(1/1010) Nitric-oxide-induced apoptosis in human leukemic lines requires mitochondrial lipid degradation and cytochrome C release.

We have previously shown that nitric oxide (NO) stimulates apoptosis in different human neoplastic lymphoid cell lines through activation of caspases not only via CD95/CD95L interaction, but also independently of such death receptors. Here we investigated mitochondria-dependent mechanisms of NO-induced apoptosis in Jurkat leukemic cells. NO donor glycerol trinitrate (at the concentration, which induces apoptotic cell death) caused (1) a significant decrease in the concentration of cardiolipin, a major mitochondrial lipid; (2) a downregulation in respiratory chain complex activities; (3) a release of the mitochondrial protein cytochrome c into the cytosol; and (4) an activation of caspase-9 and caspase-3. These changes were accompanied by an increase in the number of cells with low mitochondrial transmembrane potential and with a high level of reactive oxygen species production. Higher resistance of the CD95-resistant Jurkat subclone (APO-R) cells to NO-mediated apoptosis correlated with the absence of cytochrome c release and with less alterations in other mitochondrial parameters. An inhibitor of lipid peroxidation, trolox, significantly suppressed NO-mediated apoptosis in APO-S Jurkat cells, whereas bongkrekic acid (BA), which blocks mitochondrial permeability transition, provided only a moderate antiapoptotic effect. Transfection of Jurkat cells with bcl-2 led to a complete block of apoptosis due to the prevention of changes in mitochondrial functions. We suggest that the mitochondrial damage (in particular, cardiolipin degradation and cytochrome c release) induced by NO in human leukemia cells plays a crucial role in the subsequent activation of caspase and apoptosis.  (+info)

(2/1010) Enzymatic properties of vesicle-reconstituted human cytochrome P450SCC (CYP11A1) differences in functioning of the mitochondrial electron-transfer chain using human and bovine adrenodoxin and activation by cardiolipin.

The recently reported heterologous expression and purification of both human cytochrome P450SCC and adrenodoxin [Woods, S.T., Sadleir, J., Downs, T., Triantopoulos, T., Haedlam, M.J. & Tuckey, R.C. (1998) Arch. Biochem. Biophys. 353, 109-115] has enabled us to perform studies with the membrane-reconstituted human enzymes to better understand the side-chain cleavage reaction in humans. Human P450SCC was successfully reconstituted into dioleoylphosphatidylcholine vesicles with and without cardiolipin and its enzymatic properties characterized in the membrane-bound state. Enhancement of the P450SCC activity and significant activation by cardiolipin were observed when human adrenodoxin instead of bovine adrenodoxin was used as electron donor. In the absence of cardiolipin, Km for cholesterol was decreased twice in the case of human adrenodoxin indicating enhanced cholesterol binding. On the other hand, in the presence of cardiolipin in the membrane both Km and V for cholesterol were decreased with human adrenodoxin as electron donor. Kinetic analysis of the interaction between human P450SCC and its redox partners provided evidence for enhanced binding of the human electron donor to human P450SCC indicated by both an increased V and decreased Kd for human adrenodoxin compared with the values with bovine adrenodoxin. Because no similar effects were observed in Tween 20 micelles, these results suggest that the phospholipid membrane may play an important role in the interaction of human adrenodoxin with human P450SCC.  (+info)

(3/1010) Phospholipid composition of Rickettsia prowazeki grown in chicken embryo yolk sacs.

The phospholipid composition and phospholipid fatty acid composition of purified Rickettsia prowazeki were determined. The lipid phosphorous content was 6.8 +/- 1.3 microgram/mg of total rickettsial protein. The major phospholipid was phosphatidylethanolamine (60 to 70%); phosphatidylglycerol constituted 20%, and phosphatidylcholine constituted 15%. Small amounts of phosphatidylserine and cardiolipin were detected. The principal fatty acids were 18:1, 16:1, and 16:0. The fatty acid composition of the phosphatidylcholine in the rickettsial extracts was very different than that of the other rickettsial phosphatides and very similar to that of normal yolk sac phosphatidylcholine. The specific of the phosphatidylcholine of rickettsiae grown in the presence of 32P was markedly lower than that of phosphatidylethanolamine and phosphatidylglycerol. It is suggested that the phosphatidylcholine in the rickettsial extract is yolk sac derived and either tightly absorbed or exchanged into the rickettsial membrane.  (+info)

(4/1010) Conformationally altered beta 2-glycoprotein I is the antigen for anti-cardiolipin autoantibodies.

Anti-cardiolipin autoantibodies (aCL) induce thrombosis and recurrent fetal death. These antibodies require a 'cofactor', identified as beta 2-glycoprotein I (beta 2-GPI), to bind phospholipids. We show here that aCL can bind beta 2-GPI in the absence of phospholipid. Binding of aCL to beta 2-GPI is dependent upon the beta 2-GPI being immobilized on an appropriate surface including cardiolipin, irradiated polystyrene and nitrocellulose membrane. This effect cannot be explained by increased antigen density of beta 2-GPI immobilized on these surfaces. Rather, conformational changes that occur following the interaction of beta 2-GPI with phospholipid render this protein antigenic to aCL. Liquid-phase beta 2-GPI was not antigenic for aCL. Thus, aCL cannot bind circulating beta 2-GPI. These findings may explain why patients with aCL can remain healthy for many years but then undergo episodes of thrombosis or fetal loss without changes in their circulating aCL profile, as the triggering event for these pathologies can be predicted to be one that renders beta 2-GPI antigenic for aCL.  (+info)

(5/1010) Transbilayer movement and distribution of spin-labelled phospholipids in the inner mitochondrial membrane.

The transmembrane diffusion and equilibrium distribution of spin-labelled phosphatidylethanolamine (PE*), phosphatidylcholine (PC*) and cardiolipin (CL*) were investigated in purified mitochondrial inner membranes using electron spin resonance spectroscopy. Using the back exchange technique, we found that the outside-inside movement of PE* and PC* in beef-heart inner mitochondrial membranes was rapid (t1/2 in the range 10-15 min at 30 degrees C). The steady-state distributions in non-energised mitoplasts were approximately 30% in the inner leaflet for PC* and 39% for PE*. Within the limits of probe concentration that can possibly be used in these experiments, the initial velocity of the inward movement was not saturable with respect to the amount of analogue added to the membranes, suggesting that the spin-labelled phospholipids diffused passively between the two leaflets of the inner mitochondrial membrane. In energised mitoplasts, PC* behaviour was not affected, PE* diffused approximately two times faster toward the inner monolayer but reached the same plateau. Treatment of energised mitochondria with N-ethylmaleimide did not affect PC* diffusion, while the kinetics of PE* internalisation became identical to that of PC*. Similar results were found when PC* and PE* movements were studied in mitoplasts from beef heart, rat liver or yeast. The spin-labelled cardiolipin, which possesses four long chains, had to be introduced in the mitoplast with some ethanol. After equilibration (t1/2 of the order of 13 min at 30 degrees C), the transmembrane distribution suggested that approximately half of the cardiolipin analogue remained in the outer leaflet. These results do not allow us to determine if a specific protein (or flippase) is involved in the phospholipid transmembrane traffic within inner mitochondrial membranes, but they show that lipids can rapidly flip through the mitochondrial membrane.  (+info)

(6/1010) Involvement of Arg-328, Arg-334 and Arg-342 of DnaA protein in the functional interaction with acidic phospholipids.

We reported previously that three basic amino acids (Arg-360, Arg-364 and Lys-372) of DnaA protein are essential for its functional interaction with cardiolipin. In this study, we examined the effect of mutation of some basic amino acids in a potential amphipathic helix (from Lys-327 to Ile-345) of DnaA protein on this interaction. ATP binding to the mutant DnaA protein, in which Arg-328, Arg-334 and Arg-342 were changed to acidic amino acids, was less inhibited by cardiolipin than that of the wild-type protein, as was the case for mutant DnaA protein with mutations of Arg-360, Arg-364 and Lys-372. A mutant DnaA protein with mutations of all six basic amino acids showed the most resistance to the inhibition of ATP binding by cardiolipin. These results suggest that Arg-328, Arg-334 and Arg-342, like Arg-360, Arg-364 and Lys-372, are also involved in the functional interaction between DnaA protein and acidic phospholipids.  (+info)

(7/1010) Maternal cardiolipin, beta 2-glycoprotein-I and prothrombin antibody expression in high-risk pregnancies with bilateral abnormal uterine artery Doppler waveforms.

OBJECTIVE: To compare the frequency of maternal serum antiphospholipid antibodies (to cardiolipin, beta 2-glycoprotein I and prothrombin) in pregnancies presenting with bilateral abnormal uterine artery Doppler waveforms. DESIGN: Retrospective analysis of stored serum. SUBJECTS: Cases comprised 47 singleton pregnancies with bilateral abnormal uterine artery Doppler waveforms at 24 weeks of gestation, followed from 20 weeks, and controls were 100 healthy pregnancies with normal uterine artery Doppler waveforms. METHODS: Ultrasound examination utilized a 5-MHz curvilinear transabdominal transducer with pulsed and color Doppler facilities. Antiphospholipid antibodies were analyzed by ELISA methodology, and reference ranges were established using the geometric mean +/- 2 SD of healthy non-pregnant adults. Human chorionic gonadotropin (hCG) levels were obtained from patient notes. RESULTS: Anticardiolipin antibodies were detected in 11 (23%) of the cases (IgG, n = 7; IgM, n = 6) compared with ten (10%) of the controls (p < 0.05). Low titer anticardiolipin IgG (range, 5.5-35.3; median, 6.3 GPL units) and anticardiolipin IgM (range, 3.4-14.7; median, 5.3 MPL units) were detected in cases. Amongst the cases, adverse perinatal outcomes were more common in the presence of raised levels of anticardiolipin antibodies. Anti-beta 2-glycoprotein I IgG was not detected in any of the cases. Antiprothrombin IgG was not detected, but antiprothrombin IgM occurred in 10.6% of cases compared with 2% of controls. CONCLUSIONS: Women with persistent bilateral abnormal uterine artery. Doppler waveforms in mid-gestation were more likely to express raised levels of anticardiolipin antibodies than healthy controls with normal uteroplacental perfusion. Anticardiolipin antibodies without anti-beta 2-glycoprotein I binding may be involved in the pathogenesis of uteroplacental ischemia in a proportion of high-risk pregnancies.  (+info)

(8/1010) Polar lipids of four Listeria species containing L-lysylcardiolipin, a novel lipid structure, and other unique phospholipids.

The membrane lipids of Listeria innocua, Listeria monocytogenes, Listeria seeligeri and Listeria welshimeri were fractionated on DEAE-cellulose and purified by chromatography on silica gel and/or preparative TLC. The lipid structures were elucidated by chemical and chromatographic means. The polar lipid composition of the four listeria species was similar. Phospholipids predominated. They consisted of phosphatidylglycerol, L-lysylphosphatidylglycerol, cardiolipin [bis(phosphatidyl)glycerol] and L-lysylcardiolipin. A phospholipid more polar than cardiolipin, possibly two L-lysyl derivatives of it, sn-glycero-1-phosphoglycolipid, its D-alanyl derivative, and polyprenol phosphate were also detected. Towards the end of exponential growth, the relative amounts of cardiolipin and L-lysylcardiolipin increased, approaching 47-78% lipid phosphorus with a ratio of L-lysylcardiolipin to cardiolipin of 0.25-1.6. As shown by fast atom bombardment-mass spectrometry, cardiolipin and L-lysylcardiolipin consisted of five molecular species due to various fatty acid combinations. L-lysylcardiolipin has so far not been found in nature. It belongs to the recently discovered class of substituted cardiolipins. Its occurrence in the four listeria species tested shows that it is a characteristic lipid component of the L. monocytogenes line of descent. Further studies on the lipid pattern of members of the other descent line are required to decide whether lysylcardiolipin can serve as a genus-specific chemotaxonomic marker for listeriae.  (+info)