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In two-component phosphatidylcholine bilayers with coexisting liquid and P beta' gel phases, the distribution between phases of low concentrations of glycosphingolipids can be determined by freeze-etch electron microscopy after labeling the glycolipid with a suitable protein. We have found that the distribution depends upon the glycosphingolipid species (Rock, P. et al., (1991) Biochemistry 30, 19-25). Using this technique with cholera toxin as the protein label and bilayers formed from dipalmitoyl- and dielaidoylphosphatidylcholine (1:1) containing < 1 mol% GM1, we have studied the distribution of a family of GM1 homologues differing in the acyl chain and sphingoid base moieties. The GM1 preference for the P beta' ripple phase decreases with decreasing acyl chain length and increasing unsaturation. GM1 with either a C18:1 or C20:1 sphingoid base shows similar distributions in liquid and gel phases. When the molecules are preferentially found in the P beta' phase, they are positioned along unique loci in both A and A/2 forms of the ripple structure. This localization and acyl chain dependence reflect the volume, shape and localization of molecular packing defects in the P beta' phase. The ganglioside inclusions stabilize the P beta' phase and form compositional domains of unique topography.

We demonstrate that supported synthetic phospholipid bilayers, which are stabilized by lateral cross-linking in both leaflets, can be used for specimen preparation for atomic force microscopy of purified membrane proteins with high stability and excellent reproducibility under water or low-salt buffer. A bilayer containing 1,2-dipentacosa-10,12-diynoyl-phosphatidylcholine and 20 mol% ganglioside (GMI) was transferred onto the surface of mica from a Langmuir trough. Cholera toxin, both the B-subunit and the complete molecule randomly bound to the gangliosides, were imaged by atomic force microscopy in solution with a resolution of better than 2 nm. The pentameric structure of the B-subunit oligomers was well resolved. This result indicates that, with this preparation procedure, other membrane proteins may be studied at intermediate to high resolution under physiologically relevant conditions without the need for crystallization.

Multilamellar liposomes composed of 1:1 dielaidoylphosphatidylcholine: dipalmitoylphosphatidylcholine at 20 degrees C contain laterally separated gel and liquid-crystalline phases that can be identified by electron microscopy in freeze-etch replicas on the basis of their distinctive morphology. Visualization of marker proteins that specifically bind to glycosphingolipids included in these liposomes has revealed that, at 1 mol % or less, the ganglioside GM1 and the neutral asialo-GM1 derived from it are localized within the gel-phase regions exclusively. Increasing the mole fraction of the glycosphingolipids results in the appearance of marker in the fluid-phase regions. Another neutral glycosphingolipid, Forssman, does not display a phase preference and is found in both phases at a low mole percent. The phase preference of these three glycosphingolipids depends primarily upon interactions between the hydrophobic moieties of these molecules and the matrix phosphatidylcholines.

The techniques of ultrafast freezing and freeze-etch electron microscopy have been successfully employed to visualize IgG molecules and Fab fragments specifically bound to the neutral glycosphingolipids Forssman and asialo-GM1 incorporated into phosphatidylcholine liposomes. Monovalent Fab is the superior marker because of its small size and because it does not cause liposomal aggregation with concomitant glycolipid reorganization. Analysis of Fab labeling of liposomes containing these neutral glycosphingolipids leads to the conclusion that the Forssman glycosphingolipid is dispersed in clusters of not more than several molecules when present at low mole fraction in fluid-phase 1-palmitoyl-2-oleoylphosphatidylcholine liposomes. In contrast to this, asialo-GM1 under the same conditions is present in clusters of about 15 molecules in this phospholipid matrix.

Atomic force microscopy (AFM) has been successfully applied to image many biological specimens such as proteins, DNA molecules, membrane fragments, synthetic lipid bilayers and monolayers and even whole cells, with a resolution ranging from hundreds of nm to sub-nm. In particular, AFM has a unique capability of operating in solutions; thus, proteins in situ can be directly imaged to study their functional relationships to the structure. Because in AFM, the contrast reflects direct force interactions between the probe and the specimen surface, the resolution of AFM images is highly dependent on the specimen being studied. We, and others, found that atomic resolution images on hard crystal surfaces are among the easiest to be obtained in AFM. For example, periodic surface atomic structures with hexagonal, rhombohedral, and cubic lattices were clearly resolved by AFM [9-12]. Using the NanoScope II AFM with various home-made accessories, such as motion stages and fluid cells, we have studied several different biological materials with a resolution from tens of nm to subnm.

The development of sarcoplasmic reticulum membranes was studied in vivo and in tissue culture in chicken pectoralis muscle cells. The concentration of the calcium- and magnesium-activated ATPase measured by selective labeling of the enzyme with [32P]ATP in whole muscle homogenates was found to increase in developing chicken pectoralis muscle in vivo from 0.01 nmol/mg of protein in 12-day embryos to 0.3 to 0.4 nmol/mg of protein in 1-month-old chicks, where it constitutes about 3% of the total protein content of muscle. In cultured muscle cells the concentration of calcium-sensitive phosphoprotein increased from 0.015 nmol/mg of protein at 2 days to 0.04 to 0.05 nmol/mg of protein after 5 days of culture. This amount represents about 0.5% of the protein content of the muscle cells. The accumulation of Ca2+ transport ATPase began during fusion and continued with a linear rate during 8 days of culture. The density of 75 A intramembranous particles seen by freeze-etch electron microscopy on fracture faces of sarcoplasmic reticulum membranes is about 4,000/mum2 in adult chick pectoralis muscle but only 400/mum2 in cultured muscle cells in rough proportion to the concentration of Ca2+-sensitive phosphoprotein. The Ca2+, Na+, and K+ concentration of the medium and addition of ouabain, caffeine, or the calcium ionophores A23187 and X537A sharply influence the concentration of calcium transport ATPase in cultured muscle cells, parallel with their effect upon cell fusion and growth. These observations are consistent with the proposition that the gene expression leading to the accumulation of Ca2+ transport ATPase during development in culture may be regulated by intracellular ion concentrations.

A 37-year-old female with thrombocytopenia since childhood developed diffuse osteosclerosis followed by bone marrow aplasia 23 years after Thorotrast administration. Tissue deposition of Thorotrast was documented by radiospectroscopy (liver, bone) and autoradiography (bone). Bone marrow failure in this patient was clinically ascribed to diffuse myelofibrosis, but at autopsy marrow fibrosis was only focal. An unsuspected bone mineralization defect was manifested by thick osteoid seams. It is suggested that osteosclerosis, osteomalacia, and bone marrow aplasia represent a combined experession of Thorotrast cytotoxicity.

A radioactively-labelled glycosphingolipid, asialo-GM1, has been incorporated into phosphatidylcholine multilamellar vesicles. After incubation with ferritin-Ricinus communis agglutinin 60 (RCA 60) conjugate at different temperatures, the vesicles were separated from the conjugate by discontinuous density gradient ultracentrifugation. Measurement of the distribution of the radioactively-labelled asialo-GM1 in the pelleted conjugate fraction and freeze-etch electron microscopy of the vesicle fraction indicate that the decrease in labelling of asialo-G M 1 -containing vesicles by ferritin-RCA 60 conjugate with increasing temperatures (Tillack, T.W., Wong, M., Allietta, M. and Thompson, T.E. (1982) Biochim. Biophys. Acta 691, 261–273) reflects a decrease in apparent binding affinity rather than an ability of the conjugate to extract glycolipid from the phospholipid bilayer after binding.

A retrospective analysis of 116 primary cases of stage I, II and III-A Hodgkin's disease demonstrated 27 failures, fourteen confirmed by biopsy. Twenty of the 27 (74%) were in lymph node areas only and seven had extranodal extensions. The most frequent site of failure was the hilar nodes and contiguous lung. The majority of failures (78%) occurred within 30 months of treatment and the cause of failure determined in 23 (85%), sixteen of which were due to technical errors of irradiation. Analysis of the dosimetry in the mediastinal, hilar nodes and contiguous lung failures revealed three factors which may have contributed to a low dose. These factors are: 1) the equivalent square, 2) off-axis beam diminution, and 3) the anteroposterior dose profile. Combined radiation therapy and chemotherapy was more effective than either modality alone in the treatment of these failures.

A patient, CAL, with gamma heavy chain disease is presented who has had a complete remission lasting over 2 yr with combination chemotherapy consisting of pulsatile cyclophosphamide and prednisone. The patient exhibited many features of an atuoimmune process including a vasculitis, low serum complement levels, a positive antiglobulin (Coombs) test, Raynaud's phenomenon, and keratoconjunctivitis sicca. The CAL paraprotein was found to have several previously undescribed characteristics. It reacted with antisera to Fd, Fab, and Fab', suggesting that most of the Fd portion of the molecule was intace. CAL protein consists of two polypeptide chains of molecular weight 49,000 covalently linked to form a dimer of 95,000 molecular weight. The covalent linkage suggests that the hinge region of this gamma heavy chain is intact.

We have previously demonstrated that lentil phytohemagglutinin (lentil-PHA) binds to human platelet membranes without causing either aggregation or the release reaction. When platelets are treated with thrombin, there is an increase in lentil-PHA binding suggesting the appearance of new receptor sites on the cell surface. We prepared a lentil-PHA-ferritin conjugate using affinity chromatography which was used to saturate cell surface receptor sites. Studies using this conjugate suggest that thrombin causes a complex change in the platelet surface involving a decrease in the number of lentil-PHA receptor sites on the external platelet surface with a marked increase in sites within the center of the canalicular system. These increased sites may result from fusion of granule membranes with the canalicular membranes during the secretion process. There is no obvious relationship between lentil-PHA receptor sites and intramembranous particles.

Small sonicated dipalmitoylphosphatidylcholine vesicles when incubated at 4 degrees C and high concentrations are shown to fuse completely to vesicles about 700-A diameter in 7 days, and these further fuse to about 950 A diameter vesicles after 3-4 weeks. The 950 A diameter vesicles are spherical, homogeneous, mostly unilamellar, have an internal aqueous space about 10 times that of small vesicles, and are stable for at least 6 months. The 950-A vesicles are characterized by agarose gel chromatography, freeze-fracturing electron microscopy, trapped volume measurements, differential scanning calorimetry, and diphenylhexatriene fluorescence polarization.

Molecules of the ganglioside GM1 are randomly distributed in liquid-crystalline 1-palmitoyl-2-oleoyl phosphatidylcholine bilayers. This conclusion is based on a freeze-etch electron microscopic study using ferritin-conjugated cholera toxin and cholera toxin alone as ganglioside labels. The average number of GM1 molecules under a label is calculated by a novel method from the dependence of the fraction of bilayer area covered by the label on the mole fraction of GM1 in the bilayer.

The distribution of specific glycoprotein receptors on the external surfaces of red cells was mapped, by the freeze-etching technique, to determine if the receptors coincided with the underlying 75-A intramembranous particles. Phytohemagglutinin, ferritin-conjugated phytohemagglutinin, and influenza virus were used as labeling agents since they can be seen by freeze-etching techniques and each reacts with a different site on the same glycoprotein molecule. The distribution of these labels was studied on intact human red cells, isolated ghost membranes, and trypsin-treated ghost membranes. The results show that the receptors for these labels are distributed uniformly over the surfaces of normal red cell membranes in the same apparent distribution as that of the 75-A particles within the membrane. The association between the external receptors and the underlying particles is especially evident when trypsin-treated ghost membranes are labeled: the labeled receptor sites and the intramembranous particles both form sharply defined, reticulated networks, which overlap. These results provide further support for the idea that membrane-bound glycoproteins are oriented so that their carbohydrate-rich segments, which bear the antigenic sites and receptors, are exposed to the external medium, while hydrophobic segments of the same molecules interact with lipids, and possibly other proteins, to form the intramembranous particles.

Glycoproteins were isolated from membrane preparations of human breast cancer using lithium di-iodosalicylate followed by phenol partition as described for the isolation of CEA from colonic carcinoma. The glycoprotein isolated from breast cancer-(BCGP) differed from colonic CEA in extraction yield, antigenicity, molecular weight on SDS acrylamide gel electrophoresis and on immunoelectrophoresis. Rabbit anti-serum raised against BCGP and absorbed with red blood cells and normal breast tissue showed a reaction of identity on Ouchterlony immunodiffusion between BCGP and CEA. However, antiserum to CEA absorbed with red blood cells and normal colonic mucosa showed cross-reaction of BCGP with CEA. The anti-BCGP activity in anti-CEA could be removed by absorption with BCGP, leaving activity directed only against CEA. Therefore, BCGP appears to be a CEA-like antigen that shares antigenic determinants with CEA and may be responsible for the elevated radioimmunoassays for CEA in breast cancer patients. Whether there is a breast-cancer-specific antigen associated with the cell membrane remains to be demonstrated.

During the initial 5 minutes of T2 bacteriophage infection of Escherichia coli, there is substantial incorporation of labeled guanine into the transfer RNA (tRNA) fraction, strongly suggesting that new tRNA is synthesized early in infection. In this 5-minute period the amount of labeled guanine incorporated into the tRNA fraction of T2-infected bacteria is 23% of that in uninfected bacteria during an equivalent period. The tRNA fractions from T2-infected bacteria and uninfected bacteria were compared by pulse-labeling cultures of each for 5 minutes with 3H and 14C isotopes of guanine and partially resolving a mixture of the doubly labeled tRNA species by reversed phase chromatography. The chromatographic pattern of the labeled tRNA from uninfected cultures of E. coli was very similar to that of nonlabeled carrier tRNA from uninfected cultures. tRNA prepared from pulse-labeled T2-infected cultures of E. coli had a chromatographic pattern that was radically different from that of tRNA prepared from uninfected cultures. These results indicate either that the tRNA synthesized after T2 infection of E. coli represents species of tRNA entirely different from those made in uninfected bacteria, or that relative proportions of the different species made are greatly altered after phage infection, some being made preferentially and others in reduced amounts. Evidence is presented that a substantial amount of the material in the tRNA fraction made during T2 infection can accept amino acids.

The disappearance and reappearance of the P beta' ripple in multilamellar liposomes of dipalmitoylphosphatidylcholine (DPPC) has been examined by freeze-etch electron microscopy. The presence of less than 10 mol% of various glycosphingolipids or cholesterol in the liposomes markedly increases the time required for ripple disappearance when the vesicles are cooled from 38 degrees C to 30 degrees C, as compared to the pure phospholipid. Once the ripples have begun to disappear in the two-component vesicles, they do not uniformly reappear until the system is heated above the main transition of DPPC and allowed to cool into the pretransition region. These results suggest that the long time for ripple disappearance in the two-component systems reflects a slow molecular reorganization process which occurs when the systems are forced to change from the P beta' gel to the L beta' gel by a temperature downshift.

An affinity purified monovalent ferritin conjugate of Ricinus communis agglutinin (RCA 60) is used with freeze-etch electron microscopy to study the ultrastructural localization of the glycosphingolipid asialo-G M1 in multilamellar phosphatidylcholine liposomes. Dimyristoylphosphatidylcholine (DMPC) liposomes containing up to 20 mol% asialo-G M1 and quenched below the main transition temperature show a striking linear localization of ferritin-RCA 60 between phospholipid ridges. The glycosphingolipid localization is similar to that postulated for up to 20 mol% cholesterol in pure phosphatidylcholine bilayers by Copeland, B.R. and McConnell, H.M. (Biochim. Biophys. Acta, 599, 95–109 (1980)). Above the main phase transition temperature, asialo-G M1 appears to be organized into clusters, especially in palmitoyloleoylphosphatidylcholine (POPC) liposomes. This clustered distribution of glycosphingolipids seen above the phase transition temperature suggests that this type of lipid may exhibit compositional domain structure in biological membranes.

Glycosphingolipids, in contrast to glycerol-based lipids, are relatively minor components of mammalian cell membranes. They are, however, confined to the external surface of the plasma membrane and in this surface may collectively be a major component (1–4). In some cell types very small amounts of these lipids have been found associated with Golgi membranes, their probable site of biosynthesis (1). Their location on the trans-cytoplasmic side of the plasma membrane causes them, together with glycosylated membrane proteins, to be the primary components of the cell to interact with the molecules and other cells of the immediate environment. Thus specific glycosphingolipids have been shown to serve as receptors for toxins, viruses and some hormones (2,5–11). They have long been known to act as antigenic determinants and to mediate immune responses (11–13). There is much evidence to suggest that glycosphingolipids play a role in cell-cell interaction and recognition (7). Alterations in the amounts and types of these lipids on the cell surface are very often associated with growth, differentiation, development, aging (7,14,15), and with oncogenic transformation (11,16). Although there is considerable information about the molecular structure of many glycosphingolipids, relatively little is known about the organization of molecules of this class in phospholipid bilayers and in the bilayers of biological membranes. It seems certain that their molecular organization is a critical parameter underlying many of the functions of glycosphingolipids (17).

Freeze-etch electron microscopy has been utilized to localize the binding sites for the Ricinus communis, Agaricus bisporus and wheat germ lectins on human erythrocyte membranes and to determine the relation of these different glycoprotein receptors to the intramembranous particles. A. bisporus lectin, which could be visualized directly on the surface of erythrocyte membranes, and ferritin conjugates of wheat germ agglutinin showed a distribution that correlates exactly with the intramembranous particles at all lectin concentrations tested. The binding sites for both of these lectins are located on the major sialoglycoprotein of the membrane. The R. communis agglutinin-ferritin conjugate which binds to receptors on membrane glycoproteins that are distinct from the major sialoglycoprotein showed a close correlation with the intramembranous particles at low lectin concentrations and a poor correlation at high lectin concentrations. High concentrations resulted in virtually complete coating of the surface of trypsinized ghosts which displayed marked aggregation of the intramembranous particles. We conclude that the intramembranous particles of erythrocyte membranes contain at least two glycoproteins and that some membrane lectin receptors are not associated with the intramembranous particles.

To determine whether epithelial plasma membrane glycolipids are polarized in a manner analogous to membrane proteins, MDCK cells grown on permeable filters were analyzed for the expression of Forssman ceramide pentasaccharide, the major neutral glycolipid in these cells. In contrast to a recent report which described exclusive apical localization of the Forssman glycolipid (Hansson, G.C., Simons, K. and Van Meer, G. (1986) EMBO J. 5, 483-489), immunofluorescence and immunoelectron microscopic staining revealed the Forssman glycolipid on both the apical and basolateral surfaces of polarized cells. Immunoblots indicated that the Forssman antigen was detectable only on glycolipids and not on proteins. Analysis of metabolically labeled glycolipids released into the apical and basal culture medium, either as shed membrane vesicles or in budding viruses, also demonstrated the presence of the Forssman glycolipid on both apical and basolateral membranes of polarized cells. Quantitation of the released glycolipid indicated that the Forssman glycolipid was concentrated in the apical membrane. These results are consistent with previous reports which described quantitative enrichment of glycolipids in the apical domain of several epithelia.

Publisher Summary This chapter describes the significance and properties of tumor specific antigens. Experiments with animals gave the earliest indications that tumors had specific antigens that could not be found in normal tissues. These animal experiments showed the existence of two types of tumor antigens. Tumors induced by chemical carcinogens usually contained antigens that were specific for each tumor. It was demonstrated that transplantation of methylcholanthrene-induced sarcomas in inbred strains of mice protected the animals from subsequent challenge by the same tumor but not from other methylcholanthrene-induced tumors. Transplantation of normal tissues from a tumor-bearing mouse did not immunize the recipient against subsequent challenge with the tumor. More direct evidence for the existence of tumor specific antigens comes from studies showing that patients with tumors have humoral antibodies to their tumors or cell-mediated immunity to tumor cell surface antigens.

A new technique is described for the extraction and purification of the carcinoembryonic antigen (CEA) from human tumors. This method involves the use of lithium diiodosalicylate for dissociation of the antigen-membrane complex. The method differs from earlier procedures in that extraction conditions do not rely on an acid precipitation step and that the yield of CEA appears to be approximately 10-fold greater. A glycoprotein immunologically similar to CEA was isolated from non-tumoral human adult colonic mucosa, suggesting that the difference between tumoral and non-tumoral tissues in this system may be quantitative rather than absolute.

Developmental changes in the sarcoplasmic reticulum and surface membranes of chicken skeletal muscles were investigated by electron microscopy. A dominant feature of freeze-etched sarcoplasmic reticulum membranes isolated from adult animals is the presence of spherical intramembranous particles of 75 A apparent diameter which are more numerous on the concave than on the convex cleaved faces. The sharp increase in the Ca2+ transport and Ca2+-sensitive ATPase activity of isolated sarcoplasmic reticulum membranes with development is accompanied by an increase in the number of 75 A particles. Corresponding changes were observed in intact muscles where the sarcoplasmic reticulum elements can be readily identified on the basis of their characteristic disposition between the myofibrils. The reasonably close correlation between Ca2+ transport activity and the intramembranous particle density during development supports earlier suggestions that the intramembranous particles may represent the Ca2+ transport system. Similar 75 A particles were also observed on the surface membrane of skeletal muscle but in lesser density than the corresponding structures in sarcoplasmic reticulum. Their number also increases with development.

Developmental changes in the protein, phospholipid, cholesterol, and fatty acid composition of skeletal and cardiac microsomes were correlated with Ca2+ transport activity in chicken embryos and during the early postnatal period. In skeletal muscle microsomes isolated from leg, superficial pectoralis, or deep pectoralis muscles, the rapid increase in the rate and extent of calcium accumulation around the time of hatching was accompanied by increase in total and Ca2+-sensitive ATPase activity, the concentration of phosphoprotein intermediate and the amount of transport ATPase enzyme determined by gel electrophoresis. The increased enzyme content of the membrane is reflected in its increased density and a marked decrease in phospholipid to protein ratio with development. The concentration of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, and phosphatidylinositol changed relatively little during the first 50 days of development but the appearance of calcium transport activity was accompanied by a marked decrease in the palmitate and increase in the linoleate content of membrane phospholipids and a decrease in cholesterol concentration. These changes were clearly apparent in purified sarcoplasmic reticulum membranes isolated by calcium loading or in less pure membrane fractions obtained by differential or sucrose gradient centrifugation. Corresponding but much less pronounced changes in the enzymatic activity of cardiac microsomes were observed during development. Although the observations are consistent with nearly all possible mechanisms of membrane assembly they may suggest that the phospholipid-rich membranes of embryonic muscle are gradually converted during development into calcium-transporting structures by stepwise insertion of the ATPase enzyme and other components of the transport system.

Previous investigations have demonstrated the presence of circular negatively stained lesions in phospholipid-cholesterol liposomes and in the cell membranes of erythrocytes that have been treated with the polyene antibiotic, filipin; these lesions are morphologically similar to those found in erythrocyte cell membranes after immune lysis. Filipin has been shown also to cause release of both glucose and macromolecular enzyme markers from liposomes that contain cholesterol. In the present study, freeze-etching was employed to determine if these lesions are holes through the membrane. Structural alterations (pits, doughnut-shaped craters and protrusions), whose formation was dependent on the presence of cholesterol, were demonstrable in liposomes after incubation with the antibiotic; analogous changes were also produced by filipin in erythrocyte membranes. However, examination of outer and inner surfaces, and outer and inner fracture faces, did not indicate that these structural alterations could be equated with transverse holes. It is, therefore, unlikely that the negatively stained lesions represent stable open channels through liposomal bilayers and erythrocyte membranes which are responsible for the escape of marker compounds and cytoplasmic constituents.