Anticardiolipin antibodies (aCL) derived from the sera of individuals exhibiting the antiphospholipid syndrome (APS) directly bind to beta 2-glycoprotein I (beta 2-GPI), which is adsorbed to an oxidized polystyrene surface. Oxygen atoms were introduced on a polystyrene surface by irradiation with electron or gamma-ray radiation. X-ray photoelectron spectroscopy revealed the irradiated surfaces were oxidized to generate C-O and C = O moieties. aCL derived from either APS patients or (NZW x BXSB)F1 mice bound to beta 2-GPI coated on the irradiated plates, depending on the radiation dose. Antibody binding to beta 2-GPI on the irradiated plates was competitively inhibited by simultaneous addition of cardiolipin (CL)-coated latex beads mixed together with beta 2-GPI but were unaffected by addition of excess beta 2-GPI, CL micelles, or CL-coated latex beads alone. There was a high correlation between binding values of aCL in sera from 40 APS patients obtained by the anti-beta 2-GPI enzyme-linked immunosorbent assay (ELISA) using the irradiated plates and those by the beta 2-GPI-dependent aCL ELISA. Therefore, aCL have specificity for an epitope on beta 2-GPI. This epitope is expressed by a conformational change occurring when beta 2-GPI interacts with an oxygen-substituted solid phase surface.

A chloroform-methanol-extracted lipid of Helicobacter pylori was studied. Three kinds of glycolipids, accounting for about 25% (wt/wt) of the total lipid, were detected and identified to be cholesteryl glucosides. The structures of two of them were determined to be cholesteryl-␣-D-glucopyranoside and cholesteryl-6-O-tetradecanoyl-␣-D-glucopyranoside, and the plausible structure of the third one was identified as cholesteryl-6-Ophosphatidyl-␣-D-glucopyranoside. Cholesteryl glucosides are very rare in animals and bacteria. Furthermore, those in H. pylori had an ␣-glycosidic linkage, which is rather unusual for natural glycosides, and a phosphatelinked cholesteryl glycoside like the cholesteryl-6-O-phosphatidyl-␣-D-glucopyranoside has not been reported previously. As the cholesterol glucosides were detected in strains obtained from diverse geographical locations, the presence of cholesteryl glucosides in H. pylori is a very unique and a characteristic feature of the species. These findings add a new facet to the physiology and biochemistry, especially the cholesterol and glucose metabolism, of H. pylori. Furthermore, the cholesteryl glucosides of H. pylori showed hemolytic activities.Helicobacter pylori is a curved gram-negative bacterium which was first reported in 1983 (21, 35). The organism is now recognized as a primary cause of active chronic gastritis and is reported to be associated with peptic ulcer disease, gastric adenocarcinoma, and low-grade B-cell gastric lymphoma of mucosa-associated lymphoid tissue (4,14,36).The organism can colonize in and under the mucus layer of the human gastric mucosa without the copresence of other bacterial species. It, living in the unique gastric environment, is likely to have features distinct from other bacteria. Furthermore, it has been found to possess a unique urease having a very low K m value, which hydrolyzes urea efficiently even at the low urea concentration found in the gastric mucosa (13,24).On the other hand, the lipid composition of microorganisms can adapt in response to alterations of environmental conditions (15, 25), and those living in unique environments may have special lipids (5). It is likely that H. pylori living in the gastric niche may have a distinct lipid composition or structure. The fatty acid composition of H. pylori has been studied extensively (9, 10, 19), and it has been shown that the cellular fatty acids 3-OH C 16:0 and 3-OH C 18:0 are unique to this organism. However, little is known about the polar lipid composition of H. pylori.Therefore, we studied the lipid composition of H. pylori and detected three kinds of glycolipids, which were identified as cholesteryl glucosides (CGs). Additionally, the molecular structures of these CGs have been determined. MATERIALS AND METHODSBacterial strains and growth conditions. H. pylori NCTC 11638 was mainly used in this study. H. pylori ATCC 43504 and two clinical isolates of H. pylori obtained from Japan and Bangladesh were used for comparison. These were grown in brain heart infusion broth (Difco, D...

Cyclin-dependent kinase (Cdk) enzymes are activated for entry into the S phase of the cell cycle. Elimination of Cdk inhibitor protein p27 Kip1 during the G 1 to S phase is required for the activation process. An inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase prevents its elimination and leads to G 1 arrest. Mevalonate and its metabolite, geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate, restore the inhibitory effect of pravastatin on the degradation of p27 and allow Cdk2 activation. By the addition of geranylgeranyl pyrophosphate, Rho small GTPase(s) are geranylgeranylated and translocated to membranes during G 1 /S progression. The restoring effect of geranylgeranyl pyrophosphate is abolished with botulinum C3 exoenzyme, which specifically inactivates Rho. These results indicate (i) among mevalonate metabolites, geranylgeranyl pyrophosphate is absolutely required for the elimination of p27 followed by Cdk2 activation; (ii) geranylgeranylated Rho small GTPase(s) promote the degradation of p27 during G 1 /S transition in FRTL-5 cells.

␤ 2 -glycoprotein I ( ␤ 2 -GPI) is a major antigen for antiphospholipid antibodies (Abs, aPL) present in patients with antiphospholipid syndrome (APS). We recently reported ( J. Lipid Res ., 42: 697, 2001; J. Lipid Res. , 43: 1486, 2002) that ␤ 2 -GPI specifically binds to Cu 2 ؉ -oxidized LDL (oxLDL) and that the ␤ 2 -GPI ligands are -carboxylated 7-ketocholesteryl esters. In the present study, we demonstrate that oxLDL forms stable and nondissociable complexes with ␤ 2 -GPI in serum, and that high serum levels of the complexes are associated with arterial thrombosis in APS. A conjugated ketone function at the 7-position of cholesterol as well as the -carboxyl function of the ␤ 2 -GPI ligands was necessary for ␤ 2 -GPI binding. The ligand-mediated noncovalent interaction of ␤ 2 -GPI and oxLDL undergoes a temperature-and time-dependent conversion to much more stable but readily dissociable complexes in vitro at neutral pH. In contrast, stable and nondissociable ␤ 2 -GPI-oxLDL complexes were frequently detected in sera from patients with APS and/or systemic lupus erythematodes. Both the presence of ␤ 2 -GPI-oxLDL complexes and IgG Abs recognizing these complexes were strongly associated with arterial thrombosis. Further, these same Abs correlated with IgG immune complexes containing ␤ 2 -GPI or LDL. Thus, the ␤ 2 -GPI-oxLDL complexes acting as an autoantigen are closely associated with autoimmune-mediated atherogene-

Amphiphysin is a major dynamin-binding partner at the synapse; however, its function in fission is unclear. Incubation of large unilamellar liposomes with mice brain cytosol led to massive formation of small vesicles, whereas cytosol of amphiphysin 1 knockout mice was much less efficient in this reaction. Vesicle formation from large liposomes by purified dynamin was also strongly enhanced by amphiphysin. In the presence of liposomes, amphiphysin strongly affected dynamin GTPase activity and the recruitment of dynamin to the liposomes, but this activity was highly dependent on liposome size. Deletion from amphiphysin of its central proline-rich stretch dramatically potentiated its effect on dynamin, possibly by relieving an inhibitory intramolecular interaction. These results suggest a model in which maturation of endocytic pits correlates with the oligomerization of dynamin with either amphiphysin or other proteins with similar domain structure. Formation of these complexes is coupled to the activation of dynamin GTPase activity, thus explaining how deep invagination of the pit leads to fission.

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Accumulation of protoporphyrin IX (PpIX) in malignant cells is the basis of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy. We studied the expression of proteins that possibly affect ALA-mediated PpIX accumulation, namely oligopeptide transporter-1 and -2, ferrochelatase and ATP-binding cassette transporter G2 (ABCG2), in several tumor cell lines. Among these proteins, only ABCG2 correlated negatively with ALA-mediated PpIX accumulation. Both a subcellular fractionation study and confocal laser microscopic analysis revealed that ABCG2 was distributed not only in the plasma membrane but also intracellular organelles, including mitochondria. In addition, mitochondrial ABCG2 regulated the content of ALA-mediated PpIX in mitochondria, and Ko143, a specific inhibitor of ABCG2, enhanced mitochondrial PpIX accumulation. To clarify the possible roles of mitochondrial ABCG2, we characterized stably transfected-HEK (ST-HEK) cells overexpressing ABCG2. In these ST-HEK cells, functionally active ABCG2 was detected in mitochondria, and treatment with Ko143 increased ALA-mediated mitochondrial PpIX accumulation. Moreover, the mitochondria isolated from ST-HEK cells exported doxorubicin probably through ABCG2, because the export of doxorubicin was inhibited by Ko143. The susceptibility of ABCG2 distributed in mitochondria to proteinase K, endoglycosidase H and peptide-N-glycosidase F suggested that ABCG2 in mitochondrial fraction is modified by N-glycans and trafficked through the endoplasmic reticulum and Golgi apparatus and finally localizes within the mitochondria. Thus, it was found that ABCG2 distributed in mitochondria is a functional transporter and that the mitochondrial ABCG2 regulates ALA-mediated PpIX level through PpIX export from mitochondria to the cytosol.

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