Structure and thermotropic phase behaviour of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes.

Detergent-resistant membrane raft fractions have been prepared from human, goat, and sheep erythrocyte ghosts using Triton X-100. The structure and thermotropic phase behaviour of the fractions have been examined by freeze-fracture electron microscopy and synchrotron X-ray diffraction methods. The raft fractions are found to consist of vesicles and multilamellar structures indicating considerable rearrangement of the original ghost membrane. Few membrane-associated particles typical of freeze-fracture replicas of intact erythrocyte membranes are observed in the fracture planes. Synchrotron X-ray diffraction studies during heating and cooling scans showed that multilamellar structures formed by stacks of raft membranes from all three species have d-spacings of about 6.5 nm. These structures can be distinguished from peaks corresponding to d-spacings of about 5.5 nm, which were assigned to scattering from single bilayer vesicles on the basis of the temperature dependence of their d-spacings compared with the multilamellar arrangements. The spacings obtained from multilamellar stacks and vesicular suspensions of raft membranes were, on average, more than 0.5 nm greater than corresponding arrangements of erythrocyte ghost membranes from which they were derived. The trypsinization of human erythrocyte ghosts results in a small decrease in lamellar d-spacing, but rafts prepared from trypsinized ghosts exhibit an additional lamellar repeat 0.4 nm less than a lamellar repeat coinciding with rafts prepared from untreated ghosts. The trypsinization of sheep erythrocyte ghosts results in the phase separation of two lamellar repeat structures (d=6.00; 5.77 nm), but rafts from trypsinized ghosts produce a diffraction band almost identical to rafts from untreated ghosts. An examination of the structure and thermotropic phase behaviour of the dispersions of total polar lipid extracts of sheep detergent-resistant membrane preparations showed that a reversible phase separation of an inverted hexagonal structure from coexisting lamellar phase takes place upon heating above about 30 degrees C. Non-lamellar phases are not observed in erythrocytes or detergent-resistant membrane preparations heated up to 55 degrees C, suggesting that the lamellar arrangement is imposed on these membrane lipids by interaction with non-lipid components of rafts and/or that the topology of lipids in the erythrocyte membrane survives detergent treatment.

Comparative lipid analysis and structure of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes.

The lipid composition and structure of detergent-resistant membrane rafts from human, goat, and sheep erythrocytes is investigated. While the sphingomyelin:cholesterol ratio varied from about 1:5 in human to 1:1 in sheep erythrocytes a ratio of 1:1 was found in all raft preparations insoluble in Triton X-100 at 4 degrees C. Excess cholesterol is excluded from rafts and saturated molecular species of sphingomyelin assayed by gas chromatography-mass spectrometry determines the solubility of cholesterol in the detergent. Freeze-fracture electron microscopy shows that vesicles and multilamellar structures formed by membrane rafts have undergone considerable rearrangement from the original membrane. No membrane-associated particles are observed. Synchrotron X-ray diffraction studies showed that d spacings of vesicle preparations of rafts cannot be distinguished from ghost membranes from which they are derived. Dispersions of total polar lipid extracts of sheep rafts show phase separation of inverted hexagonal structure upon heating and this phase coexists with multilamellar structures at 37 degrees C.

Lipid composition of membrane domains.

The isolation of subfractions of cell membranes on the basis of their solubility in non-ionic detergents has led to the discovery of lipid domain structure in membranes. Detergents used for this purpose include Triton, Brij, Lubrol and CHAPS. Different lipid constituents are known to resist solubilization by different detergents and the resulting fractions may associate with different membrane proteins. In general, the detergent-resistant membrane fractions tend to be dominated by saturated molecular species of sphingomyelin and phosphatidylcholine and invariably include significant proportions of cholesterol. The lipid composition is consistent with formation of liquid-ordered phases. The present evidence favours a model in which the lateral segregation of membrane proteins takes place on the basis of their affinity for liquid-ordered lipid domains within the membrane.

Ceramides increase the activity of the secretory phospholipase A2 and alter its fatty acid specificity.

Modulation of human recombinant secretory type II phospholipase A(2) activity by ceramide and cholesterol was investigated using model glycerophospholipid substrates composed of phosphatidylethanolamine and phosphatidylserine dispersed in aqueous medium. Enzyme activity was monitored by measurement of released fatty acids using capillary GC-MS. Fatty acids from the sn-2 position of the phospholipids were hydrolysed by the enzyme in proportion to the relative abundance of the phospholipid in the substrate. Addition of increasing amounts of ceramide to the substrate progressively enhanced phospholipase activity. The increased activity was accomplished largely by preferential hydrolysis of polyunsaturated fatty acids, particularly arachidonic acid, derived from phosphatidylethanolamine. The addition of sphingomyelin to the substrate glycerophospholipids inhibited phospholipase activity but its progressive substitution by ceramide, so as to mimic sphingomyelinase activity, counteracted the inhibition. The presence of cholesterol in dispersions of glycerophospholipid-substrate-containing ceramides suppressed activation of the enzyme resulting from the presence of ceramide. The molecular basis of enzyme modulation was investigated by analysis of the phase structure of the dispersed lipid substrate during temperature scans from 46 to 20 degrees C using small-angle synchrotron X-ray diffraction. These studies indicated that intermediate structures created after ceramide-dependent phase separation of hexagonal and lamellar phases represent the most susceptible form of the substrate for enzyme hydrolysis.

Integrated dental management care.

In the early 1980s, the Federal Health Care Finance Administration (HCFA) sponsored four Medicare waiver programs nationwide to determine the economic feasibility of providing oral health services to Medicare-eligible, low-income elderly people. One such project spawned a pioneering New York-based dental care management organization, Integrated Dental Management, Inc. (IDM). IDM, as its name implies, integrates dental services in an interdisciplinary program of health care. This paper describes the history, organization, challenges and accomplishments of this unique modality for improving access to oral health care for elderly people. This will be presented in the context of the Surgeon General's report, "Oral Health in America," as the practice of dentistry enters a new century.

Oral health care for the stroke survivor.

Despite a dramatic decrease in the incidence of stroke during the past decade, cerebrovascular accidents remain the third leading cause of death among the elderly. Stroke survivors, on the other hand, often demonstrate unique and challenging oral problems. This article delineates the etiology of cerebrovascular accidents, describes its orofacial manifestations in the stroke survivor, and offers guidelines for oral rehabilitation and management of affected individuals in the community or in an institutional setting.

An unusual presentation of opalescent dentin and Brandywine isolate hereditary opalescent dentin in an Ashkenazic Jewish family.

Two children of Austrian Ashkenazic Jewish background, related as second cousins, have a variant of opalescent dentin in their deciduous teeth. This has been classified by Witkop as Brandywine isolate hereditary opalescent dentin and by Shields as dentinogenesis imperfecta type III. One of the children also has dysmorphic facial features, seizures, and severe mental retardation. Her mother has dysmorphic facial features and mild mental retardation. The mothers of both children and several other family members have classic opalescent dentin (dentinogenesis imperfecta type II). Radiographs of the deciduous and permanent dentitions of one mother showed obliterated pulp chambers. Confirmation of obliterated pulp chambers in the deciduous teeth of the mother of a child with Brandywine isolate hereditary opalescent dentin makes it unlikely that classic opalescent dentin and Brandywine isolate hereditary opalescent dentin are separate genetic disorders. Evidence from this family supports the hypothesis that Brandywine isolate hereditary opalescent dentin is a variant of opalescent dentin.