Gallium-67 localization in rat and mouse tumors.

Various nonosseous tumors in mice and rats concentrate gallium-67-ESR-586 preputial gland carcinoma, P-1798 lymphosarcoma, C3H/ HeJ mammary tumor, AKR/J thymic lymphoma, R-3259 giant cell sarcoma, Walker-256 carcinosarcoma, and a new transplantable neoplasm of rats. This last tumor showed the greatest affinity for galliun. Viable tumor cells are mainly responsible for uptake of gallium-67; autoradiographic studies indicate it is chiefly located in tumor cytoplasm.

Establishment of two parental cell lines and three clonal cell strains from rat colonic carcinoma.

Two epithelial cell lines were established from separate pools of cells that were harvested by a sequential proteolytic treatment of a single specimen of rat transplantable colonic carcinoma. The cells were small (10-micrometer diameter) and cuboidal and displayed desmosomes and light junctions. Both cell lines grew rapidly with population-doubling times of 20 to 22 hr, were near diploid in chromosome number, contained A-2 and B-7 marker chromosomes, and were tumorigenic in rats. Each was distinguishable from the other by the shape of its cell clusters in monolayer culture, its serum requirement for growth, its modal chromosome number, and its karyotypic alterations. Undifferentiated cells, cystic cells, and vacuolated cells, but not mature mucous cells, were observed in monolayer cultures. Clonal cell strains mimic the morphological and growth properties of their respective parental cell line but display unique karyotypic alterations in addition to the A-2 and B-7 marker chromosomes.

A quantitative study of the subcellular localization of 67Ga.

This paper describes a quantitative procedure for the isolation of 67 Ga-binding granules (GBG) from normal rat liver and Morris 5123C hepatoma homogenates by a combination of rate and isopyknic density gradient zonal centrifugation. Another class of GBG has been found that is much smaller than the lysosomal GBG we have previously described. These smaller particles, or microvesicles, bind the largest portion of the 67Ga found in the hepatoma whereas, in the liver, the GBG lysosomes are the major binding component. Previously, we had shown that considerably more 67Ga is taken up in hepatoma than in liver (as percentage of administered dose per g of tissue). The preferential association of 67Ga with these microvesicles in the 5123C hepatoma may be indicative of a basic difference between normal and malignant tissue.

Effects of essential fatty acid deficiency on epidermal O-acylsphingolipids and transepidermal water loss in young pigs.

Linoleate-rich O-acylglucosylceramides and acylceramides are thought to be of major significance for the physical structure and function of the epidermal permeability barrier. In the present investigation, the effects of a linoleate-free diet on O-acylsphingolipids and their associated functions were investigated. Starting at 5 days of age, male pigs were fed diets containing 12% of either lard or hydrogenated coconut oil. Transepidermal water loss was measured with an electrolytic water analyzer at weekly intervals. Pigs were killed at intervals, and epidermal lipids were isolated and analyzed. Fatty acid compositions were determined by gas-liquid chromatography (GLC). Within 2-3 weeks, pigs on the diet containing coconut oil began to display biochemical and physiological symptoms of essential fatty acid deficiency. Within 2 months, this group had extremely scaly skin and transepidermal water loss was elevated to five times that of controls. The progressive increase in transepidermal water loss correlated with replacement of linoleate by oleate in both acylceramide and acylglucosylceramide. The formation of lamellar granules and intercellular lipid sheets in the stratum corneum was not impaired in essential fatty acid deficiency as judged by electron microscopy. These results suggest that the linoleic acid normally found in the O-acylsphingolipids is not essential for formation of the epidermal membrane system. Rather, it appears that the nature of the ester-linked fatty acid in the O-acylsphingolipids regulates the permeability of this membrane system.

Covalently bound lipids in keratinizing epithelia.

Covalently bound lipids have been identified and compared in keratinizing porcine epithelia including epidermis and oral epithelium from palate and gingiva. Stratum corneum was isolated by tryptic digestion, and after extensive extraction of lipids using a series of chloroform-methanol mixtures, the residual tissue was subjected to alkaline hydrolysis to release covalently bound lipids. The lipids so released were analyzed by quantitative thin-layer chromatography. Stratum corneum from each of the three anatomical sites contained omega-hydroxyceramides, omega-hydroxyacids and fatty acids. In epidermal stratum corneum the total covalently bound lipids represented 2.4% of the dry weight of the tissue, but in the oral epithelia this figure was consistently lower: 0.24% in palatal stratum corneum and 0.20% in gingival stratum corneum. Transmission electron microscopy before and after lipid extraction confirms the presence of a lipid envelope in epidermal stratum corneum and demonstrates the absence of this structure in oral stratum corneum.

In vitro reconstitution of stratum corneum lipid lamellae.

In the final stages of differentiation in the epidermis of terrestrial mammals, lipids are extruded into the intercellular spaces. The initially extruded lipid becomes transformed into broad, multilamellar sheets that are found in the intercellular spaces throughout the stratum corneum. These lamellae display an unusual alternating broad-narrow-broad pattern of lucent bands as revealed by transmission electron microscopy (TEM). This arrangement results in two periodicities that can be measured from electron micrographs and are also evident in X-ray diffraction-5 nm (broad) and 13 nm (broad-narrow-broad). The goal of the present study was to reconstitute these lamellae in vitro. Porcine stratum corneum lipids were applied to Millipore filters. The disks were placed in water and heated to 80 degrees C for 1 h. After cooling, the disks were stored over desiccant. At each stage, the disks were prepared for TEM. TEM revealed that the application of the lipid solutions onto the disks resulted to deposition of mostly amorphous material. Heating in water resulted in the formation of many lamellae. The width of the lamellae was uniform and in the range of 5 to 6 nm with no broad-narrow-broad pattern; however, after storage under desiccating conditions, the broad-narrow-broad pattern was reproduced.

Murine keratinocyte cultures grown at the air/medium interface synthesize stratum corneum lipids and "recycle" linoleate during differentiation.

In a recent investigation we showed that murine keratinocyte cultures grown at the air/medium interface in the presence of dermis exhibit morphologic differentiation comparable to that seen in vivo, including the formation of lamellar granules and stratum corneum intercellular lipid lamellae. In the present study, lifted cultures were found to more closely reproduce the lipid composition of the parent epidermal tissue than submerged cultures grown on plastic. In addition, the specific fatty acid profile of individual lipid classes in lifted cultures was, in general, remarkably well maintained in vitro. Acylceramides, which are highly enriched in linoleic acid in vivo, remained enriched in vitro; however, the linoleic acid content of the cultures was substantially lower than that in vivo, confirming previous reports of the relative essential fatty acid deficiency of standard culture media. As the lifted cultures differentiated over time, the lipid composition changed to reflect the formation of a stratum corneum with its different complement of lipids. Label from [U-14C]linoleic acid was specifically incorporated into linoleate-containing lipids during short pulses in both submerged and lifted cultures. Changes in label distribution over a long chase period in lifted cultures indicated that linoleate was transferred from phospholipids to ceramides, providing evidence for the "recycling" of essential fatty acids in epidermis.

The role of the corneocyte lipid envelopes in cohesion of the stratum corneum.

Treatment of isolated stratum corneum with certain detergents results in complete disaggregation of the corneocytes within hours at 45 degrees C without agitation. This is prevented by prior heating of the tissue to 80 degrees C or by solvent extraction of the intercellular lipids. In the present study, electron microscopy revealed that the heated or solvent-extracted tissue was characterized by cell-to-cell contacts that appeared to involve the chemically bound hydroxyceramides which constitute the corneocyte lipid envelope. It is proposed that the irreversible bonding between corneocytes that results from heating or lipid extraction results from interdigitation of the sphingosine chains belonging to those hydroxyceramides that are bound to the corneocyte protein envelope by the omega-hydroxyl function of the 30- and 32-carbon hydroxyacid moieties. Similar interdigitation of adjacent envelopes might be involved in natural stratum corneum cohesion, limited mostly to the periphery of corneocytes where the absence of intercellular lamellae allows the appropriate cell-to-cell contact.

Presence of intact intercellular lipid lamellae in the upper layers of the stratum corneum.

The epidermal permeability barrier necessary for terrestrial life resides in the intercellular spaces of the stratum corneum and is composed of lipids. Membrane coating granules (MCGs), small intracellular organelles found in the uppermost layers of the living epidermis, contain stacks of membranous disks which are extruded into the intercellular space and undergo both biochemical and physical changes to form the lipid sheets which constitute this barrier. Using ruthenium tetroxide as a secondary fixative, we are able to demonstrate stacks of lamellae filling the intercellular spaces in the uppermost layers of the stratum corneum. The structure of these lipid lamellae is consistent with the proposed derivation of MCG lipid disks and also suggests that the lipid bilayer adjacent to the corneocyte cell envelope may be assembled from lipids not derived from MCGs.

Evidence that the corneocyte has a chemically bound lipid envelope.

The stratum corneum of mammalian epidermis contains a mixture of ceramides, free fatty acids, cholesterol, and cholesteryl sulfate, amounting to 14% of the dry weight of the tissue, that can be removed by exhaustive extraction with chloroform/methanol. Subsequent mild alkaline hydrolysis liberates additional lipid, consisting almost exclusively of C30-C34 omega-hydroxyacids in amide linkage with sphingosine, equal to 2% of the tissue mass. In the present study, transmission electron microscopy was used to demonstrate that the initial extraction removes the intercellular lamellae that constitute the epidermal water barrier but leaves the lucent band that has been termed the corneocyte plasma membrane. The subsequent alkaline hydrolysis and lipid extraction remove the lucent band, which must therefore contain the omega-hydroxyacylsphingosines. From the results of in situ derivatization of these lipids and the construction of molecular models, it is inferred that the bound lipids exist in ester linkage with protein on the surface of the corneocyte envelope. The tightly packed hydroxyacylsphingosine molecules thus form a lipid envelope for each corneocyte.

Composition and morphology of epidermal cyst lipids.

The contents of epidermal cysts were used as a source of desquamated human keratinocytes uncontaminated by sebaceous, subcutaneous, or bacterial lipids. Lipids extracted with chloroform:methanol mixtures included six series of ceramides (41% of the total extractable lipid), cholesterol (27%), cholesteryl esters (10%), fatty acids (9%), cholesteryl sulfate (1.9%), a novel class of ceramide esters (3.8%), and a sterol diester (0.9%). Electron microscopy revealed that the lipids in the cyst contents existed as multiple intercellular lamellae, as in stratum corneum. One lamella, adjacent to the horny cell protein envelope, was resistant to lipid extraction and is thought to represent covalently bound lipid on the outer surface of the keratinocyte. The results indicate that the degradation of intercellular lipid lamellae is not required for desquamation.

Lamellar granule extrusion and stratum corneum intercellular lamellae in murine keratinocyte cultures.

Lamellar granules are specialized epidermal organelles containing stacks of membranous disks that are extruded into the intercellular spaces in the upper portion of the granular layer. The extruded disks are believed to undergo biochemical and biophysical changes to form the stratum corneum intercellular lipid sheets that constitute the epidermal permeability barrier. Little is known about this important component of epidermal differentiation, in part due to lack of a suitable in vitro model. We have demonstrated microscopically the presence of characteristic lipid membrane structures in a primary keratinocyte culture system which shows morphologic differentiation comparable to that seen in vivo. A basal cell-enriched fraction of isolated neonatal mouse keratinocytes was plated into Vitrogen-coated 30 mm Millicell (Millipore, Bedford, Massachusetts) wells, fed daily with Medium 199 containing 10% fetal bovine serum, 10 micrograms/ml each of insulin and hydrocortisone, and kept at 32 degrees C in a 5% CO2/95% air atmosphere in a humidified incubator. Three days after plating, cultures were placed on living, epidermis-free mouse dermis at the air/liquid interface. At 2 wk, histologic examination showed multiple well-organized cell layers, including a distinct granular layer and a well-developed stratum corneum. Transmission electron microscopy demonstrated numerous lamellar granules and extrusion of their contents into the intercellular space. After fixation with ruthenium tetroxide, stacked intercellular lamellae in the stratum corneum were seen. Both the presence of dermis and growth at the air/liquid interface were necessary to achieve complete differentiation. This system conclusively demonstrates the formation of complex epidermal lipid structures in vitro and should allow the mechanisms and regulation of their synthesis to be elucidated.

Sphingolipid metabolism in organotypic mouse keratinocyte cultures.

Ceramides are the dominant component of the stratum corneum intercellular lipid lamellae, which constitute the epidermal permeability barrier. Only pig and human epidermal ceramides have been extensively characterized and the structures of the ceramides of cultured keratinocytes have not been previously investigated. In the present studies, we have characterized the ceramides synthesized by organotypic lifted mouse keratinocyte cultures for the first time and compared them to the ceramides of intact mouse epidermis. Both mouse epidermis and cultures contained five ceramides, ceramide 1 being the least polar and ceramide 5 the most polar. Ceramide 1 was a group of acylceramides, i.e., very-long-chain omega-hydroxyceramides with an ester-linked nonhydroxy fatty acid. Ceramide 2 contained medium-length saturated nonhydroxy fatty acids. (In culture, the ceramide 2 band was split into two parts with the slightly more polar ceramide 2' containing short-chain saturated nonhydroxy fatty acids.) Ceramide 5 contained short-chain alpha-hydroxy fatty acids. The structures of ceramides 1, 2, and 5 were analagous to those of pig and human epidermis. Mouse epidermal ceramide 3 was quite unusual, containing beta-hydroxy fatty acids, a structure not previously identified among mammalian ceramides. In contrast, culture ceramide 3 was composed of omega-hydroxy fatty acids with a chain-length distribution similar to that of ceramide 1. Mouse ceramide 4 was composed of fatty acids with chromatographic mobility similar to hydroxy fatty acids but with different chemical reactivity; it remains only partially characterized. Culture ceramide 4 was present in quantities too small for analysis. All ceramides in mouse epidermis and cultures contained only sphingosine bases, whereas pig and human ceramides also contain phytosphingosine. These results indicate that considerable diversity of ceramide structures occurs among mammalian species and that cultured keratinocytes may only partially reproduce the in vivo complement of ceramides. Using labeled serine in keratinocyte cultures, we have also demonstrated the de novo synthesis of ceramides and the transfer of label from glucosylceramides to ceramides during terminal differentiation of lifted cultures. The covalently bound corneocyte lipid envelope, which has recently been characterized in pig and human epidermis, was also present in mouse epidermis and was reproduced by the lifted cultures. Very-long-chain omega-hydroxyceramides were the dominant bound lipid and labeling studies in culture indicated that they were derived from ceramides synthesized in the viable epidermis.

Molecular models of the intercellular lipid lamellae in mammalian stratum corneum.

Intercellular lipid lamellae in the stratum corneum constitute the barrier to water diffusion and may also play a role in cohesion between corneocytes. The lamellae arise from stacks of lamellar disks that are extruded from the granular cells and then fuse edge-to-edge to form sheets. It has been proposed that each lamellar disk is formed from a flattened vesicle, and therefore consists of two lipid bilayers in close apposition. In the present study, electron microscopic examination of ruthenium-tetroxide-fixed stratum corneum from mouse, pig, and human skin revealed that the double bilayer pattern persists in the intercellular lamellae. In addition, distinctive patterning of the intercellular lamellae has led us to propose novel molecular arrangements of the intercellular lipids. These include interlamellar sharing of lipid chains to produce lipid monolayers between pairs of bilayers. The pattern reflects the provenance of the intercellular lamellae from lamellar granule disks and the nonrandom orientation of the lamellar lipids.

Osmium tetroxide and ruthenium tetroxide are complementary reagents for the preparation of epidermal samples for transmission electron microscopy.

Ruthenium tetroxide and osmium tetroxide were compared as post-fixatives in the preparation of human epidermis for transmission electron microscopic examination. Both reagents revealed characteristic lamellar granules within the granular layer and extruded lamellar granule contents in the upper granular layer. The transformation of the granule contents into multilamellar sheets at the interface between the granular and cornified layers and the persistence of these sheets through all levels of the stratum corneum were demonstrated only with ruthenium tetroxide fixation. Therefore, the reactivity of osmium tetroxide with isolated epidermal lipids was examined. The failure of osmium tetroxide to reveal membrane structures in the stratum corneum can be explained by its inability to react with many of the lipid components of these membranes, rather than to selective removal of lipids during tissue processing, as was formerly believed. Ruthenium tetroxide, a stronger oxidizing agent than osmium tetroxide, overcomes this problem but has other severe limitations as a post-fixative.

Essential fatty acids and epidermal integrity.

The intercellular spaces of the stratum corneum contain multilamellar lipid sheets derived from the extruded contents of lamellar granules. In the absence of linoleic acid, lamellar granules appear empty, and only fragmentary extracellular sheets are found. This defective differentiation is attributable to substitution of oleate for linoleate in O-acylsphingolipids. Normally, linoleate is ester-linked to 30- to 34-carbon omega-hydroxyacids, which, in turn, are amide-linked to sphingosine. Acylglucosylceramides, bearing a beta-D-glucosyl moiety on the sphingosine, may provide the driving force for lamellar granule assembly. The omega-hydroxyacyl chains are long enough to span a lipid bilayer, while the linoleate inserts into an adjacent bilayer. This interaction could promote assembly of lamellar granules. It has also been proposed that acylceramides may stabilize the extracellular sheets by a similar mechanism. In addition, the horny cell has been found to possess a covalently bound lipid envelope consisting principally of omega-hydroxyacylsphingosines derived from O-acylsphingolipids.

Isolation of corneocyte envelopes from porcine epidermis.

Sheets of porcine stratum corneum were dispersed into individual corneocytes after 4 h in a solution consisting of 8 mM N,N-dimethyldodecylamine oxide and 2 mM sodium dodecylsulfate in phosphate-buffered isotonic saline, at 45 degrees C. With continued detergent treatment and moderate sonication, most of the cells lost their keratin contents and were then separated from the remaining intact cells by centrifugation in cesium chloride solution of density 1.280. Electron microscopy showed that the cell envelopes retained both the crosslinked protein envelope and its attached lipid envelope. The dry weight of envelopes was approximately 7% of the estimated dry weight of the original stratum corneum, while the corneocytes surviving intact also amounted to 7% of the starting weight. Mild alkaline hydrolysis of the corneocyte envelopes allowed the extraction of hydroxyceramides amounting to 10% of the dry weight of the envelopes. The procedure therefore provides isolated corneocyte envelopes suitable for studying both the protein and lipid components of this compound sheath.

Regional variation in content, composition and organization of porcine epithelial barrier lipids revealed by thin-layer chromatography and transmission electron microscopy.

Epidermis and oral epithelia provide permeability barriers that limit penetration of potentially harmful agents. Barrier function is determined by lipids in the superficial epithelial layers and varies regionally by more than 10-fold. The purpose of this study was to determine whether differences in lipid content, composition or organization could account for this variation in barrier function. Stratum corneum from skin, gingiva and palate and superficial layers from buccal regions and the floor of the mouth were isolated, and lipids were extracted and analysed by thin-layer chromatography. Tissue from each region was examined by electron microscopy. There was an inverse correlation between permeability and ceramide content and a direct correlation with triglyceride content. Electron microscopy revealed that the intercellular space in epidermal stratum corneum contained multiple lipid lamellae displaying an alternating broad-narrow-broad spacing. In palatal and gingival stratum corneum, uniformly spaced lamellae were present at the periphery of dilations of the intercellular space, but the interiors of the dilations contained disorganized lamellae and electron-dense material. In the non-keratinized barriers, there was a single, broad lamella at the cell periphery and occasional short stacks of lamellae traversing the intercellular space. These intercellular lamellae may be derived from a population of membrane-coating granules that contain internal lamellae. The results suggest that ceramides may be important barrier components, even in non-keratinizing epithelia where they are very minor components. Regional differences in the physical organization of barrier lipids may also contribute to differences in barrier function.

Lipids of hamster cheek pouch epithelium.

The hamster cheek pouch is a much used but incompletely understood experimental model. In particular, the cheek pouch epithelial lipids, which are important for permeability barrier function as well as other aspects of epithelial biology, have not been completely characterized. In the present study, the complete lipid class composition has been determined by thin-layer chromatography in conjunction with photodensitometry. The major lipid classes were phospholipids, free sterols, and ceramides. Minor amounts of monohexosylceramides, sterol esters, fatty acids, and triglycerides were also present. Significant amounts of covalently bound omega-hydroxyceramide was also detected. Transmission electron micrographs reveal extensive, largely paired, lipid bilayers in the intercellular spaces of the stratum corneum.