Involvement of apoptosis-inducing factor during dolichyl monophosphate-induced apoptosis in U937 cells.

Dolichyl monophosphate (Dol-P) has been found to induce apoptosis in human leukemia U937 cells. During this apoptotic execution, the increase of plasma membrane fluidity (5-20 min), caspase-3-like protease activation (2-4 h), chromatin condensation and DNA ladder formation (3-4 h) were observed successively. Here, we report that reduction in mitochondrial transmembrane potential and translocation of apoptosis-inducing factor (AIF) are early events (1-3 h) in the apoptotic process induced by Dol-P in U937 cells. The AIF was concentrated around nuclei and partly translocated to the nuclei, which was confirmed by immunocytochemistry using specific anti-AIF antibody. Both caspase-8 and caspase-3 inhibitors blocked only DNA fragmentation but not mitochondrial processes, AIF migration and chromatin condensation. These results indicate that mitochondrial changes are an early step in the apoptosis induced by Dol-P and AIF is one of the important factors which induce chromatin condensation in nuclei.

Induction of apoptosis in human hematopoietic U937 cells by granulocyte-macrophage colony-stimulating factor: possible existence of caspase 3-like pathway.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) induced apoptosis in human hematopoietic U937 cells by itself and in a synergistic manner with tumor necrosis factor (TNF). GM-CSF-induced apoptosis was not inhibited by caspase inhibitors YVAD-CMK, DEVD-CHO and z-VAD-FMK, under the condition that these inhibitors potently suppressed TNF-induced apoptosis. Both GM-CSF and TNF induced caspase 3-like activity in this cell line though the time course was distinct between two cytokines, and combined stimulation of cells with GM-CSF plus TNF induced additive or synergistic activation of caspase 3-like activity. Amount of immunoreactive cleaved forms of caspase 3 recognized by specific antibody was completely dissociated with its enzymatic activity when the cells were stimulated with GM-CSF, but not with TNF. These results indicate that GM-CSF induces apoptosis of U937 cells via unknown pathway, which seems to be mediated by caspase 3-like activity, yet not caspase 3 itself, resistant to the caspase inhibitors, and synergistically interacts with conventional caspase 3 pathway of TNF. Possible involvement of caspases 1 and 8 (-like activity) but not caspase 7 in this pathway was also suggested.

Control of cell death by the smart polymeric vehicle.

We report a novel drug delivery system for apoptosis induction by a "smart" polymer vehicle possessing thermosensitivity and bioaffinity. The polymer chain was prepared by copolymerization of N-isopropylacrylamide and N-methacryloyloxysuccinimide. Cell-adhesive RGDS peptide was conjugated with the copolymer as a ligand model for bioaffinity. When the temperature was increased, nanoscale aggregates precipitated from a copolymer aqueous solution. Either dolichyl phosphate (dol-p), which is an apoptotic inducer, or dolichol was added to aggregates at around the precipitation temperature (31 degrees C), and the temperature was raised to 37 degrees C for incorporation. Aggregates incorporating dol-p or dolicol were added to a human promonocytic leukemia U937 cell suspension at 37 degrees C. When the temperature was lowered to 25 degrees C, cells underwent apoptosis in the presence of Ca2+. Probably, copolymer vehicles were concentrated on a cell surface through the binding of RGDS and integrin and the release of lipid inducers was caused by the disruption of vehicles in response to temperature.

Serum fatty acids, lipoprotein(a) and apolipoprotein composition of rural, suburban and urban populations in North Vietnam.

This study was conducted to investigate the concentrations of serum fatty acids, lipoprotein(a) and apolipoprotein of three populations in North Vietnam: rural area with low income (n = 101), suburban with average income (n = 97), and urban with high income (n = 95). The results showed the suburban and urban populations had higher fat intake than the rural. The fat intake in quality was different in these three populations. The suburban had the highest consumption of fatty foods rich in n-6 polyunsaturated fatty acid (PUFA). The rural consumed more fatty foods rich in monounsaturated fatty acid (MUFA), but less fatty foods rich in n-3 PUFA than the two other populations. The high index of thrombogenicity (IT) of the Vietnamese diet may result from their low intake of fish and vegetable oils. Risk factors for premature cardiovascular disease (CVD) assessed by serum lipoprotein(a) and apolipoprotein levels were not observed in all three populations. However, coronary heart disease (CHD) and stroke are problems that should be monitored because the increase of CVD morbidity has been reported in Vietnamese people. From a nutritional point of view, the increase of fish and vegetable oils consumption is necessary for the prevention of CVD and CHD in these Vietnamese populations.

Phospholipid turnover in the inflamed intestinal mucosa: arachidonic acid-rich phosphatidyl/plasmenyl-ethanolamine in the mucosa in inflammatory bowel disease.

Cytosolic phospholipase A2 (PLase A2) is activated by low Ca2+ concentrations and translocates from the cytosol to the cell membrane, releasing arachidonic acid; the arachidonic acid cascade then leads to the production of many inflammatory mediators. The aim of this study, accordingly, was to investigate the role of phospholipid metabolism in the intestinal mucosa in inflammatory bowel disease (IBD). Surgically resected specimens from patients with Crohn's disease (CD), ulcerative colitis (UC), and colrectal cancer (non-cancerous tissue; as a control) were submitted to phospholipid analysis and a PLase A2 assay, which measures the degradation of endogenous mucosal phospholipids. A high percentage of plasmenylethanolamine (plas.E) was detected in the glycerophospholipid fraction of CD mucosa. The arachidonic acid content of the phosphatidylethanolamine plus plas.E subfraction was higher in inflamed than in intact mucosa in CD. PLaseA2 activity, resulting in lysophosphatidyl ethanolamine production, was detected only in inflamed mucosa from CD and UC patients, but not in normal mucosa from controls. PLaseA2 activity was highest in moderately inflamed mucosa adjacent to a severely ulcerated area. The PLaseA2 that reacts with endogenous phosphatidylcholine (PC) to form lysoPC was found irrespective of the presence of inflammation. The PLaseA2 that reacts with ethanolamine-containing phospholipids is more closely related to inflammation than other PLaseA2 isoenzymes in IBD mucosa.

Cell membrane dynamics and the induction of apoptosis by lipid compounds.

To investigate the induction of apoptosis by some lipid compounds which are a potent inducer of apoptosis, the plasma membrane fluidity of U937 cells was measured using the fluorescent probe, pyrene. The increase of the membrane fluidity was observed immediately after the treatment of cells with lipid inducers. We also found that the trigger of apoptosis was pulled within 30 min after treatment. Data from the dynamic light scattering experiment indicated that lipid inducers were dissolved to form the emulsion. At the very early stage of apoptosis, possibly, the well-controlled transfer of lipid inducers from the emulsion to the lipid layer of cells can bring about the increase of membrane dynamics which might lead to the induction of apoptosis.

Dihydroheptaprenyl and dihydrodecaprenyl monophosphates induce apoptosis mediated by activation of caspase-3-like protease.

Dolichyl phosphate, an essential carrier lipid in the biosynthesis of N-linked glycoprotein, has been found to induce apoptosis in rat glioma C6 cells and human monoblastic leukemia U937 cells. In the present study, dolichyl phosphate and structurally related compounds were examined regarding their apoptosis-inducing activities in U937 cells. Dihydroheptaprenyl and dihydrodecaprenyl phosphates, of which isoprene units are shorter than that of dolichyl phosphate, induced apoptosis in U937 cells. This phenomenon occurred in a dose- and time-dependent manner, as seen with dolichyl phosphate-induced apoptosis. Derivatives of the same isoprene units of dolichyl phosphate, such as dolichol, dolichal or dolichoic acid, did not induce DNA fragmentation. Farnesyl phosphate and geranylgeranyl phosphate also failed to induce apoptosis. During apoptosis, the caspase family of cysteine proteases play important roles. We observed that apoptosis induced by dihydroprenyl phosphate was mediated by caspase-3-like (CPP32-like) activation but not by caspase-1-like (ICE-like) activation. This caspase-3-like activation was inhibited by a specific inhibitor of caspase-3, DEVD-CHO, but not by an caspase-1 inhibitor YVAD-CHO. We interpret these results to mean that dihydroprenyl phosphates with more than seven isoprene units have apoptosis-inducing activity and that their signal is mediated by caspase-3-like activation.

CPP32 activation during dolichyl phosphate-induced apoptosis in U937 leukemia cells.

Treatment of U937 cells with dolichyl phosphate led to an increase in the activity of the ICE family protease CPP32, accompanied with cleavage of pre-CPP32 to generate p17. Peptide inhibitors YVAD-cmk and Z-Asp-CH2-DCB (specific to ICE) and DEVD-CHO (specific to CPP32) blocked the dolichyl phosphate-induced apoptosis. The dolichyl phosphate-induced increase of CPP32 activity was inhibited by adenylate cyclase inhibitors, SQ 22536 and 2',5'-dideoxyadenosine. Dolichyl phosphate caused a transient increase of intracellular cAMP concentration. The results suggest that modulation of cAMP synthesis due to the stimulation of adenylate cyclase by dolichyl phosphate plays a critical role in CPP32 activation and apoptosis.

Activation of mitogen activated protein kinase in dolichyl phosphate-induced apoptosis in U937 cells.

Exogenous dolichyl phosphate (Dol-P) induced apoptosis in the human monoblastic leukemia cell line U937 within 4 hours. Phosphorylation of p42 mitogen-activated protein kinase (MAP kinase) increased prior to DNA fragmentation. MAP kinase activation occurred within 5 min, and the maximum response was observed at 30 min. Inhibition of tyrosine phosphorylation of MAP kinase by herbimycin A resulted in complete inhibition of DNA fragmentation and partial inhibition of cell death. These results suggested that Dol-P-induced apoptosis is mediated by the MAP kinase cascade.

Biochemical bases in differentiation of a mouse cell line GSM06 to gastric surface cells.

A mouse gastric surface cell line GSM06 established from a transgenic mouse harboring temperature-sensitive simian virus 40 large T-antigen gene was subjected to the lipid and glycoprotein analysis. When GSM06 cells were cultured for a long time after formation of a confluent monolayer, they differentiated to resemble foveolar epithelial cells morphologically. Biochemical changes during culture were studied in cells harvested just when a monolayer had formed (day 0), on day 7, and on day 21. Content of total phospholipids, cholesterol, cholesterol sulfate, total sugar and sialic acid increased about 1.5-fold from day 0 to 7 and remained elevated till day 21. The fatty acid composition of phospholipids revealed increased relative levels of oleic acid in phosphatidylcholine and phosphatidylethanolamine, and an increased level of plasmenylethanolamine from day 0 to 7. The level of dolichylphosphate continued to increase in a time-dependent manner. Glycosylation of various proteins, detected with lectins, was enhanced from day 7. In addition, greater resistance to taurodeoxycholate and acetylsalicylic acid was observed on days 7 and 21 than on day 0. Thus, enhanced glycosylation of proteins and an overall increase in the area of cellular membranes were the major changes in GSM06 cells during culture, and they were accompanied by an enhancement of cytoprotective potential.

Dolichyl phosphate, a potent inducer of apoptosis in rat glioma C6 cells.

Exposure of rat glioma C6 cells to dolichyl phosphate resulted in cell shrinkage followed by nuclear fragmentation and internucleosomal cleavage of genomic DNA, yielding ladder patterns of oligonucleosomal fragments, all characteristics of apoptosis. This phenomenon occurred in a dose and time dependent manner. Dolichol and prenol failed to induce apoptosis. The inhibitors of N-glycosylation, tunicamycin and swainsonine had no apparent effect on dolichyl phosphate-induced apoptosis. Apoptotic changes were also observed in HL-60 cells, SIRC cells and HeLa cells. Thus, dolichyl phosphate functions as a potential apoptosis inducer as well as an essential carrier lipid in the biosynthesis of N-linked glycoprotein.

Plasmenylethanolamine in human intestinal mucosa detected by an improved method for analysis of phospholipid.

Analysis of phospholipid and their fatty acid composition of human intestinal mucosa was performed by an method elaborated to analyze the limited amount of sample with 2-dimensional TLC followed by lipid-phosphorus determination. Using this method, plasmenylethanolamine was detected in human intestinal mucosa and accounted for about 7% of phospholipid in small and large intestinal mucosa. The amounts of polyunsaturated fatty acids of phosphatidylethanolamine were higher than those of other phosphoglycerides in intestinal mucosa, hence, inflammation-related eicosanoids may originate from ethanolamine containing phospholipid.

Major defect of carbohydrate-deficient-glycoprotein syndrome is not found in the synthesis of dolichyl phosphate or N-acetylglucosaminyl-pyrophosphoryl-dolichol.

The contents of dolichyl phosphate and UDP-N-acetylglucosamine:dolichyl phosphate N-acetylglucosamine 1-phosphate transferase (GlcNAc-1-P transferase) activity in fibroblasts from patients with carbohydrate-deficient-glycoprotein (CDG) syndrome were analyzed. The amount of dolichyl phosphate and GlcNAc-1-P transferase activity in CDG syndrome fibroblasts were similar to those in normal fibroblasts, suggesting that CDG syndrome may not be due to a deficiency of a biosynthetic enzyme for dolichol-oligosaccharide intermediates, but to a metabolic error in assembly of asparagine-linked oligosaccharide.

Sequential microanalyses of free dolichol, dolichyl fatty acid ester and dolichyl phosphate levels in human serum.

Sequential microanalyses of free dolichol, dolichyl fatty acid ester and dolichyl phosphate in human serum were made. To determine the level of each dolichol, samples were pretreated using three different methods prior to fluorescent derivatization. To estimate the concentrations of free dolichol, samples were added to alkaline methanol and kept at room temperature for 1 h. In case of dolichyl fatty acid ester, samples were saponified at 100 degrees C for 1 h. To estimate dolichyl phosphate, saponified lipid extracts were treated with acid phosphatase. Each pretreated dolichol was reacted with anthracene-9-carboxylic acid and amounts of 9-anthroyl derivatives were determined fluorometrically by HPLC. This method is simple and three types of dolichols can be estimated using the same HPLC system. This analysis is also sufficiently sensitive for measurement of serum dolichol levels. The contents of free dolichol, dolichyl fatty acid ester and dolichyl phosphate in human serum were found to be 44.9 +/- 10.5 ng/ml (n = 32), 76.4 +/- 24.2 ng/ml (n = 32) and 43.5 +/- 15.1 ng/ml (n = 13), respectively. These levels had apparently no correlation to age or serum total cholesterols. A linear correlation between dolichols and high-density lipoprotein cholesterols reflects the fact that the dolichols are associated with the high-density lipoprotein fraction.

Composition of long chain bases in ceramide of the guinea pig Harderian gland.

Ceramide of the guinea pig Harderian gland was isolated and characterized. The purified ceramide gave two spots on thin-layer chromatography. Ceramide with the higher Rf value (NHCer) contained non-hydroxy fatty acids and that with the lower Rf value (HCer) contained 2-hydroxy fatty acids. The ratio of NHCer to HCer was 6:1. The non-hydroxy fatty acids of NHCer were composed of straight-chain acids (94.9%) and branched-chain acids (5.1%). The 2-hydroxy fatty acids were also composed of straight-chain acids (94.2%) and branched-chain acids (5.8%). The ratio of straight-chain acids to branched-chain acids was similar in NHCer and HCer. The long chain bases of NHCer and HCer consisted of straight chain sphinganines and sphingenines, and methyl-branched long chain bases. In NHCer, 59.9% of the total bases were methyl branched, and in HCer, 48.3%. The characteristics of ceramide, that is, the large amount of methyl-branched long chain bases and relatively small amount of methyl-branched fatty acids, are similar to those of cerebroside and sphingomyelin isolated from the same organ, although the ratios of constituents are different among these sphingolipids.

Composition of long-chain bases in sphingomyelin of the guinea pig Harderian gland.

Sphingomyelin from the guinea pig Harderian gland was isolated and characterized. The purified sphingomyelin gave a broad spot on thin-layer chromatography. The fatty acid composition of the whole sphingomyelin was 71% nonhydroxy acids and 29% 2-hydroxy acids. Methyl-branched fatty acids were only 2% of the total acids. The long-chain bases were composed of straight-chain sphingenines (50%) and sphinganines (6%). Methyl-branched long-chain bases were 44% of the bases. The sphingomyelin was further separated into four fractions (I, II, III, IV) by high-performance liquid chromatography. The ratio of fractions I, II, III, and IV was approximately 2:5:2:1, respectively. The fatty acids of fractions I and II consisted of nonhydroxy acids and those of fractions III and IV were 2-hydroxy acids. The long-chain bases of fractions I and III were sphinganines including 10-, 9-, and 8-methylsphinganines and anteiso-sphinganines. These methyl-branched bases occupied about 70% of the total sphinganines. The long-chain bases of fractions II and IV consisted of sphingenines. The methyl-branched unsaturated bases were only 30% of the total sphingenines, all in the anteiso-form. Thus, the sphingomyelin obtained from guinea pig Harderian gland had complex compositions of fatty acids and long-chain bases, and half the number of long-chain bases had methyl branches. The methyl-branched fatty acids were only a minor component. These characteristics are similar to those of cerebrosides isolated from the same source.

Identification of 10-methylsphinganine in cerebrosides of the guinea pig harderian gland.

A large amount of branched long chain bases was detected in the cerebrosides of guinea pig Harderian gland. The long chain bases of cerebrosides were analyzed by GLC as trimethylsilyl derivatives. The branched long chain bases were separated into four peaks (I, II, III, IV) according to the number of carbon atoms and the position of branching. In the present work, the structures of long chain bases in the four peaks were analyzed by GLC and GC-MS after conversion of them to aldehydes, alcohols, and fatty acids. Furthermore the main component of long chain bases (Peak II) was isolated by HPLC as N-acetyl derivatives and analyzed by NMR. The structures of branched long chain bases in Peaks I, II, III, and IV are as follows. Branched long chain bases of Peak I are 2-amino-10- (main component), 2-amino-9-, and 2-amino-8-methylhexadecane-1,3-diol. Branched long chain bases of Peak II also consist of a mixture of 2-amino-10-, 2-amino-9-, and 2-amino-8-methyl-heptadecane-1,3-diol. The branched long chain base of Peak III is 2-amino-10-methyl-octadecane-1,3-diol, while that of Peak IV is 2-amino-16-methyloctadecane-1,3-diol. Among these branched long chain bases, 10-methylsphinganines are dominant though the chain lengths are different. These branched long chain bases, in which the substituted positions exist in the middle part of aliphatic chain (10-, 9-, or 8-methylsphinganine) are novel long chain bases in mammals.

Branched long chain bases in cerebrosides of the guinea pig Harderian gland.

Cerebrosides obtained from the guinea pig Harderian gland were analyzed. The purified cerebrosides gave a single spot on thin-layer chromatography, the Rf value being similar to that of phrenosine obtained from whale brain. The cerebrosides consisted of 74.7% of glucosylceramide and 25.3% of galactosylceramide. The fatty acid composition of these cerebrosides was 0.7% of non-hydroxy fatty acids and 99.3% of alpha-hydroxy fatty acids. Among these alpha-hydroxy fatty acids, a small amount of methyl branched acids was detected. The substituted position of methyl branching of alpha-hydroxy fatty acids was the 16th carbon atom from the carboxyl end irrespective of the carbon chain length. The long chain bases were composed of sphinganine (78%) and sphingenine (22%). 4-D-Hydroxysphinganine was not found. The most remarkable feature of the long chain bases of cerebrosides in the Harderian gland was the presence of a large amount of methyl branched sphinganine. The cerebrosides obtained from the cerebrum and cerebellum of the same animal were also analyzed. The sugar, fatty acid, and long chain base compositions of these cerebrosides were similar to those of whale brain cerebrosides. Methyl branched sphinganine was not found in guinea pig brain.

Fatty acid composition of cardiolipin from guinea pig Harderian gland.

The fatty acid composition of cardiolipin from the Harderian gland of guinea pig was examined by gas chromatography and gas chromatography-mass spectrometry. At least 33 kinds of fatty acids were detected. Oleic acid was the most prominent component, accounting for 18.2 mol% of the total fatty acids. About 70.2 mol% of fatty acids had methyl branches. Ethyl branches were also detected (1.3 mol%). Straight chain saturated acids comprised only 10.3 mol%. On the other hand, linoleic, linolenic, and arachidonic acids were not found in this lipid. The 2-(2'-) acyl moieties contained larger amounts of oleic acid and smaller amounts of branched chain acids than the 1-(1'-)acyl moieties, but the saturated straight chain acids showed even distribution between the 1-(1'-) and 2-(2'-)positions. The fatty acids of cardiolipin from the liver of the same animal were also examined. Linoleic acid was the most abundant component (66.9 mol%), and saturated straight chain acids occupied 21.9 mol%. Branched chain acids were detected but comprised only 11.2 mol%.