Occurrence of galactosylceramide in pig epidermal cells.

Monoglycosylceramides were isolated from pig epidermal cells which had been prepared free from dermal elements. The most polar glycolipid among the five isolated monoglycosylceramides was galactosylceramide. The galactosylceramide was composed of alpha-hydroxypalmitic acid and 16- and 18-carbon chain sphingenine, being quite different from epidermal glucosylceramides. This is the first report demonstrating the occurrence of galactosylceramide in mammalian epidermal cells.

Structure determination of glycosphingolipids of cultured human keratinocytes.

From cultured human keratinocytes, seven glycolipid fractions were isolated by DEAE and silica-gel column chromatographies, and further by HPLC on a silica-gel column. By means of 1H-NMR spectroscopy, fast atom bombardment mass spectrometry and GLC-mass spectrometry, one fraction was determined to contain acylglucosylceramides, which consist of amide linked omega-hydroxy fatty acids (C30:0, C30:1, C32:1 and C34:1), fatty acids linked to the omega-hydroxy fatty acids through ester linkages (C14:1, C16:1, C18:1 and C18:2), a long-chain base (d18-sphingenine), and beta-glucose. Five of the other fractions contained glucosylceramides, and the seventh fraction contained a mixture of glucosylceramides and galactosylceramides. Glucosylceramides containing long-chain omega-hydroxy fatty acids, which are assumed to be immediate precursors of the acylglucosylceramides, were hardly detected in these glycolipid fractions. Six glucosylceramide fractions were separated due to differences in their fatty acids and sphingosines. On comparison with the results reported in our previous paper, the acylglucosylceramide content of the cultured human keratinocytes was about half that of human epidermis. Under the culture conditions used, the human keratinocytes did not differentiate into granular or horny cells. Taken together, the results suggest that the synthesis of acylglucosylceramides is not activated much in the cultured keratinocytes, but would be more activated in differentiated cells.

Glycosphingolipid compositions of human T-lymphotropic virus type I (HTLV-I) and human immunodeficiency virus (HIV)-infected cell lines.

Glycolipid compositions of cells infected by human retroviruses (human immunodeficiency virus, HIV and/or human T-cell lymphotropic virus type I, HTLV-I) have been studied. Eight cell lines, comprising two HTLV-I-infected T-cell lines (MT-2 and MT-4), two HTLV-I-negative T-cell lines (Jurkat and MF), a macrophage cell line (U937), and three HIV-infected counterpart cell lines (MT-4/HIV, Jurkat/HIV and U937/HIV) were used. The neutral glycolipids and gangliosides isolated from these cell lines were compared. Among them, the HTLV-I-infected T-cell lines, MT-2 and MT-4, showed similar patterns for both neutral glycolipids and gangliosides. Neutral glycolipids (GlcCer and LacCer) of MT-2 and MT-4 cells were markedly decreased, and a ganglioside, GM3, of theirs was decreased to only a trace amount compared to that in other cell lines. Gangliosides of MT-4 and MT-4/HIV were further separated on an Iatrobeads column, and were identified as GM2, GM1a and GD1a by methylation and liquid secondary ion mass spectrometric analyses. Since the patterns of neutral glycolipids and gangliosides of MT-2 and MT-4 are unique, as compared to those of HTLV-I-negative cells, it is suggested that these changes are related to HTLV-1 infection. No prominent differences in the ganglioside compositions between HIV-infected and non-infected cell lines could be observed. But it is noteworthy that the contents of asialo-GM2 in Jurkat/HIV and MT-4/HIV cells were increased as compared to those in the parental cell lines.

Direct measurement of nitric oxide during experimental cardiopulmonary bypass.

We developed a system to measure nitric oxide (NO) concentration during cardiopulmonary bypass in anaesthetized pigs (n = 6). A T-shaped connector, attached to an NO sensor, was mounted in the extracorporeal circuit at two measuring sites: proximal to the membrane oxygenator (venous side) and distal to the arterial line filter (arterial side). After performing a preliminary validation study, we measured plasma NO concentration before and during total cardiopulmonary bypass circulation (non-pulsatile flow 1.5 l/min) and without pulmonary ventilation. After establishing bypass, PaO2 was 318 - 393 mmHg; when PaO2 was decreased to 80 - 100 mmHg, plasma NO concentration in the arterial circuit fell by 39.2 +/- 15.6 nM. There was no observable change in plasma NO concentration at the venous circuit. This new system could be useful in monitoring NO concentration during cardiac surgery with cardiopulmonary bypass, and for understanding the possible pathophysiological roles of hyper-nitric oxaemia in cardiopulmonary bypass-related cardiovascular complications.

Relationship between cholesterol sulfate and intercellular cohesion of the stratum corneum: demonstration using a push-pull meter and an improved high-performance thin-layer chromatographic separation system of all major stratum corneum lipids.

To investigate the role of cholesterol sulfate (CS) as an intercellular glue or cement in the stratum corneum, we compared the relationship between CS levels and magnitude of the intercellular cohesion of the stratum corneum between the palm and the upper arm. Using a push-pull meter, the palm displayed approximately seven times the magnitude of cohesion of the stratum corneum as the upper arm (n = 11). CS and other stratum corneum lipids were extracted from the palm and the upper arm (n = 22) by a cup method and determined by our improved high-performance thin-layer chromatography (HPTLC). Despite a great difference in the magnitude of cohesion (p less than 0.01), CS levels and ratios of CS to ceramides and CS to cholesterol in the stratum corneum showed no significant differences between the palm and the upper arm. Our results suggest that differences in CS cannot account for the differences in cohesion between palm and upper arm.

Monoclonal antibody against polyglucosan isolated from the myocardium of a patient with Lafora disease.

Polyglucosan was extracted from the myocardium of a patient with Lafora disease. Gas chromatographic analysis of the isolated polyglucosan revealed glucose as the only sugar component. Monoclonal antibodies (MAb) were raised against the isolated polyglucosan. With the MAb, immunocytochemistry showed positive staining of Lafora bodies, corpora amylacea, basophilic degeneration of the cardiac muscle cells, and inclusion bodies within cardiac muscle cells and the hepatocytes of patients with Lafora disease and type IV glycogenosis (Andersen's disease). These findings indicate that cells of the latter group share antigenicity with Lafora bodies.

Ganglioside antigen of DU-PAN-2 in a human pancreatic cancer.

DU-PAN-2 reactive gangliosides were isolated from the tumor of a patient with pancreatic cancer (duct cell carcinoma, moderately differentiated adenocarcinoma), having a negative Lewis blood phenotype, and were analyzed by means of TLC-immunostaining, enzyme-linked immunosorbent assay (ELISA), permethylation study, 1H NMR spectroscopy and fast atom bombardment mass spectrometry. The structures of the gangliosides were found to be NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer, containing normal and hydroxy fatty acids. By TLC-immunostaining and ELISA with chemically synthesized gangliosides, DU-PAN-2 was demonstrated to react strongly with IV3 alpha NeuAc-Lc4Cer, weakly with IV3 alpha NeuAc-nLc4Cer, and moderately with IV6 alpha NeuAc-Lc4Cer and IV6 alpha NeuAc-nLc4Cer. Thus it was concluded that the DU-PAN-2 reactive ganglioside in the tumor is IV3 alpha NeuAc-Lc4Cer and that DU-PAN-2 has a rather broad specificity.

Intramyocardial angiomyolipoma.

We report a case of cardiac angiomyolipoma in a 48-year-old woman who went to the hospital because of shortness of breath. Cardiac ultrasonography showed a right atrial mass, which was surgically removed. Pathologic examination revealed a 6-cm-diameter, dome-shaped mass composed of a mixture of blood vessels, smooth muscle, and fat. Because of its distinctive morphology and location, we diagnosed it as an intramyocardial angiomyolipoma. There was no evidence of tuberous sclerosis. Since excision of the mass, the patient has remained well without recurrence for 20 months. Angiomyolipomas usually develop in the kidney; extrarenal occurrence is rare. To date, no case of a cardiac angiomyolipoma has been reported in the English literature. The histogenesis of angiomyolipoma is uncertain, but it is most likely hamartomatous in nature.

Galactocerebroside and not glucocerebroside or ceramide stimulate epidermal beta-glucocerebrosidase activity.

Glycosphingolipids including glucocerebroside (GluCer) and galactocerebroside (GalCer) have been recognized as bioreguratory lipids by our group and others. In addition, our recent study demonstrated that GalCer corrects dry skin conditions in humans. The processing of stratum corneum lipids, which occurs when beta-glucocerebrosidase (beta-GluCer'ase) changes GluCer to ceramide (Cer), is required to form the epidermal permeability barrier. We herein investigated the effects of GluCer, GalCer and Cer on the processing of GluCer to Cer by assaying epidermal beta-GluCer'ase in mice (155%, P < 0.01) when compared to vehicle treated controls, while neither GluCer nor Cer had this effect. Studies using inhibitors of beta-GluCer'ase or beta-galactosidase and measuring the optimum pH of the enzyme verified that GalCer specifically activated beta-GluCer'ase. We confirmed that GalCer significantly increased beta-GluCer'ase activity in the outer epidermal fraction (172%, P < 0.01) and that the activation of beta-GluCer'ase is not due to a direct activating effect of GalCer on the enzyme. Furthermore, the induction of beta-GluCer'ase activity by GalCer was also observed in cultured normal human deratinocytes (123%, P < 0.01). Finally, acylceramide content in stratum corneum was increased in mice treated with GalCer (194%, P < 0.0005). These results indicate that GalCer appears to affect the Cer construct in the stratum corneum by the activation of beta-GluCer'ase, which ultimately contribute to an enhancement of barrier formation.

Effect of a chemically-synthesized acylglucosylceramide, epidermoside, on normal human keratinocyte differentiation.

Epidemosides (N-(0-linoleoyl)-(1)-hydroxy fatty acyl sphingosyl glucose) are found exclusively in the epidermis not in dermis, and are thought to play important role in forming the mammalian epidermal permeability barrier. A species of epidermoside isolated from guinea pig epidermis and named lipokeratinogenoside has been shown to enhance fetal rat keratinocyte differentiation. In the present investigation, we studied the effects of a chemically synthesized equivalent of human epidermoside on the viability and differentiation of cultured human keratinocytes (HK Cells). The chemically-synthesized epidermoside was not toxic to cultured HK Cells at concentrations of 0.01 to 10 micrograms/ml. When 10 micrograms/ml of the chemically-synthesized epidermoside was added to keratinocyte growth medium containing 1.2 mM Ca2+, HK Cells showed a 5.6-fold increase of keratin content compared to the vehicle treated control at 144 h of cultivation, and they also displayed morphological changes suggestive of differentiation. A similar increase of cellular keratin content was observed in HK cells treated with tetradecanoyl phorbol-13 myristyl-12 acetate (TPA), an agent known to enhance the differentiation of keratinocytes. Lipokeratinogenoside also increased the keratin content of cultured HK cells. These results suggest that epidermosides have an ability to enhance keratinocyte differentiation. Epidermoside could thus be a key molecule, not only as a constituent of the epidermal permeability barrier, but also as a regulator of keratinocyte differentiation.

Porokeratosis large skin lesions are susceptible to skin cancer development: histological and cytological explanation for the susceptibility.

Porokeratosis (PK), an autosomal dominant inherited skin disorder, is known to develop malignant skin tumors on its skin lesions. Our recent literature survey has revealed that large PK skin lesions are frequently a precursor of malignant changes. In the study, large and small PK skin lesions were investigated in terms of histological features of the epidermis and of the cellular DNA content of epidermal cells. Large PK lesions frequently showed hypertrophic epidermis with many mitotic cells, while small lesions usually presented atrophic epidermis without such mitotic cells. Abnormal cells, like those containing hyperchromatic, large, and/or irregularly shaped nuclei, were present in the epidermis of both large and small lesions with a preponderance in the former over the latter. DNA polyploidy was seen more frequently in large PK lesions than in small ones. DNA index values were significantly higher in large lesions than in small ones. The histological features and DNA ploidy abnormalities probably reflect the higher proliferation and the greater potential for malignant changes of large PK skin lesions. Our study helps to explain the clinical evidence that large PK skin lesions are frequently a precursor of malignant skin tumors.

Ganglioside compositions of erythrocytes from various strains of inbred mice. Ocurrence of sialosylgalactosylceramide in red blood cells of inbred mice.

The gangliosides from the erythrocytes of various strains of inbred mice were analyzed. The ganglioside compositions differed in different strains and the strains could be divided into 4 types on the basis of this difference. The main ganglioside is GM4 or sialosyl galactosyl ceramide in Type 1, and unidentified ganglioside in Type 2, GM4 and GM2 in almost equal amounts in Type 3, and GM2 in Type 4.

Occurrence of hematoside with two moles of N-acetyl-neuraminic acid in a certain breed of Persian cat.

The glycolipids of erythrocytes from individual cats were examined. The main glycolipid of cat erythrocytes was generally NeuGc-NeuGc-Gal-Glc-ceramide (NeuGc-GD3), but among 41 cats of 5 breeds and 2 mongrels examined, 2 Persian cats were found to have NeuAc-NeuAc-Gal-Glc-ceramide (NeuAc-GD3). This is the first report of the occurrence of NeuAc-GD3 in cat erythrocyte membranes.

Further studies on gangliosides of erythrocytes from horses and cattle.

The ganglioside patterns of erythrocytes from individual horses and cattle were examined. Variations in the ganglioside patterns were found in both horses and cattle. In the erythrocytes of most horses examined, NeuGc-Gal-Glc-ceramide (NeuGc-GM3) of 25 horses examined had only NeuGc-GM3 with no 4-O-Ac-NeuGc-GM3. The erythrocytes of various breeds of cattle had a characteristic ganglioside pattern, but they could be divided into 4 types on the basis of the composition of their gangliosides.

Structure determination of glucosyl beta 1-N-(omega-O-linoleoyl)-acylsphingosines of human epidermis.

Human epidermis gave two glycolipid bands that migrated faster than glucosylceramide and two bands that migrated like glucosylceramide and galactosylceramide, respectively, on TLC. The two faster migrating glycolipids (GL-I and GL-II), which exhibited alkalilability, were purified by conventional DEAE and silica gel column chromatographies, and further by HPLC on a silica gel column. Structure determination of the two components, named GL-I3 and GL-II3, which were finally purified from GL-I and GL-II, respectively, by HPLC on a reversed phase column, was performed by means of 1H-NMR spectroscopy, fast atom bombardment mass spectrometry, and component analysis involving GLC-mass spectrometry. GL-I3 was determined to be a mixture of glucosyl beta 1-N-(omega-O-linoleoyl)-triacontanoyl- and -dotriacontamonoenoyl-eicosasphingenine, and one of the two components of GL-II3 was determined to be glucosyl beta 1-N-(omega-O-linoleoyl)triacontanoyl-trihydroxyeicosasphingenin e. GL-I3 and GL-II3 were the major components of GL-I and GL-II, respectively, and both the latter contained additional four components, which were heterogeneous as to the ceramide portion. This paper reports the structures of acylglucosylceramides isolated from human epidermis together with 1H-NMR spectra and mass spectra demonstrating their molecular weights. The structure of molecular species containing trihydroxysphingosine having a double bond is novel.

Sialyl Lewis(a) ganglioside in pancreatic cancer tissue correlates with the serum CA 19-9 level.

CA 19-9 ganglioside antigen was extracted from the tumors of 16 patients with pancreatic cancer and the antigen was confirmed by mass spectrometry to be a sialyl Lewis(a) ganglioside. Ductal adenocarcinomas from four patients, two whose Lewis blood phenotype was Le(a- b-), one with undifferentiated carcinoma, and one with an acinar cell carcinoma, did not contain detectable amounts of sialyl Le(a) ganglioside. A significant correlation was found between serum CA 19-9 levels and the recovery of ganglioside antigen from respective tumors, but not tumor burden.

Lipid composition and fatty acid analysis of Helicobacter pylori.

Lipids extracted from Helicobacter pylori were separated into lipid classes by thin-layer chromatography. Simple H. pylori lipids consisted of cholesterol esters, triglycerides, free fatty acids, cholesterol, diacylglycerols, and monoacylglycerols. Fatty acids were released from each lipid class by acid methanolysis, and analyzed by gas liquid chromatography and mass spectrometry. Unique methoxy fatty acids, including 11-methoxy heptadecanoic and 11-methoxy nonadecanoic acids, were the major components of the cholesterol esters and triglycerides. The predominance of methoxy fatty acids in the cholesterol esters of H. pylori may contribute to the acid-resistant characteristic of this bacillus.

Tissue accumulation of sulfatide and GM3 ganglioside in a patient with variant Farber disease.

We analyzed the lipids in the tissues of a patient with an atypical form of Farber disease who developed several clinical symptoms not seen in patients with typical Farber disease (acid ceramidase deficiency). Lipids were extracted from formalin-fixed brain, liver and kidney and purified by ion exchange and silica gel column chromatographies and further by high-performance liquid chromatography on a silica gel column. We performed structural and quantitative analyses of three lipids named lipids X, Y and Z. Lipid X accumulated in the liver but not in the brain. Accumulation of lipids Y and Z was observed in liver and kidney. The content of lipid Y in the patients liver was more than ten times that in a control. The structures of lipids X, Y and Z were confirmed by means of 1H-nuclear magnetic resonance spectroscopy, fast atom bombardment mass spectrometry, infrared absorption spectroscopy, and component analysis involving gas liquid chromatography and gas chromatography-mass spectrometry. The structures of lipids X, Y and Z were identified as those of ceramide, sulfatide and GM3 ganglioside, respectively. These results suggest two possibilities. One is that the accumulation of glycolipids such as sulfatide and GM3 ganglioside is a secondary event produced by the accumulation of ceramide due to ceramidase deficiency. The other is that the accumulation of glycolipids other than ceramide is due to a deficiency of sphingolipid activator proteins which may affect the degradation of sulfatide and GM3 ganglioside as well as ceramide.

Identification of cDNA encoding cytochrome c oxidase subunit 5c (COX5c) from rice: comparison of its expression with nuclear-encoded and mitochondrial-encoded COX genes.

Little is presently known about the nuclear-encoded genes for cytochrome c oxidase (COX) in higher plants. In rice, only the nuclear-encoded COX5b gene has been reported. To understand the relationship between the expression of nuclear-encoded and mitochondrial-encoded COX genes in rice, we first characterized a cDNA encoding one of the other nuclear COX genes, COX5c, which encodes 63 amino acids. The deduced amino acid sequence of COX5c from rice was highly homologous to that from sweet potato. Genomic Southern hybridization indicated that the rice COX5c subunit is encoded by a single copy of the COX5c gene. Furthermore, we compared the expression patterns of the nuclear-encoded COX5c and COX5b genes with the expression pattern of the mitochondrial-encoded COX1 gene among several organs by Northern blot analysis. The results suggested that regulatory systems of expression between the nuclear-encoded and the mitochondrial-encoded COX genes are different among different organs in rice.

Bound lipids liberated by alkaline hydrolysis after exhaustive extraction of pulverized clavus.

In the present study, covalently bound lipids were found in clavus material and their lipid classes were determined by high-performance thin-layer chromatography (HPTLC). Clavus material, pulverized completely in a Mikro-Dismembrator II, was exhaustively extracted three times with chloroform/methanol (2:1, 1:1 and 1:2 v/v) at 80 degrees C for 1 h each time and this sequence of extractions was repeated to obtain the unbound lipid-free residue which was saponified and then extracted with chloroform. The extract proved to comprise several bands of lipids covalently bound through an ester-like linkage. These were identified as free fatty acids, cholesterol, ceramides and glucocerebrosides by HPTLC. However, omega-hydroxy fatty acids were not detected in the lipids. To analyse the fatty acids amide-linked to the bound ceramides, the latter were isolated by preparative HPTLC and subjected to mild acid hydrolysis. Since the bound ceramides constituted neither omega-hydroxy fatty acids nor alpha-hydroxy fatty acids, they were not identified as hydroxyl-acylsphingosines.