Mieko Oshima

The lipid composition of the prepubertal and adult rat testis

Abstract A quantitative study of the lipid components of maturing rat testes which includes data on the fatty acid components of individual phosphatides, triglycerides and cholesteryl esters has been made. Testicular lipids of prepubertal and adult rats have been isolated and separated into individual neutral lipid and phosphatide classes by silicic acid column chromatography. The lipid representing 2.2% of the tissue wet weight was about 80% phosphatide and 20% neutral lipid. Phospholipid distribution was as follows: phosphatidyl choline, 47.5%; phosphatidyl ethanolamine, 26.2%; phosphatidyl serine, 7.2%; phosphatidyl inositol, 2.3%; and sphingomyelin, 6%. Plasmalogens were found primarily in the phosphatidyl ethanolamine fraction. Free cholesterol, about 95% of total cholesterol, and triglyceride were the predominant classes found in the neutral lipid fraction; monoglyceride, trace amounts of diglyceride and other components were also detected. No conspicuous differences in the relative distributions of the lipid classes were observed between the immature and adult animal. The fatty acid composition of each of the isolated lipid classes was determined by gas-liquid chromatography. Comparable fractions of the neutral lipids and of phospholipid classes of both young and mature testes had similar fatty acid contents. Major fatty acids of the triglyceride fraction were palmitic, oleic, and docosapentaenoic; unsaturation increased in the older animal. About half of the fatty acids of the phosphatides were unsaturated; palmitic, stearic, oleic, arachidonic and docosapentaenoic acids represented most of the fatty acids found, but their relative distributions varied in individual phosphatides. β-Position fatty acids of phosphatidyl choline were predominantly oleic and arachidonic in the prepubertal animal but the concentration of docosapentaenoic in the adult increased to equal that of arachidonic. A similar relative distribution of unsaturated fatty acids was found in the phosphatidyl ethanolamine fractions, but large amounts of palmitic and stearic were also present. A marked increase in the concentration of docosapentaenoic acid was found in the triglyceride fraction as well as in individual phosphatide classes of the adult testes.

Detection of N-acetylgalactosaminyltransferase mRNA which determines expression of Sda blood group carbohydrate structure in human gastrointestinal mucosa and cancer

The Sda blood group carbohydrate structure, GaINAcβl‐4[NeuAcα2–3]Galβl‐4GlcNAc‐R, is expressed on glycolipid and glycoprotein in human gastrointestinal mucosa. The expression of the Sda determinant dramatically decreases in cancer tissue. The activity of the βl,4N‐acetylgalactosaminyltransferase (Sda‐GaINAcT), which transfers GaINAc to NeuAcα2–3Galβl‐4Glc(NAc)‐R, correlates with the expression of the Sda immuno‐epitope. From the total RNA fraction of human gastric mucosa, we have amplified a cDNA segment by reverse‐transcription‐polymerase‐chain reaction (RT‐PCR), using primers designed according to the cDNA sequence of a murine βl,4GalNAcT which synthesizes the Sda determinant. An RT‐PCR product of 390 bp shared 85% nucleotide identity with the murine Sda‐related βl,4GalNAcT. This RT‐PCR product hybridized to a transcript in mRNA prepared from human gastric mucosa. In RT‐PCR using specific primers to this PCR product, Sda‐GaINAcT mRNA was detected in all samples of normal stomach and small intestine examined and the majority of normal colonic specimens. Six out of nine cases of gastric cancer, and 9 out of 13 cases of colonic cancer failed to produce the target DNA. These results correlate with the βl,4GalNAcT activity measured in the same samples. In conclusion, a segment of the cDNA for βl,4GalNAcT which determines expression of the Sda carbohydrate structure was obtained, and reduced transcription of this βl,4GalNAcT resulted in the disappearance of the Sda epitope in gastrointestinal cancer.

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.

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

Abstract: 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 Crohns 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.

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.

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.

Urinary neutral glycosphingolipid analysis of patients with Fabry's disease; rapid isocratic elution from high-performance liquid chromatography as per-o-benzoyl derivatives

Neutral glycosphingolipids from urinary sediments of six patients with Fabrys disease and 11 members of the family of one propositus were analyzed using high-performance liquid chromatography. Per-o-benzoyl derivatives of GlcCer, LacCer, GbOse3Cer and GbOse4Cer were clearly resolved by a solvent mixture of hexane/dioxane/isopropanol (75:25:1, v/v) on a normal-phase silica column. Using our isocratic solvent system, the analysis was completed within 15 min. The smallest amount of glycolipid that could be detected by HPLC was 50 pmol and a linear response was shown at 230 nm up to 400 pmol. The calculated peak area of GbOse4Cer was higher than those of GlcCer, LacCer and GbOse3Cer. The molar ratios of GbOse3Cer to monohexosyl ceramide (CMH) in the urinary sediments were: Fabry hemizygotes, 36.33 +/- 25.54 (n = 6); heterozygotes, 0.94 +/- 0.50 (n = 4); and controls, 0.11 +/- 0.06 (n = 5). The molar ratio CDH/CMH was also higher in patients (8.42 +/- 6.23) than in controls (0.77 +/- 0.23). The female H was a rare example of a carrier with typical clinical manifestations. From the urinary sediment analysis, females E, G and J were suspected to be Fabry heterozygotes, although no clinical signs were observed at the time of examination.

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.

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.