Cholesterol metabolism and sterol carrier protein-2 (non-specific lipid transfer protein)

Hepatic sterol carrier protein-2 significantly enhances the microsomal conversion of cholesterol to 7 alpha-hydroxy-cholesterol. In the present work we have attempted to correlate the hepatic content of sterol carrier protein-2 with bile acid formation. We have determined the amount of this protein in a variety of physiological and experimental conditions, in which the rate of bile acid synthesis varies over a wide range, viz. during fetal development, in inbred strains of rats with different rates of bile acid synthesis, and in rats fed diets containing drugs which modify the rate of bile acid synthesis. The outcome of these experiments does not support the idea that sterol carrier protein-2 has any association with bile acid synthesis. From our data we further conclude that hepatic sterol carrier protein-2 is an adaptable protein because its level increases during development from the fetal to the post-weaning stage of the rat and since it can be modulated by oral administration of certain drugs. Furthermore, it is demonstrated that the level of sterol carrier protein-2 varies between six inbred strains of rats.     

Intracellular redistribution of SCP2 in Leydig cells after hormonal stimulation may contribute to increased pregnenolone production

Sterol carrier protein2 (SCP2) also designated non specific lipid transfer protein (nsL-TP), added to tumour Leydig cell mitochondria as a pure compound or in cytosolic preparations, stimulates pregnenolone production two- to three-fold. This stimulation can be abolished by addition of anti rat SCP2 but not by preimmune IgG-antibodies. SCP2- levels in the cytosol are increased in less than two minutes after addition of lutropin (LH). This increased SCP2 level may contribute to stimulation of steroid production in intact cells. After hormonal stimulation the subcellular distribution of SCP2 changes. A two-fold increase of SCP2- levels in the supernatant fraction and four-fold decrease in extracts of the particulate fraction was observed 30 min after stimulation of tumour Leydig cells with LH and subsequent fractionation. This apparent shift of SCP2 can be explained by an altered association with membranes or a true relocation of the protein from the particulate to the supernatant fractions under the influence of the hormone.     

Differentiation of keratinocytesin vitro: a new culture vessel mimicking thein vivo situation

A culture vessel consisting of two independent chambers separated only by the growth substrate is described. Cells may be cultured on both sides of the growth substrate. Culture medium and gas exposure can independently be controlled in both compartments. Human hair follicles have been used as source of keratinocytes and the bovine eye lens capsule has been explored as growth substrate. The presence of 5% CO₂ in air in the lower compartment appears to have a significant effect on the morphology of the cultures. When the cultures are being exposed to air with 5% CO₂, the culture medium being applied in the lower compartment, formation of corneocytes characteristic for adult stratum corneum is induced, as evidenced by light and electron microscopy. To the knowledge of the authors, this stage of differentiationin vitro has not been obtained with previously described systems. Differentiation of the lower cell layers has been characterised with specific antibodies. The possible use of the system for applied and pure scientific research is discussed.     

An improved technique for the demonstration of glycogen depleted skeletal muscle fibres

Several technical difficulties have been overcome in the use of Lowicryl 4KM resin. In order to embed and section tissue satisfactorily in the resin, it has been found necessary to thoroughly degass the resin before infiltration and polymerisation. After irradiation with UV light, the blocks are further polymerised by exposure to daylight for 2-3 weeks and then stored under partial vacuum over dessicant.     

Characterization of the non-specific lipid transfer protein EP2 from carrot (Daucus carota L.)

The extracellular protein EP2 was previously identified as non-specific lipid transfer protein based on its cDNA-derived amino acid sequence. Here, the purification of the EP2 protein from the medium of somatic embryo cultures is described. After two cycles of ion-exchange and gel permeation chromatography, a single silver-stained protein band with an apparent molecular mass of 10 kDa was observed on SDS-PAGE. This protein band was recognized by the antiserum raised against a EP2--galactosidase fusion-protein. Employing a fluorescent phospholipid analog, it was shown that the purified EP2 protein is capable of binding phospholipids and is able to enhance their transfer between artificial membranes. Employing a gel permeation assay, it could be demonstrated that the EP2 protein is also capable of binding palmitic and oleic acid as well as oleyl-CoA. Because in plants these fatty acids are used as precursor molecules for cutin, these results are in support of the proposed role of the EP2 protein to transport cutin monomers from their site of synthesis through the cell wall of epidermal cells to sites of cutin polymerization.     

The protein-mediated net transfer of phosphatidylinositol in model systems

The phospholipid monolayer technique has been used to study the transfer activity of the phospholipid exchange protein from beef brain. In measuring the transfer between a monolayer consisting of equimolar amounts of phosphatidylcholine and phosphatidylinositol and liposomes consisting of 98 mol% phosphatidylcholine and 2 mol% phosphatidylinositol, the beef brain protein demonstrates an 8-fold higher transfer activity for phosphatidylinositol than for phosphatidylcholine. Under similar conditions the phosphatidylcholine exchange protein from beef liver showed a great preference for phosphatidylcholine. Phosphatidylcholine liposomes devoid of phosphatidylinositol still functioned as receptors of phosphatidylinositol when the beef brain exchange protein was present. This indicates that this protein can catalyse a net transfer of phosphatidylinopsitol. Binding of both phosphatidylinositol and phosphatidylcholine to the beef brain protein was shown.     

The Social Structure of Inachus phalangium,a Spider Crab Associated with the Sea Anemone

The behaviour of the spider crab Inachus phalangium (Fabricius, 1775), which lives in association with the sea anemone Anemonia sulcata (Pennant), was studied in the field. The crab was found in the littoral zone of the Mediterranean Sea near Banyuls sur Mer, France, in the whole depth range studied (0.5–25 m). The crabs had a long-lasting association with individual Anemonia sulcata, occasionally with Aiptasia mutabilis. Most crabs were found in association with the same anemone for several days, some crabs were found in association with the same anemone for longer than one month. In the areas studied, on average 65 Inachus phalangium were found on 100 anemones. Crabs released in the vicinity of anemones moved towards them and entered them. Inachus phalangium could walk between the tentacles of Anemonia sulcata and Aiptasia mutabilis without eliciting feeding reactions of the anemone. The crabs left the anemones for moulting. After moulting masking material was removed from the exuvia and used again. The animals returned into an anemone while still soft. Material used for masking, usually algae, could be picked off the body and eaten. Masking material may be a food reservoir in addition to providing camouflage. Anemones were left only during night-time. The crabs left their anemone to moult, to feed in the vicinity, fleeing from larger conspecifics, and to migrate to a different anemone. Outside the anemone's protection Inachus was eaten by several species of fish. Individuals appeared to avoid each other. 57% of all animals were found alone on an anemone. Large males and females were more frequently found alone than were small males and females. Fights were observed between members of the same and of the opposite sex. During fights, legs and claws could be torn off. Adult males migrated more often between anemones and moved over larger distances when migrating than did adult females. Adult males probably migrated in search of sexually mature females. Such a roving strategy is evolutionarily stable only when the higher costs (in terms of energy expenditure and mortality) are compensated for by a higher number of offspring than produced in the alternative, pair-bonding strategy.     

Affinity of phosphatidylcholine molecular species for the bovine phosphatidylcholine and phosphatidylinositol transfer proteins. Properties of the sn-1 and sn-2 acyl binding sites

Both the phosphatidylcholine transfer protein (PC-TP) and the phosphatidylinositol transfer protein (PI-TP) act as carriers of phosphatidylcholine (PC) molecules between membranes. To study the structure of the acyl binding sites of these proteins, the affinity of 32 distinct natural and related PC molecular species was determined by using a previously developed fluorometric competition assay. Marked differences in affinity between species were observed with both proteins. Affinity vs lipid hydrophobicity (determined by reverse-phase HPLC) plots displayed a well-defined maximum indicating that the acyl chain hydrophobicity is an important determinant of binding of a phospholipid molecule by these transfer proteins. However, besides the overall lipid hydrophobicity, steric properties of the individual acyl chains contribute considerably to the affinity, and PC-TP and PI-TP respond differently to modifications of the acyl chain structure. The affinity of PC-TP increased steadily with increasing unsaturation of the sn-2 acyl moiety, resulting in high affinity for species containing four and six double bonds in the sn-2 chain, whereas the affinity of PI-TP first increased up to two to three double bonds and then declined. These data, as well as the distinct effects of sn-2 chain double bond position and bromination, indicate that the sn-2 acyl chain binding sites of the two proteins are structurally quite different. The sn-1 acyl binding sites are dissimilar as well, since variation of the length of saturated sn-1 chain affected the affinity differently. The data are discussed in terms of the structural organization of the sn-1 and sn-2 acyl binding sites of PC-TP and PI-TP.(ABSTRACT TRUNCATED AT 250 WORDS)