Changes in Lipid Composition During Manganese-Induced Apoptosis in PC12 Cells.

Lipid composition of membranes is fundamental to modulate signaling pathways relying on lipid metabolites and/or membrane proteins, thus resulting in the regulation of important cell processes such as apoptosis. In this case, membrane remodeling is an early event important for the activation of signaling leading to cell death and removal of apoptotic cells. In the present study, we analyzed phospholipid, cholesterol and fatty acid content during apoptosis induced by manganese in PC12 cells. Lipid analysis of whole cells and detergent-resistant membranes was carried out by HPLC/GC. Results showed that apoptosis is associated with changes in lipid composition detectable in whole cell extracts, namely cholesterol, phosphatidylserine and phosphatidylethanolamine decreases. Noteworthy, phosphatidylserine level reduction was detectable before to the detection of apoptosis, in correlation with our previous study carried out by radioactive labelling. By contrast, phosphatidylserine and phosphatidylethanolamine changes were not detected in detergent resistant membranes, which instead showed an altered composition in phosphatidylinositol, phosphatidylcholine and sphingomyelin in apoptotic cells.

Atomic force microscopy imaging of lipid rafts of human breast cancer cells.

Several studies suggest that the plasma membrane is composed of micro-domains of saturated lipids that segregate together to form lipid rafts. Lipid rafts have been operationally defined as cholesterol- and sphingolipid-enriched membrane micro-domains resistant to solubilization by non-ionic detergents at low temperatures. Here we report a biophysical approach aimed at investigating lipid rafts of MDA-MB-231 human breast cancer cells by coupling an atomic force microscopy (AFM) study to biochemical assays namely Western blotting and high performance thin layer chromatography. Lipid rafts were purified by ultracentrifugation on discontinuous sucrose gradient using extraction with Triton X-100. Biochemical analyses proved that the fractions isolated at the 5% and 30% sucrose interface (fractions 5 and 6) have a higher content of cholesterol, sphingomyelin and flotillin-1 with respect to the other purified fractions. Tapping mode AFM imaging of fraction 5 showed membrane patches whose height corresponds to the one awaited for a single lipid bilayer as well as the presence of micro-domains with lateral dimensions in the order of a few hundreds of nanometers. In addition, an AFM study using specific antibodies suggests the presence, in these micro-domains, of a characteristic marker of lipid rafts, the protein flotillin-1.

Long chain omega 3 polyunsaturated fatty acids supplementation in the treatment of elderly depression: effects on depressive symptoms, on phospholipids fatty acids profile and on health-related quality of life.

OBJECTIVE: Recent observations showed that long chain omega 3 polyunsaturated fatty acids (n-3 LCPUFA) could represent a potential treatment for elderly depression. To determine if a n-3 LCPUFA containing supplement improves depressive symptoms, changes phospholipids acids profile and ameliorates Health related quality of life (HRQoL) in depressed elderly patients. DESIGN: Two-months, randomized, double-blind, placebo-controlled trial. SETTING: Nursing home in Pavia, Italy. SUBJECTS: Forty-six depressed females, aged 66-95 years. INTERVENTION: 22 depressed females were included in the intervention group (n-3 group, that received 2.5 g/day of n-3 LCPUFA, with 1.67 grams of EPA and 0.83 grams of DHA), and 24 patients in the placebo group. The primary endpoint was the improvement of depressive symptoms as evaluated by Geriatric Depression Scale (GDS). Secondary endpoints were the evaluation of modifications of erythrocyte membrane phospholipids fatty acid profile and of of HRQoL, by using the Short-Form 36-Item Health Survey (SF-36). All parameters were assessed before and after the treatment period of 8 weeks. RESULTS: The mean GDS at 2 months was significantly lowered only for the n-3 group. SF-36 physical and mental components were significantly increased in the intervention group. Compliance was good, as confirmed by erythrocyte membrane phospholipid FA concentrations, with significant increase of EPA and DHA in the intervention group. CONCLUSION: The supplementation of n-3 LCPUFA in elderly female patients reduces the occurrence of depressive symptoms, improves phospholipids fatty acids profile and health-related quality of life.

Mare's colostrum globules stimulate fibroblast growth in vitro: a biochemical study.

The wound repair function of mare's milk and colostrum was investigated. Mare's colostrum improved wound healing in vivo; thus fibroblast growth activation by mare's milk and colostrum was examined. As expected, colostrum was more effective than milk. To establish the biochemical nature of the bioactive molecules involved, colostrum was fractionated into whey, casein, and fat globules, and the efficacy of these fractions on fibroblast proliferation was studied. The fat globule fraction provided the strongest stimulation; its composition was studied and compared with the less-active milk fat globule fraction. The lipid pattern highlighted several differences between mare's colostrum and milk; in particular, total lipid, linoleic acid, linolenic acid, ganglioside, and glycolipid contents were higher in colostrum. A proteomic investigation revealed some differences between the protein composition of colostrum and milk fat globules. Adipophylin and lactadherin were significantly overexpressed in colostrum fat globules. The role of specific lipids on skin wound repair and that of the epidermal growth factor-like domain, embedded within the lactadherin molecule and probably released in conditions stimulating proteolysis, are discussed.

Blood profiles, body fat and mood state in healthy subjects on different diets supplemented with Omega-3 polyunsaturated fatty acids.

BACKGROUND: Diets and Omega-3 polyunsaturated fatty acids have been considered as important factors to reduce the risk of cardiovascular and inflammatory diseases, but there are few details on the effects on healthy subjects. The aim of the present study was to examine the variation of several physiological parameters in healthy subjects on different diets supplemented with Omega-3 fatty acids. MATERIALS AND METHODS: The experiment was carried out on 33 subjects divided into four groups according to a double-blind cross-over design with a 1 : 1 ratio for Omega-3 (vs. placebo) and open-label parallel-groups with a 1 : 1 ratio for the Zone diet (vs. the diet suggested by the Italian National Research Institute for Nutrition and Foods). Blood samples were collected at the beginning of the experiment and after 35 (cross-over) and 70 days. The Profile of Mood States test (POMS) was also performed. RESULTS: The arachidonic acid/eicosapentaenoic acid ratio (AA/EPA) was strongly reduced by Omega-3 with a supplementary effect of the diet and in particular the Zone diet. The AA/EPA reduction was correlated with a concomitant decrease of insulin and homocysteine levels. The Zone diet reduced skinfold thickness and body fat percentage and also showed antioxidant effects. The mood state changed after Omega-3 supplementation, with an increased POMS index. This was related to a concomitant reduction of AA/EPA and was particularly evident in the Zone diet. CONCLUSION: AA/EPA and mood state are differently influenced by diet and Omega-3, body fat is particularly reduced by Zone diet, while blood parameters such as triglycerides/HDL ratio, insulin and homocysteine are related to AA/EPA variations. These findings are discussed in terms of differences in the composition of the diets and the influences of Omega-3 on physiological functions.

Glycolipid analysis of different tissues and cerebrospinal fluid in type II Gaucher disease.

The lipid composition or the liver, spleen, brain, cerebellum and cerebrospinal fluid of a Gaucher disease type II patient who died at the age of 5 months was examined. The glycolipid analysis demonstrated a marked increase of total amounts not only in the peripheral tissues but also in the brain cerebellum and cerebrospinal fluid, with a prevalence of glucosylceramide. A reduction in gangliosides was observed in all the analysed tissues with a relative increase of GD3 in the nervous tissue. The fatty acid composition of glucosylceramide showed a prevalence of stearic acid in the central nervous system, while in the peripheral tissues palmitic acid was prevalent. This result suggests a different origin of the glucosylceramide stored in different tissues. The generalized reduction of gangliosides and their modified distribution together with the central nervous system GD3 increment represent a new observation. These data could be useful in the effort to clarify the pathophysiological mechanism of brain damage in neuronopathic Gaucher disease.

Effect of ethanol exposure on Xenopus embryo lipid composition.

Exposure to ethanol is known to alter cell membranes both chemically and biophysically; these effects may be related to the development of tolerance and physical dependence. In the present study, the effects of various concentrations of ethanol (1-250 mM) on the lipid composition after the first 6 days of Xenopus embryo development were investigated, using an in vitro fertilization technique. Lipid analysis revealed: (1) a decrease of the cholesterol/phospholipid molar ratio mainly derived from a higher content of phospholipids; (2) an increase of phospholipid unsaturated fatty acids, especially C20:4 and C20:5, with ethanol concentration of 150-250 mM; (3) a decrease of lipid-bound sialic acid with ethanol concentrations of > or =5 mM. These results underline that sialoglycoconjugates are a more sensitive target of alcohol in comparison with other lipid components. The cultured embryo method certainly represents a useful model for investigation of the direct effects of ethanol on lipid metabolism, excluding maternal interference which can lead to misinterpretation of data.

Comparison between extra virgin olive oil and oleic acid rich sunflower oil: effects on postprandial lipemia and LDL susceptibility to oxidation.

The aim of our study was to determine whether the minor polar components of virgin olive oil could have favorable effects (1) on fasting and postprandial lipid profile and (2) on low-density lipoprotein (LDL) composition and susceptibility to oxidation in vitro. Ten normolipidic subjects were included in a crossover study (two diet periods of 3 weeks) and received either virgin olive oil (OO diet) or oleic acid rich sunflower oil. An oral fat load was performed at the end of each period. The plasma lipid levels were not significantly different after both diets in the fasting and postprandial states. A few minor variations of the LDL composition were observed only in the postprandial lipemia, and they were different after both diets. The LDL oxidation susceptibility was evaluated by the formation of conjugated dienes. With LDL isolated in the fasting state, the diene production decreased (p = 0.0573) only after the OO diet. The dienes determined at time 0 and the maximal dienes obtained during the oxidation reaction decreased (p = 0.0145 and p = 0.0184, respectively) only after the OO fat load. Nevertheless, the diene production decrease was not significant (p = 0.0848). Our results suggest a mild effect of minor components of virgin olive oil related to a decrease of LDL susceptibility to oxidation; further analyses are necessary to give clear conclusions about their role.

Rat erythrocyte susceptibility to lipid peroxidation after chronic ethanol intake.

Erythrocytes, from 2- and 7-month-old rats chronically fed with a liquid diet containing ethanol, were analyzed for their susceptibility to lipid peroxidation estimated as thiobarbituric acid reactive substance (TBARS) products. In spite of a lower alcohol consumption, adult rats were the more affected by the ethanol treatment. Erythrocyte membranes prepared from alcoholic animals were more sensitive to lipid peroxidation than those prepared from control rats. In both age groups lipid analysis revealed similar changes: 1) an increased cholesterol/phospholipid molar ratio mainly derived from a higher content of cholesterol that accounts for the lower membrane fluidity and the higher tolerance to the disordering effect exerted by ethanol in vitro; 2) an increase of phospholipid unsaturated fatty acids, especially C20:4; 3) a modification of the phospholipid pattern, characterized in the ethanol group by an increase of PE and decrease of PE and decrease of PC levels; moreover, significant increases of the anionic phospholipids were detected in the adult group.

Vitamin D metabolites activate the sphingomyelin pathway and induce death of glioblastoma cells.

1 alpha, 25-dihydroxyvitamin D3 was previously shown to induce cell death in brain tumour cell lines when added to the medium at micromolar concentration. In this paper we show that Cholecalciferol, a poor ligand of the vitamin D receptor, also induces cell death of HU197 human glioblastoma cell line and early passages cultures derived from a recurrent human glioblastoma. This finding suggests that the effects of vitamin D metabolites on brain tumour cells are at least partially independent from the activation of the classic nuclear receptor pathway. Vitamin D metabolites have been shown to activate the sphingomyelin pathway inducing an increase in cellular ceramide concentration. We determined the levels of sphingomyelin ceramide and ganglioside GD3 in Hu197 cells after treatment with cholecalciferol. A significant increase in ceramide concentration and a proportional decrease in sphingomyelin was already present after 6 hours of cholecalciferol treatment when no morphological changes were visible in the cultures. Treatment with ceramides (N-acetylsphingosine or natural ceramide from bovine brain) of the same cells also induces cell death. Similarly, treatment of the same cells with bacterial Sphingomyelinase also results in cell death. The demonstration of an increase in intracellular ceramide after cholecalciferol treatment and the ability of ceramide to induce cell death suggest that the sphingomyelin pathway may be implicated in the effect of vitamin D metabolites on human glioblastoma cells. Inhibition of ceramide biosynthesis by fumonisin B1 treatment did not alter the dose response curve of HU197 cells to cholecalciferol. Insensitivity to fumonisin B1 together with a decrease in sphingomyelin content after cholecalciferol treatment indicate that activation of sphingomyelinase should be responsible for the increase in intracellular ceramide concentration.

Prolonged in vitro culture modifies the surface lipid composition of murine melanoma cell lines.

Lipid composition of two murine melanoma cell variants (B16, without malignant properties and B16-F10, with high metastatic activity), has been examined at different stages of growth. The aim of the work was to identify cell surface modifications due to the time length of in vitro culture, that could be one variable to consider when metastatic potential is studied. Some of the analyzed parameters (ganglioside- and glycoprotein-bound neuraminic acid, cholesterol, neutral glycolipids, phospholipids, triacylglycerols) undergo statistically significant variations at the various passages in B16-F10 line. Fatty acids composition of the phospholipidic fraction was changed only at the last observed passage (100) in B16 line. No one of the examined parameters justifies the ability of B16-F10 cells to invade distant districts and to originate new tumors. Probably detailed lipid analysis on cellular subfractions, as already performed in this study on total lipid extract of the whole cell, could be a valuable tool to identify differences related with metastatic potential.

Glycosphingolipid expression in solid tumours and transformed cell lines.

Glycosphingolipids are assumed to play a crucial role in cell-cell and cell-substrate interactions, including cell adhesion, proliferation, differentiation and apoptosis. Furthermore, cell surface glycolipid profile changes in the so called "social disorders", such as malignant transformation. To better investigate these modifications, the ganglioside composition in different solid tumours and in two transformed cell lines was analyzed. In some of these models we also tried to correlate the pattern of gangliosides to the key enzymes involved in their metabolism. The results we obtained can be summarized as follows:(1), meningiomas with or without chromosome 22 deletion: predominance of ganglioside GD3 in the former and of ganglioside GM3 in the latter. Correlation between GM3/GD3 ratio and SAT-2 activity; (2), mammary carcinomas developed in MMTV/c-neu transgenic mice: accumulation of GM3-derived species. The different ganglioside distribution seems to correlate with the tumour size; (3), Sarcoma Galliera-strain cells SGS/3A and normal syngenic murine fibroblasts FG: transformed cells exhibit a lower activity of sialyltransferases (SAT-1, SAT-2, SAT-4) compared to normal fibroblasts, suggesting a possible correlation with the ganglioside pattern. The neuraminidase activity seems to correlate to the glycoprotein sialic acid content; (4), 3T3 normal murine fibroblasts and SVT2 transformed cells: GM3 is absent in 3T3, while it accounts for the main ganglioside species in SVT2. On the contrary, GM2 present in a large amount in normal fibroblasts, is practically absent in transformed cells. No correlation has been observed between ganglioside profile and glycosyltransferase activities so far examined.

Prolonged in vitro culture modifies the surface lipid composition of murine melanoma cell lines.

Lipid composition of two murine melanoma cell variants (B16, without malignant properties and B16-F10, with high metastatic activity), has been examined at different stages of growth. The aim of the work was to identify cell surface modifications due to the time length of in vitro culture, that could be one variable to consider when metastatic potential is studied. Some of the analyzed parameters (ganglioside- and glycoprotein-bound neuraminic acid, cholesterol, neutral glycolipids, phospholipids, triacylglycerols) undergo statistically significant variations at the various passages in B16-F10 line. Fatty acids composition of the phospholipidic fraction was changed only at the last observed passage (100) in B16 line. No one of the examined parameters justifies the ability of B16-F10 cells to invade distant districts and to originate new tumors. Probably detailed lipid analysis on cellular subfractions, as already performed in this study on total lipid extract of the whole cell, could be a valuable tool to identify differences related with metastatic potential.

Age-related effects of chronic ethanol intake on physical properties, lipid composition and galactosyltransferase activity of rat small intestine microsomes.

The effect of a 4-week ethanol administration on: (1) glycoprotein content of brush border membrane (BBM): (2) galactosyltransferase activity; (3) lipid composition and fluidity of intestinal microsomes prepared from young and adult rats was investigated. In spite of a lower alcohol consumption, the more dramatic effects of treatment have been observed in the older rats, where BBM protein-bound hexoses and microsomal galactosyltransferase activity were significantly decreased. On the contrary, these parameters were unaffected in young rats. However, both rat groups were similarly affected in having their microsomal cholesterol contents significantly increased. Microsomal membranes from ethanol-fed adult rats were less fluid compared to control rats: the high fluorescence anisotropy value could be related to the high cholesterol/phospholipid ratio and to the decrease of the unsaturated fatty acids C22:4 and C22:6.

Glycosyltransferase activities in human meningiomas. Preliminary results.

The biosynthesis of a given glycosphingolipid is under the control of specific glycosyltransferases, while its catabolism is catalyzed by step-wise action of glycosidases. The net amount of glycolipids apparently result from the difference between these two processes. However, other parameters should be taken into consideration, such as intracellular recycling of catabolic products, membrane insertion, and membrane turnover. In order to establish a possible correlation between ganglioside expression in brain tumor and the activities of the enzymes involved in their metabolism, we analyzed the activities of specific sialyltransferases (SAT-1 and SAT-2), galactosyltransferase (GalT-4), N-acetylgalactosaminyltransferase (GalNAcT-1), and N-acetylglucosaminyltransferase (GlcNAcT-1) in 9 human meningiomas whose ganglioside pattern was characterized either by the predominance ganglioside GM3 (4 out of 9) or ganglioside GD3 (5 out of 9). The results indicated a strong correlation between the GM3/GD3 ratio and SAT-2 activity; to the contrary, SAT-1 activity did not show any correlation if compared with the Lc2/GM3 ratio. In all the samples where GM3 was the main ganglioside, little or no activity of GalNAcT-1 and GlcNAcT-1 was detectable.

Phospholipid distribution and fatty acid composition in different brain regions during chick embryo development.

The phospholipid profile of different chick embryo brain regions was studied from 11 to 21 days of development, revealing interesting changes in content and distribution. Total phospholipid phosphorus (P), in micrograms of P per microgram of DNA, increases significantly during development of cerebral hemispheres (CHs), optic lobes (OLs), and brainstem (BS). Compared with CH and OL, the BS shows at all stages a significantly higher concentration of phospholipid P, which in contrast decreases in the cerebellum (CB) during development. Moreover, the data show interesting differences between the right and the left portion of the brain. The distribution of phospholipid P and the fatty acid composition of phospholipids were asymmetric between left and right OL and CH, as were the concentrations of DNA and cholesterol, demonstrating lateralized neurochemical development in these structures, i.e., left OL, right OL, left CH, and right CH. The data are discussed also in relation to the potential importance of neurochemical lateralization for determining lateralized embryonic and postnatal behavior of this species.

Altered membrane lipid composition in a human meningosarcoma.

In a sample of meningosarcoma, obtained at the time of surgery, the amount of total gangliosides and phospholipids was examined, together with the cholesterol content and the distribution of different ganglioside and phospholipid species. The phosphatidylinositol, phosphatidylinositol-4-phosphate, phosphatidylinositol-4, 5-bisphosphate and phosphatidylcholine fatty acid composition was also analyzed. The ganglioside pattern in the meningosarcoma was different from the previously reported pattern in meningiomas of different histological origin, showing a higher concentration of GD3, indicating that the so-called b pathway of ganglioside biosynthesis was the preferred one in this type of tumor; moreover the percentage content of polysialylated gangliosides was very low. Cholesterol and phospholipid content was lower than in meningiomas; the phosphatidylcholine increase and the sphingomyelin decrease would indicate a lower membrane microviscosity, a characteristic of tumor cells. Phosphoinositide and phosphatidylcholine fatty acid analysis revealed a considerable amount of docosahexaenoic acid. This abnormal presence of this fatty acid could lead to the production, after receptor stimulation, of a diacylglycerol containing docosahexaenoic acid, which, in turn, could be responsible for an altered activation pattern of protein kinase C, in this way promoting carcinogenesis.

Treatment of sphingomyelinase deficiency by repeated implantations of amniotic epithelial cells.

Five young patients with Niemann-Pick disease type B were treated with repeated implantations of amniotic epithelial cells, as a source of exogenous sphingomyelinase. This treatment abolished the recurrent infections, mainly of the respiratory tract, and led to other improvements of the general conditions of the patients. In particular, we noticed a disappearance of vomiting, a recovery from muscular hypotrophy, and significantly reduced pulmonary distress. In four subjects, who were in a prepuberal state, there was a puberal spurt with a concomitant burst of growth. In two cases, characterized by a greater than normal content of sphingomyelin in urinary sediments, a single implantation caused a sustained normalization of sphingomyelin and total phospholipids in the urine. Finally, sphingomyelinase activity of peripheral leukocytes, when assayed 0.5 to 4 months after some of the implantations, showed a rise to heterozygous values in 30-40% of the assays.

A new procedure for gangliosidic N-acetylneuraminic acid analysis in serum.

In this paper a method is presented which is suitable for the extraction, purification and analysis of serum gangliosides. The advantage in comparison with other previously published procedures is the complete extraction of sialoglycolipids without contamination of sialoglycoproteins and/or sialoglycopeptides. The method could be used as a second-level test for the diagnosis and follow-up of cancer patients, and also could be potentially used for pharmaco-kinetic studies after ganglioside treatment.