining in-situ proteolysis and microseed matrix screening to promote crystallization of PrPc-nanobody complexes

Prion proteins (PrPs) are difficult to crystallize, probably due to their inherent flexibility. Several PrPs structures have been solved by nuclear magnetic resonance (NMR) techniques; however, only three structures were solved by X-ray crystallography. Here we combined in-situ proteolysis with automated microseed matrix screening (MMS) to crystallize two different PrP(C)-nanobody (Nb) complexes. Nanobodies are single-domain antibodies derived from heavy-chain-only antibodies of camelids. Initial crystallization screening conditions using in-situ proteolysis of mouse prion (23-230) in complex with a nanobody (Nb_PrP_01) gave thin needle aggregates, which were of poor diffraction quality. Next, we used these microcrystals as nucleants for automated MMS. Good-quality crystals were obtained from mouse PrP (89-230)/Nb_PrP_01, belonged to the monoclinic space group P 1 21 1, with unit-cell parameters a = 59.13, b = 63.80, c = 69.79 ?, β = 101.96° and diffracted to 2.1 ? resolution using synchrotron radiation. Human PrP (90-231)/Nb_PrP_01 crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 131.86, b = 45.78, c = 45.09 ?, β = 96.23° and diffracted to 1.5 ? resolution. This combined strategy benefits from the power of the MMS technique without suffering from the drawbacks of the in-situ proteolysis. It proved to be a successful strategy to crystallize PrP-nanobodies complexes and could be exploited for the crystallization of other difficult antigen-antibody complexes.     

The effects of dietary phospholipids enriched with phosphatidylethanolamine on bile and red cell membrane lipids in humans

The role of phospholipids in biliary cholesterol solubilization and crystallization has only recently begun to be appreciated. Phospholipid vesicles are believed to be the metastable carrier from which cholesterol nucleates. Cholesterol crystallization is influenced by the phospholipid species in bile. Feeding rats and hamsters with diets enriched in phospholipids or their precursors, especially ethanolamine, resulted in reduced cholesterol saturation of bile. Although whole phospholipids are normal dietary constituents, the effects and safety of phospholipid components have not been tested in humans. In the present study, we have evaluated the effects of a dietary phospholipid mixture, enriched with phosphatidylethanolamine, on human bile and red blood cell membrane lipid composition. Five ambulatory volunteers having a chronic indwelling T-tube, with an intact enterohepatic circulation, were investigated. Thirty-six grams of phospholipids (54% phosphatidylethanolamine, 54% linoleyl acyl chains) were added to their daily diet for fourteen days. Biliary nucleation time, cholesterol carriers, as well as plasma, red blood cell membrane, and bile lipid compositions, were monitored. Following phospholipid supplementation, the proportion of linoleyl chains (18:2) in biliary phospholipids increased significantly from 31.1±1.2 to 37.7±5.3%, while that of oleyl chains (18:1) decreased from 11.4±1.6 to 9.6±1.1%. These changes were accompanied by an increase of linoleate and its metabolite, arachidonate, in red cell membranes. Phospholipid feeding did not cause any side effects, and no significant changes in biliary nucleation time, cholesterol, phospholipid, or bile salt concentrations, or in the distribution of cholesterol within micelles or vesicles. We conclude that phospholipid feeding is safe, and can be effective as a vehicle for lecithin fatty acyl chain modulation of bile and lipid membranes. These findings may provide a basis for a controlled modulation of biliary phospholipids to increase cholesterol solubility in bile.     

Disruption of the X-loop turn of the prion protein linked to scrapie resistance

The prion diseases are a class of neurodegenerative diseases caused by the misfolding and aggregation of the prion protein (PrP(C)) into toxic and infectious oligomers (PrP(Sc)). These oligomers are critical to understanding and combating these diseases. Differences in the sequence of PrP affect disease susceptibility, likely by shifting the tolerance of the protein for adaptation to PrP(Sc) conformations and/or the recognition event between PrP(Sc) and PrP(C) prior to conversion of the PrP(C). We selected two sets of PrP(Sc)-resistant mutant sequences for solvated atomistic molecular dynamics simulation to investigate the structural basis of resistance. The first group involved mutation in the X-loop (residues 164-171) resulting from selective breeding of sheep. The second group included eight mutants in mice identified by random mutagenesis targeting helix C followed by screening in cell cultures. Multiple simulations were performed of 14 different mutant and control constructs under different pH conditions for a total of 3.6 μs of simulation time. The X-loop formed a stable turn at neutral pH in wild-type PrP from both species. PrP(Sc)-resistant mutations disrupted this turn even though only one of the mutants is in the X-loop. The X-loop is compact and buried in our previously described spiral models of PrP(Sc)-like oligomers. On the basis of the findings presented here and in the context of the spiral oligomer model, we propose that expansion of the X-loop disrupts protofibril packing, providing a structural basis for resistance.     

How an Infection of Sheep Revealed Prion Mechanisms in Alzheimer’s Disease and Other Neurodegenerative Disorders

Although it is not yet universally accepted that all neurodegenerative diseases (NDs) are prion disorders, there is little disagreement that Alzheimer’s disease (AD), Parkinson’s disease, frontotemporal dementia (FTD), and other NDs are a consequence of protein misfolding, aggregation, and spread. This widely accepted perspective arose from the prion hypothesis, which resulted from investigations on scrapie, a common transmissible disease of sheep and goats. The prion hypothesis argued that the causative infectious agent of scrapie was a novel proteinaceous pathogen devoid of functional nucleic acids and distinct from viruses, viroids, and bacteria. At the time, it seemed impossible that an infectious agent like the one causing scrapie could replicate and exist as diverse microbiological strains without nucleic acids. However, aggregates of a misfolded host-encoded protein, designated the prion protein (PrP), were shown to be the cause of scrapie as well as Creutzfeldt–Jakob disease (CJD) and Gerstmann–Sträussler–Scheinker syndrome (GSS), which are similar NDs in humans. This review discusses historical research on diseases caused by PrP misfolding, emphasizing principles of pathogenesis that were later found to be core features of other NDs. For example, the discovery that familial prion diseases can be caused by mutations in PrP was important for understanding prion replication and disease susceptibility not only for rare PrP diseases but also for far more common NDs involving other proteins. We compare diseases caused by misfolding and aggregation of APP-derived Aβ peptides, tau, and α-synuclein with PrP prion disorders and argue for the classification of NDs caused by misfolding of these proteins as prion diseases. Deciphering the molecular pathogenesis of NDs as prion-mediated has provided new approaches for finding therapies for these intractable, invariably fatal disorders and has revolutionized the field.     

Nonmembrane associated dolichol in rat liver

The distribution of dolichol in rat liver was studied. Upon high-speed centrifugation, 9% of the total tissue dolichol was recovered in the supernatant. Dolichol was enclosed in vesicles and in lipidic particles which were isolated by gel filtration and density gradient centrifugation. The particles had a diameter of 20 nm and contained dolichol, ubiquinone, cholesterol, phospholipid and some protein. Similar particles were recovered upon incubation of isolated hepatocytes with liposomes containing dolichol. From the lysosomal lumen, lipid particles containing dolichol, ubiquinone, cholesterol and phospholipid, but no protein, were isolated. The diameter of the particles was 20–40 nm with a molecular weight of 130 kDa. Puromycin treatment inhibited protein synthesis, but did not affect dolichol transfer from the endoplasmic reticulum to lysosomes, suggesting that the transfer is not mediated by newly synthesized apoprotein. The results indicate that a sizeable portion of the total cellular dolichol is present in cytoplasm and in lysosomal lumen. Furthermore, dolichol probably participates in the translocation process.     

Niemann-Pick C2 Protein Expression Regulates Lithogenic Diet-Induced Gallstone Formation and Dietary Cholesterol Metabolism in Mice

Niemann-Pick C2 protein (NPC2) is a lysosomal soluble protein that is highly expressed in the liver; it binds to cholesterol and is involved in intracellular cholesterol trafficking, allowing the exit of lysosomal cholesterol obtained via the lipoprotein endocytic pathway. Thus, this protein may play an important role in controlling hepatic cholesterol transport and metabolism. The aim of this work was to study the relevance of NPC2 protein expression in hepatic cholesterol metabolism, biliary lipid secretion and gallstone formation by comparing NPC2 hypomorph [NPC2 (h/h)] and wild-type mice fed control, 2% cholesterol, and lithogenic diets. NPC2 (h/h) mice exhibited resistance to a diet-induced increase in plasma cholesterol levels. When consuming the chow diet, we observed increased biliary cholesterol and phospholipid secretions in NPC2 (h/h) mice. When fed the 2% cholesterol diet, NPC2 (h/h) mice exhibited low and high gallbladder bile cholesterol and phospholipid concentrations, respectively. NPC2 (h/h) mice fed with the lithogenic diet showed reduced biliary cholesterol secretion, gallbladder bile cholesterol saturation, and cholesterol crystal and gallstone formation. This work indicates that hepatic NPC2 expression is an important factor in the regulation of diet-derived cholesterol metabolism and disposal as well as in diet-induced cholesterol gallstone formation in mice.     

An improved assay for cholesterol 7α-hydroxylase activity using phospholipid liposome solubilized substrate

A persistent problem in measurement of cholesterol 7α-hydroxylase (7α-OHase) activity by isotope incorporation has been solubilization of cholesterol substrate. Solubilization with Tween 20, for example, resulted in a 75% reduction in 7α-OHase activity after a 60 min incubation of substrate with microsomes. Incorporation of cholesterol substrate into small, unilamellar phospholipid vesicles (liposomes) prevented this effect, resulting in a 50% increase in activity over the same 60 min incubation at optimal concentrations. Using cholesterol in liposomes as substrate, standard assay conditions were determined to be: preparation of liposomes with 180 μM cholesterol substrate and 0.5 mg phospholipid/assay; incubation of these liposomes with 0.5 mg microsomal protein at 37 C for 60 min; addition of a NADPH generating system to start the reaction, and incubation at 37 C for 30 min before stopping the reaction and determining the amount of 7α-hydroxycholesterol formed. In addition to preventing the detergent-related inhibition of the enzyme, liposome-solubilized substrate also reduced the variation among replicates from a coefficient of 45% with Tween 20 to 4.2% with phospholipid. This method provides a sensitive and reliable alternative to methods which require more sophisticated equipment and allows total control of substrate concentration in a form readily accessible to the enzyme. This work was reported in part at the American Society of Biological Chemists Meeting, St. Louis, Missouri, June 1984.     

Removal of lipid from intact erythrocytes and ghosts by aqueous solutions and its relevance to membrane structure

Molar values for cholesterol, total phospholipid, and individual phospholipid classes of intact erythrocytes and their membranes (ghosts) washed with various aqueous solutions are presented. The data show that lipid can be washed from erythrocyte ghosts prepared rapidly from freshly drawn blood but that lipid is not removed from intact erythrocytes under the same conditions. Thus, it appears that the polar groups of lipids of intact cells are not exposed as they are in ghosts. In the preparation of hemoglobin-free ghosts, up to 25% cholesterol and phospholipid can be removed, while loss of ca. 50% cholesterol and phospholipid from ghosts can be achieved with aqueous solutions containing ethylenediamine tetraacetate. No significant loss of membrane protein was encountered even when almost half of the lipid had been removed from the ghosts. Phospholipid classes were removed to different extents with different wash solutions. Lipid loss from ghosts can be prevented, in part, by adding 0.5% albumin or calcium to wash solutions containing ethylenediamine tetraacetate. These findings contrast a report where insignificant lipid loss was noted in the preparation of hemoglobin-free human erythrocyte membranes, but agree with results reported for bovine red cell ghosts.     

A role for phospholipid polyunsaturation in modulating membrane protein function

Visual transduction is one of the best characterized G protein—coupled signalling systems. In addition, about 50% of the disk membrane phospholipid acyl chains are 22:6n-3, making this system ideal for determining the role of polyunsaturation in modulating membrane-signalling systems. The extent of formation of metarhodopsin II (MII), the G protein—activating photointermediate of rhodopsin, was studied in phospholipid vesicles composed of a variety of phosphatidylcholines, differing in their acyl chain composition at thesn-2 position. The amount of MII formed increased progressively with the level of acyl chain unsaturation at thesn-2 position. The effect of added cholesterol was to reduce the amount of MII formed. The acyl chain packing free volume of the rhodopsin containing lipid vesicles was characterized by a fractional volume parameter f_v derived from measurements of the time-resolved fluorescence anisotropy decay of the hydrophobic membrane probe 1,6-diphenyl-1,3,5-hexatriene. The relationship amongsn-2 acyl chain unsaturation, cholesterol content, and MII formation is explained on the basis of variation in f_v with bilayer lipid composition and a novel model for the packing of phospholipids containing polyenoic acyl chains, such as 22:6n-3.     

Nutritional effects of mustard seed protein detoxified with aqueous isopropanol in young rats

The effect of removing anti‐nutritional factors from n‐hexane‐extracted mustard meal using 80% isopropanol (to reduce thioglucosides, phenolics, etc.) on growth, food efficiency ratio, serum and liver lipid profiles and protein content of young rats was examined. For this n‐hexane‐extracted mustard meal was extracted with 80% isopropanol giving a fraction with 68% protein and low residual thioglucoside (0.5%) as well as phenolic (0.3%) content. This isopropanol‐extracted mustard seed protein fraction reduced the growth of young rats slightly when compared with casein. The food efficiency ratio between rats fed isopropanol‐extracted mustard seed protein or casein did not differ, nor did the protein composition affect serum total cholesterol, triglyceride, HDL‐cholesterol, LDL‐cholesterol, VLDL‐cholesterol and LDL‐C/HDL‐C ratio. However, rats fed isopropanol‐extracted mustard seed protein showed a significantly lower (p<0.05) liver cholesterol concentration than rats fed casein. Liver triglyceride and phospholipid concentrations did not differ between rats fed the two proteins, nor was serum protein affected. This study indicates that extraction of hexane‐extracted mustard meal with 80% isopropanol reduced a number of anti‐nutritional factors like thioglucoside and phenolics. Furthermore the nutritional quality of mustard seed protein fraction is comparable to casein in respect to growth, food efficiency ratio, serum lipid and protein concentrations and organ weights.     

Intestinal cholesterol uptake from phospholipid vesicles and from simple and mixed micelles

This study was undertaken in vitro to examine the rat jejunal uptake of cholesterol from phospholipid vesicles and from mixed bile salt micelles, under conditions of low effective resistance of the intestinal unstirred water layer. Cholesterol uptake J_d, occurred from vesicles only when the cholesterol: phospholipid ratio was high. The addition of phospholipid (PL) to micelles comprising 20 mM taurodeoxycholic acid (TDC) extended the concentration of cholesterol, beyond which the relationship between cholesterol concentration and uptake remained linear. When the concentration of cholesterol in the bulk phase was held constant and the concentration of TDC or of PL added to the TDC was increased, there was a decline in cholesterol uptake; this effect was masked when the concentration of TDC was high, or when higher concentrations of PL were added to the mixed micelle. When increasing concentrations of palmitic acid were added to mixed micelles composed of cholesterol, TDC and PL, the uptake of cholesterol decreased; in contrast, cholesterol uptake progressively increased when palmitic acid was added to simple TDC micelles. The results suggest that the mechanism responsible for cholesterol uptake may vary, depending on the nature of the constituents of the micelle, and it is proposed that PL inhibits the intestinal uptake of cholesterol by altering the partitioning of cholesterol out of the micelle.     

Cholesterol blocks the disordering effects of ethanol in biomembranes

To assess the relation between the physical order of a membrane and its sensitivity to ethanol, we enriched biomembranes with cholesterol, both in vivo and in vitro. Japanese quail of the SEA line (selectively bred for susceptibility to experimental atherosclerosis) were treated for 9 to 16 weeks with a diet that contained 2% cholesterol. This regimen increased the cholesterol content of serum and erythrocytes. The cholesterol content of brain synaptosomal plasma membranes (SPM) was unaffected by the high cholesterol diet. In other experiments, isolated mouse synaptosomal plasma membranes were incubated with cholesterol/phospholipid (C/P) vesicles; different amounts of cholesterol were transferred according to the sterol content of the donor vesicles. Membrane order was determined in both types of membranes by a sensitive electron paramagnetic resonance (EPR) technique. The order parameter with 5- and 12-doxylstearic acid increased along with the cholesterol content. As expected, ethanol disordered membranes (decreased the order parameter) in a concentration-related manner. The slope of the concentration response curve was less steep in high cholesterol than low cholesterol membranes, indicating that cholesterol enrichment partially blocks the membrane action of ethanol in both types of membranes.