Occurrence of 2- and 3-hydroxy fatty acids in high concentrations in the extractable and bound lipids of Flavobacterium meningosepticum and Flavobacterium IIb

The major hydroxy fatty acids of cellular lipids in Flavobacterium meningosepticum and Flavobacterium sp. King's group UUb were identified as 2-hydroxy 13-methyltetradecanoic, 3-hydroxy 13-methyltetradecanoic, 3-hydroxy palmitic, and 3-hydroxy 15-methylhexadecanoic acids using gas chromatography-mass spectrometry and GC-mass fragmentography. The concentration of these hydroxy fatty acids comprised up to 30-40% of the total extractable and 20-30% of the bound lipid fatty acids, respectively. From the stability for mild alkaline hydrolysis, 2-hydroxy fatty acids seemed to be attached with ester linkage, and 3-hydroxy fatty acids with amide linkage.     

"Cross talk" between the bioactive glycerolipids and sphingolipids in signal transduction

Hydrolysis of phosphatidylcholine via receptor-mediated stimulation of phospholipase D produces phosphatidate that can be converted to lysophosphatidate and diacylglycerol. Diacylglycerol is an activator of protein kinase C, whereas phosphatidate and lysophosphatidate stimulate tyrosine kinases and activate the Ras-Raf-mitogen-activated protein kinase pathway. These three lipids can stimulate cell division. Conversely, activation of sphingomyelinase by agonists (e.g., tumor necrosis factor-alpha) causes ceramide production that inhibits cell division and produces apoptosis. If ceramides are metabolized to sphingosine and sphingosine 1-phosphate, then these lipids can stimulate phospholipase D and are also mitogenic. By contrast, ceramides inhibit the activation of phospholipase D by decreasing its interaction with the G-proteins, ARF and Rho, which are necessary for its activation. In whole cells, ceramides also stimulate the degradation of phosphatidate, lysophosphatidate, ceramide 1-phosphate, and sphingosine 1-phosphate through a multifunctional phosphohydrolase (the Mg(2+)-independent phosphatidate phosphohydrolase), whereas sphingosine inhibits phosphatidate phosphohydrolase. Tumor necrosis factor-alpha causes insulin resistance, which may be partly explained by ceramide production. Cell-permeable ceramides decrease insulin-stimulated glucose uptake in 3T3-L1 adipocytes after 2-24 h, whereas they stimulate basal glucose uptake. These effects do not depend on decreased tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 or the interaction of insulin receptor substrate-1 with phosphatidylinositol 3-kinase. They appear to rely on the differential effects of ceramides on the translocation of GLUT1-and GLUT4-containing vesicles. It is concluded that there is a significant interaction and "cross-talk" between the sphingolipid and glycerolipid pathways that modifies signal transduction to control vesicle movement, cell division, and cell death.     

Modification of cystatin C activity by bacterial proteinases and neutrophil elastase in periodontitis

AIM: To study the interaction between the human cysteine proteinase inhibitor, cystatin C, and proteinases of periodontitis associated bacteria. METHODS: Gingival crevicular fluid samples were collected from discrete periodontitis sites and their cystatin C content was estimated by enzyme linked immunosorbent assay (ELISA). The interaction between cystatin C and proteolytic enzymes from cultured strains of the gingival bacteria Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans was studied by measuring inhibition of enzyme activity against peptidyl substrates, by detection of break down patterns of solid phase coupled and soluble cystatin C, and by N-terminal sequence analysis of cystatin C products resulting from the interactions. RESULTS: Gingival crevicular fluid contained cystatin C at a concentration of approximately 15 nM. Cystatin C did not inhibit the principal thiol stimulated proteinase activity of P gingivalis. Instead, strains of P gingivalis and P intermedia, but not A actinomycetemcomitans, released cystatin C modifying proteinases. Extracts of five P gingivalis and five P intermedia strains all hydrolysed bonds in the N-terminal region of cystatin C at physiological pH values. The modified cystatin C resulting from incubation with one P gingivalis strain was isolated and found to lack the eight most N-terminal residues. The affinity of the modified inhibitor for cathepsin B was 20-fold lower (Ki 5 nM) than that of full length cystatin C. A 50 kDa thiol stimulated proteinase, gingipain R, was isolated from P gingivalis and shown to be responsible for the Arg8-bond hydrolysis in cystatin C. The cathepsin B inhibitory activity of cystatin C incubated with gingival crevicular fluid was rapidly abolished after Val10-bond cleavage by elastase from exudate neutrophils, but cleavage at the gingipain specific Arg8-bond was also demonstrated. CONCLUSIONS: The physiological control of cathepsin B activity is impeded in periodontitis, owing to the release of proteinases from infecting P gingivalis and neutrophils, with a contribution to the tissue destruction seen in periodontitis as a probable consequence.     

Apoptosis and activation of the sphingomyelin-ceramide pathway induced by oxidized low density lipoproteins are not causally related in ECV-304 endothelial cells

Oxidized low density lipoproteins (oxLDL) are thought to play a central role in the development of atherosclerosis. Toxic concentrations of mildly oxidized LDL elicit massive apoptosis of endothelial cells (Escargueil-Blanc, I., Meilhac, O., Pieraggi, M. T. , Arnal J. F., Salvayre, R., Nègre-Salvayre, A. (1997) Arterioscler. Thromb. Vasc. Biol. 17, 331-339). Since the lipid mediator ceramide emerged as a potent inducer of apoptosis, we aimed at investigating the occurrence of ceramide formation and its potential role in oxLDL-induced apoptosis. In ECV-304 endothelial cells, toxic concentrations of oxLDL triggered an early activation of the sphingomyelin-ceramide pathway, as shown by both sphingomyelin hydrolysis and ceramide formation. N-Tosyl-L-phenylalanyl chloromethyl ketone (TPCK) and dichloroisocoumarin (DCIC), two serine-protease inhibitors (serpins), blocked the oxLDL-induced ceramide generation but, unexpectedly, did not inhibit the oxLDL-induced apoptosis. Conversely, treatment of endothelial cells by bacterial sphingomyelinase, under conditions effectively generating ceramide, did not induce apoptosis. In contrast, short-chain permeant C2- and C6-ceramides elicited apoptosis of ECV-304. However, the mechanisms of apoptosis triggered by C2-ceramide and by oxLDL were (at least in part) different, because C2-ceramide-induced apoptosis was calcium-independent, whereas oxLDL-induced apoptosis was calcium-dependent. In conclusion, it is suggested that oxLDL-induced apoptosis is calcium-dependent but independent of the activation of the sphingomyelin-ceramide pathway and that the toxic effect of short chain permeant ceramides is calcium-independent and does not mimic the effect of natural ceramides induced by oxLDL.     

A murine model of accelerated periodontal disease in diabetes

Diabetes is a risk factor for periodontal disease in humans. In hyperglycemia, glycoxidation of proteins and lipids results in the formation of advanced glycation endproducts, or AGEs. The accumulation of AGEs in the plasma and tissues, and their interaction with their cellular receptor for AGE (RAGE), has been implicated in diabetic complications. In order to establish a model with which to delineate the specific host response factors that underlie the development of periodontal disease in diabetes, male C57BL/6J mice were rendered diabetic with streptozotocin. One month after documentation of diabetes or control state, mice were inoculated with the human periodontal pathogen Porphyromonas gingivalis, strain 381 (P. gingivalis) or treated with vehicle. Infection with P. gingivalis was achieved, as demonstrated by infiltration of gingival tissue with granulocytes, presence of DNA specific for P. gingivalis as well as increased serum antibody titer to P. gingivalis. At 2 and 3 months after infection, increased alveolar bone loss was demonstrated in P. gingivalis-inoculated diabetic vs. non-diabetic mice, along with enhanced tissue-destructive capacity, as demonstrated by increased collagenolytic activity in gingival extracts. Consistent with an important role for AGE-RAGE interaction, increased AGE deposition and expression of vascular and monocyte RAGE were demonstrated in diabetic gingiva compared with non-diabetic controls. Taken together, these data indicate that we have established a murine model of enhanced periodontal disease in diabetes. This model will serve to delineate molecular mechanisms which account for the increased susceptibility of diabetic patients to periodontal disease.     

Stratum corneum lipids: the effect of ageing and the seasons

Stratum corneum lipids play a predominant role in maintaining the water barrier of the skin. In order to understand the biological variation in the levels and composition of ceramides, ceramide 1 subtypes, cholesterol and fatty acids, stratum corneum lipids collected from tape strippings from three body sites (face, hand, leg) of female Caucasians of different age groups were analysed. In addition, we studied the influence of seasonal variation on the lipid composition of stratum corneum from the same body sites. The main lipid species were quantified using high-performance thin-layer chromatography and individual fatty acids using gas chromatography. Our findings demonstrated significantly decreased levels of all major lipid species, in particular ceramides, with increasing age. Similarly, the stratum corneum lipid levels of all the body sites examined were dramatically depleted in winter compared with spring and summer. The relative levels of ceramide 1 linoleate were also depleted in winter and in aged skin whereas ceramide 1 oleate levels increased. The other fatty acid levels remained fairly constant with both season and age, apart from lignoceric and heptadecanoic acid which showed a decrease in winter compared with summer. The decrease in the mass levels of intercellular lipids and the altered ratios of fatty acids esterified to ceramide 1, are likely to contribute to the increased susceptibility of aged skin to perturbation of barrier function and xerosis, particularly during the winter months.     

Relationship between hydroxy fatty acids and prostaglandin E2 in gingival tissue

Bacterial hydroxy fatty acids and alpha-hydroxy fatty acids have been demonstrated in complex lipid extracts of subgingival plaque and gingival tissue. However, little is known about the relationship between these hydroxy fatty acids in plaque and gingival tissues or the significance of these complex lipids in promoting inflammatory periodontal disease. The present study determined the percentages of ester-linked and amide-linked hydroxy fatty acids in complex lipids recovered from plaque and gingival tissue samples and the relationship between bacterial hydroxy fatty acids and alpha-hydroxy fatty acids in the lipid extracts. To evaluate a potential role for these hydroxy fatty acids in inflammatory periodontal disease, gingival tissue samples were examined for a relationship between prostaglandin E2 (PGE2) and hydroxy fatty acids recovered in gingival lipid. This investigation demonstrated that alpha-hydroxy fatty acids are only ester linked in plaque lipids but are largely amide linked in gingival tissue lipids. Furthermore, the level of alpha-hydroxy fatty acid in gingival lipid is directly related to the level of the bacterial hydroxy fatty acid 3-OH iso-branched C17:0 (3-OH iC17:0) in the same lipid extract. However, the relationship between hydroxy fatty acids in gingival lipids does not parallel the fatty acid relationship observed in plaque lipids. Finally, alpha-hydroxy fatty acid levels in gingival tissue lipids correlate directly with the recovery of PGE2 in the same tissue samples. These results demonstrate that alpha-hydroxy fatty acid levels in gingival lipids are directly related to both 3-OH iC17:0 bacterial lipid levels and PGE2 levels. These results indicate that in periodontal tissues there are unusual host-parasite interactions involving penetration of bacterial lipid in association with an altered gingival lipid metabolism and prostaglandin synthesis.     

Angiotensin AT1 receptor antagonism and protection against cardiovascular end-organ damage

Little is known regarding the molecules expressed by gingival epithelial cells that are involved in initiating and maintaining inflammation following the interaction with periodontal pathogens. Thus, we investigated the effect of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis infection on the expression of neutrophil chemoattractant interleukin 8 (IL-8) and the adhesion molecule intercellular adhesion molecule-1 by gingival epithelial cells. The data revealed that both IL-8 and intercellular adhesion molecule-1 expression increased after infection with A. actinomycetemcomitans (IL-8: 2- to 7-fold; intercellular adhesion molecule-1: 2.5- to 3.7-fold). IL-8 secretion reached a maximal level 6 h after the infection and the expression subsequently decreased to basal level. The increased cell surface intercellular adhesion molecule-1 expression started at 4 h after infection and reached a maximal level 14 h after the infection. In contrast, the expression of both molecules rapidly decreased 2 h after challenge with P. gingivalis. This opposite influence of A. actinomycetemcomitans and P. gingivalis infection on the expression of IL-8 and intercellular adhesion molecule-1 by gingival epithelial cells suggests that A. actinomycetemcomitans infection may initiate the recruitment of neutrophils, whereas the P. gingivalis infection may retard this process and therefore demonstrate a distinct perspective of virulence.     

Cellular lipid and fatty acid compositions of Burkholderia pseudomallei strains isolated from human and environment in Viet Nam

Viet nam is known as an endemic area of melioidosis but its etiologic agent originated in Viet nam was not extensively studied. For the first time, we analyzed the cellular lipid and fatty acid compositions of 15 Vietnamese isolates of Burkholderia pseudomallei, 10 from humans and 5 from the environment. Cellular lipid compositions were analyzed by two-dimensional thin-layer chromatography on silica gel G plates. Cellular fatty acid methyl esters were analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The major lipids in all the isolates were phosphatidylglycerol (PG), two forms of phosphatidylethanolamine (PE-1 and PE-2), and two forms of ornithine-containing lipid (OL-1 and OL-2). PE-1 contained non-hydroxy fatty acids at both sn-1 and -2 positions, while PE-2 possessed 2-hydroxy fatty acids and non-hydroxy fatty acids in a ratio of 1:1. Since snake venom phospholipase A2 digestion of PE-2 liberated 2-hydroxy fatty acids, it was confirmed that these acids are at the sn-2 position of glycerol moiety. In both OL-1 and OL-2, amide-linked fatty acid was 3-hydroxy palmitic acid (3-OH-C16:0), while ester-linked fatty acids were non-hydroxy acids in OL-1 and 2-hydroxy acids in OL-2. The total cellular fatty acid compositions of the test strains were characterized by the presence of 2-hydroxy palmitic (2-OH-C16:0), 2-hydroxy hexadecenoic (2-OH-C16:1), 2-hydroxy octadecenoic (2-OH-C18:1), 2-hydroxy methylene octadecanoic (2-OH-C19CPA), 3-hydroxy myristic (3-OH-C14:0) and 3-hydroxy palmitic (3-OH-C16:0) acids. There were significant differences in the concentration of hexadecenoic (C16:1), methylene hexadecanoic (C17CPA), octadecenoic (C18:1) and methylene octadecanoic (C19CPA) acids among the Vietnamese isolates of B. pseudomallei. However, no significant difference was observed in cellular lipid and fatty acid components between strains of human and environmental origins.     

Phosphatidate phosphohydrolase and signal transduction

A Mg(2+)-independent and N-ethylmaleimide-insensitive phosphatidate phosphohydrolase (PAP-2) has been identified in the plasma membrane of cells and it has been purified. The enzyme is a multi-functional phosphohydrolase that can dephosphorylate phosphatidate, lysophosphatidate, sphingosine 1-phosphate and ceramide 1-phosphate and these substrates are competitive inhibitors of the reaction. The action of PAP-2 could terminate signalling by these bioactive lipids and at the same time generates compounds such as diacylglycerol, sphingosine and ceramide which are also potent signalling molecules. In relation to phosphatidate metabolism, sphingosine (or sphingosine 1-phosphate) stimulates phospholipase D and thus the formation of phosphatidate. At the same time sphingosine inhibits PAP-2 activity thus further increasing phosphatidate concentrations. By contrast, ceramides inhibit the activation of phospholipase D by a wide variety of agonists and increase the dephosphorylation of phosphatidate, lysophosphatidate, sphingosine 1-phosphate and ceramide 1-phosphate. These actions demonstrate "cross-talk' between the glycerolipid and sphingolipid signalling pathways and the involvement of PAP-2 in modifying the balance of the bioactive lipids generated by these pathways during cell activation.     

Heat-induced elevation of ceramide in Saccharomyces cerevisiae via de novo synthesis

Sphingolipid-related metabolites have been implicated as potential signaling molecules in many studies with mammalian cells as well as in some studies with yeast. Our previous work showed that sphingolipid-deficient strains of Saccharomyces cerevisiae are unable to resist a heat shock, indicating that sphingolipids are necessary for surviving heat stress. Recent evidence suggests that one role for the sphingolipid intermediate ceramide may be to act as a second messenger to signal accumulation of the thermoprotectant trehalose. We examine here the mechanism for generating the severalfold increase in ceramide observed during heat shock. As judged by compositional analysis and mass spectrometry, the major ceramides produced during heat shock are similar to those found in complex sphingolipids, a mixture of N-hydroxyhexacosanoyl C18 and C20 phytosphingosines. Since the most studied mechanism for ceramide generation in animal cells is via a phospholipase C-type sphingomyelin hydrolysis, we examined S. cerevisiae for an analogous enzyme. Using [3H]phytosphingosine and [3H]inositol-labeled yeast sphingolipids, a novel membrane-associated phospholipase C-type activity that generated ceramide from inositol-P-ceramide, mannosylinositol-P-ceramide, and mannose(inositol-P)2-ceramide was demonstrated. The sphingolipid head groups were concomitantly liberated with the expected stoichiometry. However, other data demonstrate that the ceramide generated during heat shock is not likely to be derived by breakdown of complex sphingolipids. For example, the water-soluble fraction of heat-shocked cells showed no increase in any of the sphingolipid head groups, which is inconsistent with complex sphingolipid hydrolysis. Rather, we find that de novo ceramide synthesis involving ceramide synthase appears to be responsible for heat-induced ceramide elevation. In support of this hypothesis, we find that the potent ceramide synthase inhibitor, australifungin, completely inhibits both the heat-induced increase in incorporation of [3H]sphinganine into ceramide as well as the heat-induced increase in ceramide as measured by mass. Thus, heat-induced ceramide most likely arises by temperature activation of the enzymes that generate ceramide precursors, activation of ceramide synthase itself, or both.     

Membrane-destabilizing properties of C2-ceramide may be responsible for its ability to inhibit platelet aggregation

We have studied the effects of short-chain ceramides on platelet structure and function. N-Acetylsphingosine (C2-ceramide), a cell-permeable short-chain analogue, and N-acetyldihydrosphingosine (C2-dihydroceramide), which lacks the 4-5 double bond, have been investigated. C2-Ceramide (15 microM) inhibited ADP-induced aggregation by 50% at a platelet concentration of 1.25 x 10(8)/mL, while it took twice that concentration to inhibit aggregation by 50% when the platelet concentration was doubled. This indicates that the effect of C2-ceramide on ADP-induced platelet aggregation depends on the ratio of ceramide to total platelet lipid, with a ratio of 0.2 giving significant inhibition. C2-Ceramide at a ceramide: lipid ratio of 0.2 caused platelets to form fenestrations and pseudopodia which were longer and thinner than those caused by agonists such as ADP or thrombin. C2-Dihydroceramide had no effect on ADP-induced aggregation or platelet morphology at any ceramide:lipid ratio. Platelet lysis was induced by C2-ceramide at higher ceramide:lipid ratios (0.5), whereas C2-dihydroceramide did not induce lysis, suggesting that C2-ceramide is able to destabilize membranes. This was tested directly by assessing whether the ceramides induced leakage of 6-carboxyfluorescein from lipid vesicles. C2-Ceramide caused nearly total leakage of dye from the vesicles at a ceramide:lipid ratio of 10. The leakage caused by C2-dihydroceramide at a ceramide:lipid ratio of 10 was equal to that induced by C2-ceramide at a ratio of 0.2 (approximately 3%). The ability of the ceramides to destabilize membranes was also examined by measuring changes in fluorescence anisotropy of the fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH) incorporated into lipid vesicles. C2-Ceramide induced a larger decrease in anisotropy than a detergent (Triton X-100) which is known to lyse membranes. C2-Dihydroceramide did not alter membrane fluidity. The ability of C2-ceramide to cause platelet fenestrations, formation of irregular platelet pseudopodia, platelet lysis, lipid vesicle leakage, and increases in the fluidity of lipid vesicles all suggest that C2-ceramide inhibits platelet aggregation because it destabilizes the platelet membrane. C2-Dihydroceramide did not inhibit platelet aggregation and lacked the nonspecific effects on membranes that C2-ceramide possessed, suggesting that C2-dihydroceramide is not an appropriate control for the nonspecific effects of C2-ceramide.     

Sphingomyelinase induces lipid microdomain formation in a fluid phosphatidylcholine/sphingomyelin membrane

The behaviors of two chemically well-defined sphingolipids, N-palmitoyl-sphingomyelin (C16:0-SM) and the corresponding ceramide (C16:0-Cer), in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) matrix were compared. Minor attenuation of lateral diffusion upon increasing the mole fraction of C16:0-SM (XSM, up to 0.25) was indicated by the slight decrement in the excimer/monomer intensity ratio (Ie/Im) for a trace amount (mole fraction X = 0.01) of a pyrene-labeled ceramide analogue (N-[(pyren)-1-yl]decanoyl-sphingosine, PDCer) in keeping with the miscibility of C16:0-SM in POPC. Increasing membrane order was revealed by the augmented polarization P for diphenylhexatriene (DPH). In contrast, when C16:0-Cer was substituted for C16:0-SM an approximately 1.6-fold increase in Ie/Im for PDCer was evident upon increasing Xcer, with parallel increment in DPH polarization. In agreement with our recent data on natural ceramides in dimyristoylphosphatidylcholine (DMPC) bilayers [Holopainen et al. (1997) Chem. Phys. Lipids 88, 1-13], we conclude that C16:0-Cer becomes enriched into microdomains in the fluid POPC membrane. Interestingly, enhanced formation of microdomains by ceramide was observed when the total sphingolipid content in tertiary alloys with POPC was maintained constant (Xcer + XSM = 0.25) and the SM/Cer stoichiometry was varied. Finally, when ceramide was generated enzymatically in POPC/C16:0-SM (3:1, molar fraction) LUVs by sphingomyelinase (SMase, Bacillus cereus), maximally approximately 85% of hydrolysis of sphingomyelin was measured within <3 min at 30 degreesC. The formation of ceramide was accompanied by a closely parallel increase in DPH polarization. There was also an increase in Ie/Im for PDCer; however, these changes in Ie/Im were significantly slower, requiring approximately 105 min to reach a steady state. These data show that the rapid enzymatic formation of ceramide under these conditions is followed by much slower reorganization process, resulting in the formation of microdomains enriched in this lipid.     

Purification and characterization of a novel ceramidase from Pseudomonas aeruginosa

We report here a novel type of ceramidase of Pseudomonas aeruginosa AN17 isolated from the skin of a patient with atopic dermatitis. The enzyme was purified 83,400-fold with an overall yield of 21.1% from a culture supernatant of strain AN17. After being stained with a silver staining solution, the purified enzyme showed a single protein band, and its molecular mass was estimated to be 70 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme showed quite wide specificity for various ceramides, i.e. it hydrolyzed ceramides containing C12:0-C18:0 fatty acids and 7-nitrobenz-2-oxa-1, 3-diazole-labeled dodecanoic acid, and not only ceramide containing sphingosine (d18:1) or sphinganine (d18:0) but also phytosphingosine (t18:0) as the long-chain base. However, the enzyme did not hydrolyze galactosylceramide, sulfatide, GM1, or sphingomyelin, and thus was clearly distinguished from a Pseudomonas sphingolipid ceramide N-deacylase (Ito, M., Kurita, T., and Kita, K. (1995) J. Biol. Chem. 270, 24370-24374). This bacterial ceramidase had a pH optimum of 8.0-9.0, an apparent Km of 139 microM, and a Vmax of 5.3 micromol/min/mg using N-palmitoylsphingosine as the substrate. The enzyme appears to require Ca2+ for expression of the activity. Interestingly, the 70-kDa protein catalyzed a reversible reaction in which the N-acyl linkage of ceramide was either cleaved or synthesized. Our study demonstrated that ceramidase is widely distributed from bacteria to mammals.     

X-ray diffraction analysis of isolated skin lipids: reconstitution of intercellular lipid domains

Low- and wide-angle X-ray diffraction were used to determine the structural organization of lipids isolated from the stratum corneum extracellular matrix that forms the major water permeability barrier in mammalian epidermis. Hydrated pig skin ceramides gave a single low-angle reflection of about 62 angstroms and a wide-angle-reflection at 4.15 angstroms. The addition of either cholesterol or fatty acid, the other major lipid components of the skin stratum corneum extracellular matrix, modified this diffraction pattern, depending on the lipid mole ratios. In the absence of water, lipid mixtures exhibited lipid phase separation, as shown by low- and wide-angle reflections typical of a separate cholesterol phase. However, a hydrated 2:1:1 mole ratio of ceramide:cholesterol:palmitic acid (similar to that found in stratum corneum) produced a diffraction pattern with a single sharp wide-angle reflection at 4.10 angstroms and low-angle reflections which indexed as the first eight orders of a single repeat period of 130 angstroms. The repeat period and intensity distribution of the low-angle data were similar to those found in intact stratum corneum [White et al. (1988) Biochemistry 27, 3725-3732; Bouwstra et al. (1994) Biochim. Biophys. Acta 1212, 183-192]. Higher concentrations of cholesterol or palmitic acid resulted in lipid phase separations. The 130 angstrom repeat period decreased only about 3 angstroms as water was removed by incubation in low-relative humidity atmospheres. The 130 angstrom repeat period depended on the presence of a particular ceramide, N-(omega-acyloxy)-acylsphingosine, which is found only in the epidermis. In contrast, 2:1:1 mixtures of brain ceramide:cholesterol:palmitic acid gave reflections of 56 and 34 angstroms. These results indicate that a structure with dimensions similar to those of the lamellar repeating unit found in skin stratum corneum does not depend on the presence of protein but does depend on the presence of specific skin ceramides and appropriate concentrations of cholesterol and fatty acid.     

Porphyromonas gingivalis genes isolated by screening for epithelial cell attachment

Porphyromonas gingivalis is associated with chronic and severe periodontitis in adults. P. gingivalis and the other periodontal pathogens colonize and interact with gingival epithelial cells, but the genes and molecular mechanisms involved are unknown. To dissect the first steps in these interactions, a P. gingivalis expression library was screened for clones which bound human oral epithelial cells. Insert DNA from the recombinant clones did not contain homology to the P. gingivalis fimA gene, encoding fimbrillin, the subunit protein of fimbriae, but showed various degrees of homology to certain cysteine protease-hemagglutinin genes. The DNA sequence of one insert revealed three putative open reading frames which appeared to be in an operon. The relationship between P. gingivalis attachment to epithelial cells and the activities identified by the screen is discussed.     

Deletion of the putative effector region of Era, an essential GTP-binding protein in Escherichia coli, causes a dominant-negative phenotype

Gangliosides are highly immunosuppressive molecules but the mechanism(s) by which they act upon cells remains to be fully defined. Several metabolic products of exogenous gangliosides, including ceramide, have recently been suggested as second messengers in programmed cell death (PCD). Therefore, we have probed the role of gangliosides and ceramides in the induction of PCD and in the inhibition of in vitro lymphoproliferation. PCD was caused only by exogenous ceramides with short fatty acyl groups-d18:1-C2:0 (C2-ceramide, where d18:1 is sphingosine and C2:O is an acetyl group) and d18:1-C6:0 (C6-ceramide, where C6:O is a hexanoyl group). None of the gangliosides studied induced PCD, including naturally occurring GM3, synthetic d18:1-C18:0 GM3 (C18-Cer GM3, where C18:0 is a stearoyl group), or even d18:1-C2:0 GM3 (C2-Cer GM3), which itself contains a PCD-causing ceramide. However, these gangliosides were highly immunosuppressive, inhibiting antigen-induced lymphoproliferation at micromolar concentrations. We conclude that exogenous sphingolipids cause inhibition of lymphoproliferation and PCD by two separate and distinct mechanisms of action.     

Molecular arrangements in sphingolipids. Conformation and hydrogen bonding of ceramide and their implication on membrane stability and permeability

The preferred conformation of the ceramide part of sphingolipids has been deduced from single crystal structures of a series of sphingolipid constituents: N-tetracosanoylphytosphingosine, glycosylphytosphingosine hydrochloride, sphingosine hydrochloride, triacetylsphingosine, DL-2-hydroxytetradecanoic acid and N-stearoylethanolamine. The amide group of the ceramide, which serves as a link between the hydrocarbon chains, has a basic significance for the contormation of the entire molecule. This rigid group, which comprises six atoms in a planar conformation, adopts a perpendicular orientation towards the axes of the two hydrocarbon chains. The carbonyl oxygen thereby turns into an eclipsed position with the hydrogen atoma at carbon atom 2 of the sphingosine. A parallel chain stacking is achieved by a sharp perpendicular bend of the fatty acid. This bend is produced by a sequence of two --60 degrees rotations about the C-C bonds at both sides of the alpha-carbon atom. The orientation of the hydrogen bond donors and acceptors of the amide group and the hydroxyl groups allow lateral interaction with other lipid molecules. The proposed models are supported by infrared spectra, thin-layer chromatographic behaviour and monolayer studies of synthetic model ceramides. The functional role of the hydrogen bonding groups in the ceramide part of sphingolipids is emphasized and their significance for the formation of lateral hydrogen bonds within the membrane layer and thereof arising effects on membrane stability and permeability are discussed.     

Influence of cardiopulmonary bypass surgery on cancer-specific survival rate of patients with colorectal cancer

We investigated whether cystatins and cystatin-derived peptides, encompassing sequences of secondary structures of cystatin S and papain binding domains of cystatin C, display antimicrobial properties. Of the different microorganisms tested, only the growth of P. gingivalis was inhibited by chicken cystatin and cystatin C. Cystatin S, cystatin S:1-14, cystatin S:61-73 and cystatin S:108-121 also inhibited its growth, whereas cystatin S:21-38, cystatin S:39-55, cystatin S:81-95, cystatin S:94-109, and cystatin C: 9-12/55-60/106-107 did not. No inhibition of the cysteine proteinase activity of P. gingivalis was observed for all cystatin-derived peptides. On the other hand, leupeptin and antipain inhibited P. gingivalis proteinase activity, but had no effect on the growth. These data suggest that cystatins contain antibacterial sequences active against P. gingivalis and that the growth inhibition does not depend on the inhibition of P. gingivalis cysteine proteinases.     

Definition of conditions for the detection of genotoxic chemicals in the adult rat-liver epithelial cell/hypoxanthine-guanine phosphoribosyl transferase (ARL/HGRPT) mutagenesis assay

1. Fresh human erythrocytes were treated with lytic and non-lytic combinations of phospholipases A2, C and sphingomyelinase. The 31P-NMR spectra of ghosts derived from such erythrocytes show that, in all cases, the residual phospholipids and lysophospholipids remain organized in a bilayer configuration. 2. A bilayer configuration of the (lyso)phospholipids was also observed after treatment of erythrocyte ghosts with various phospholipases even in the case that 98% of the phospholipid was converted into lysophospholipid (72%) and ceramides (26%). 3. A slightly decreased order of the phosphate group of phospholipid molecules, seen as reduced effective chemical shift anisotropy in the 31P-NMR spectra, was found following the formation of diacyglycerols and ceramides in the membrane of intact erythrocytes. Treatment of ghosts always resulted in an extensive decrease in the order of the phosphate groups. 4. The results allow the following conclusions to made: a. Hydrolysis of phospholipids in intact red cells and ghosts does not result in the formation of non-bilayer configuration of residual phospholipids and lysophospholipids. b. Haemolysis, which is obtained by subsequent treatment of intact cells with sphingomyelinase and phospholipase A2, or with phospholipase C, cannot be ascribed to the formation of non-bilayer configuration of phosphate-containing lipids. c. Preservation of bilayer structure, even after hydrolysis of all phospholipid, shows that other membrane constitutents, e.g. cholesterol and/or membrane proteins play an important role in stabilizing the structure of the erythrocyte membrane. d. A major prerequisite for the application of phospholipases in lipid localization studies, the preservation of a bilayer configuration during phospholipid hydrolysis, is met for the erythrocyte membrane.