Formation of palmitic acid/Ca2+ complexes in the mitochondrial membrane: a possible role in the cyclosporin-insensitive permeability transition.

A possible role of palmitic acid/Ca2+ (PA/Ca2+) complexes in the cyclosporin-insensitive permeability transition in mitochondria has been studied. It has been shown that in the presence of Ca2+, PA induces a swelling of mitochondria, which is not inhibited by cyclosporin A. The swelling is accompanied by a drop in membrane potential, which cannot be explained only by a work of the Ca2+ uniporter.

Calcium-binding properties of the mitochondrial channel-forming hydrophobic component.

A hydrophobic, low-molecular weight component extracted from mitochondria forms a Ca2+-activated ion channel in black-lipid membranes (Mironova et al., 1997). At pH 8.

Palmitic and stearic acids bind Ca2+ with high affinity and form nonspecific channels in black-lipid membranes. Possible relation to Ca2+-activated mitochondrial pores.

A mitochondrial hydrophobic component that forms Ca2+-induced nonspecific ion channels in black-lipid membranes (Mironova et al., 1997) has been purified and its nature elucidated. It consists of long-chain saturated fatty acids--mainly palmitic and stearic.

Inactivation of wild-type p53 by a dominant negative mutant renders MCF-7 cells resistant to tubulin-binding agent cytotoxicity.

The present study was performed to gain insight into the role of p53 on the cytotoxicity of tubulin-binding agents (TBA) on cancer cells. Drug sensitivity, cell cycle distribution and drug-induced apoptosis were compared in 2 lines derived from the mammary adenocarcinoma MCF-7: the MN-1 cell line containing wild-type p53 (wt-p53) and the MDD2 line, containing a dominant negative variant of the p53 protein (mut-p53). The MDD2 cell line was significantly more resistant to the cytotoxic effects of vinblastine and paclitaxel than the MN1 cell line.

Phospholipids reacylation and palmitoylcoa control tumour necrosis factor-alpha sensitivity.

From the hypothesis that in TNF-alpha-resistant cells the activity of mitochondrial phospholipase A2 could be reversed by a lysophospholipid acyltransferase, we report that the mitochondrial reacylation of phosphatidylcholine as phosphatidylethanolamine was considerably higher in C6 (TNF-alpha-resistant) than in WEHI-164 (TNF-alpha-sensitive) cells. TNF-alpha did not modify the phospholipids' reacylation in C6, while in WEHI-164 it was increased several-fold. These results suggest that TNF-alpha is not sufficient to restore the barrier permeability in sensitive cells, but may be enough to explain the absence of permeability change in resistant cells.

A possible involvement of endogenous polyamines in the TNF-alpha cellular sensitivity.

A critical step in the cytotoxic action mechanism of tumor necrosis factor-alpha (TNF-alpha) involves, among mitochondrial dysfunctions, an early change of the inner membrane permeability displaying the characteristics of permeability transition. Cytosolic polyamines, especially spermine, are known to inhibit it. Our results show that spermine is only detectable in the TNF-alpha resistant C6 cells while N1-acetylspermidine is present in the TNF-alpha sensitive WEHI-164 cells, and putrescine and spermidine are found in both.

Increase of mitochondrial PLA2-released fatty acids is an early event in tumor necrosis factor alpha-treated WEHI-164 cells.

We have previously reported that TNF induced changes in mitochondrial enzymes, one of which, succinate-dehydrogenase, is specifically activated in various TNF-sensitive cell lines. In an attempt to further characterize the mechanism of trans-membrane signalling at the mitochondrial level, we have oriented our investigation to the study of phospholipase A2 activity localized in this organelle isolated from TNF-treated WEHI-164 cells. Under physiological conditions, this enzyme has a very low basal activity near the resting state, while under TNF treatment its activity is dramatically increased.

Study of the succinate dehydrogenase activation in permeabilized mitochondria through the Ca(2+)-stimulated phospholipase A2.

The development of a mitochondrial membrane permeability triggered by the Ca(2+)-stimulation of PLA2 (phospholipase A2; EC 3.1.1.

Dual localization of the mitochondrial phospholipase A2: outer membrane contact sites and inner membrane.

Mitochondria were fractionated according to a procedure which allowed to get free outer and inner membrane plus two distinct contact sites between the two membranes. The data indicate that phospholipase A2 is localized in outer membrane contact sites and in inner membrane. The enzyme activity is twice higher in the contact site fraction than in the free membrane.

Tumor necrosis factor induces activation of mitochondrial succinate dehydrogenase.

We have studied TNF-induced changes in mitochondrial enzymes. One enzyme, succinate dehydrogenase (SDH), is specifically activated in TNF sensitive cells including U937 (human monocytic), WEHI-164 (murine fibrosarcoma), and ME-180 (human cervical carcinoma). SDH is activated by TNF concentrations which also cause cytolysis, however the enzyme activity is elevated several hours before maximum cytotoxicity is observed.

Mitochondrial contact sites. Lipid composition and dynamics.

Two membrane fractions of intermediate density between inner and outer mitochondrial membranes were isolated by density gradient centrifugation from osmotically lysed mitochondria and mitoplasts of liver. These fractions were characterized by the presence of both monamine oxidase and cytochrome c oxidase activities and bound hexokinase. 1) The content of the fractions in proteins and lipids was assessed by biochemical determination.

Comparative study of the N-glycoprotein synthesis through dolichol intermediates in mitochondria, Golgi apparatus-rich fraction and endoplasmic reticulum-rich fraction.

1. Glycosylation of endogenous dolichol acceptors was higher in mitochondria than in C 30,000 g (Golgi apparatus-rich fraction) and C 100,000 g (endoplasmic reticulum-rich fraction). 2.

Biosynthesis of glycoconjugates in mitochondrial outer membranes. Preliminary characterization of the oligosaccharide moiety of a N-glycoprotein.

Outer mitochondrial membranes synthesize a N-glycoprotein by a direct incorporation of sugars from their nucleotide-donors into an endogenous protein acceptor. To characterize the oligosaccharide moiety of this N-glycoprotein, we sequentially incorporated [14C]-sugars into the protein acceptor. After pronase digest, the released [14C]-glycopeptides were fractionated on a QAE-Sephadex column which gave rise to 9% of neutral glycopeptides and 91% of charged glycopeptides.

Characterization of the submitochondrial compartments: study of the site of synthesis of dolichol and dolichol-linked sugars.

The fractionation of mitochondrial membranes on discontinuous sucrose gradient leads to the obtaining of free outer membranes, free inner membranes and two distinct membrane contact site populations characterized as follows. Only outer membrane contact sites and inner membrane contact sites bind hexokinase. Outer membranes and outer membrane contact sites are cholesterol-rich fractions.

Topological investigations. Study of the trypsin sensitivity of the N-acetylglucosaminyl and mannosyl-transferase activities located in the outer mitochondrial membrane.

The trypsin sensitivity of the mitochondrial N-acetylglucosaminyl and mannosyltransferase activities involved in the N-glycoprotein biosynthesis through dolichol intermediates as well as the N-acetylglucosaminyl-transferase activity involved in direct N-glycosylation were examined in mitochondria and isolated outer mitochondrial membrane preparations. The trypsin action on mitochondrial membrane was checked by measuring the activities of marker enzymes (rotenone-insensitive NADH cytochrome c reductase, adenylate kinase, and monoamine oxidase). Glycosyl-transferase activities of both N-glycosylation pathways were insensitive to trypsin action and consequently were located in the outer mitochondrial membrane.

Distribution of glycosyltransferase activities in different compartments of mitochondria.

The liver mitochondria were submitted to a first swelling which allowed to get outer membranes. The mitoplasts obtained in these conditions were subject to a second swelling. The separation of submitochondrial membranes on a discontinuous sucrose gradient revealed three membrane fractions, an outer membrane rich fraction, an inner membrane rich fraction and a fraction enriched with contact sites between the two membranes.

Study of the N-glycoprotein biosynthesis through dolichol intermediates in the mitochondrial membranes.

1. In the mitochondria, the biosynthesis of N-glycoprotein products, through the dolichol intermediates pathway, appears in the outer and in the inner membranes. 2.

Biosynthesis of dolichyl pentasaccharide diphosphate in calf pancreas microsomes.

Incubation of synthetic dolichyl pyrophosphate tetrasaccharide and GDP-[14C]mannose with calf pancreas microsomes gave three lipid-linked oligosaccharides, which could be extracted with chloroform/methanol (2:1) and separated on silica gel plates. The fastest migrating product was characterized as dolichyl pyrophosphate pentasaccharide based on gel filtration and high pressure liquid chromatography. The formation of the pentasaccharide-lipid was greatly stimulated by addition of synthetic tetrasaccharide-lipid and required the presence of Triton X-100.

Biosynthesis of pulmonary glycoconjugates--V. Biosynthesis of oligosaccharide inner core region of N-glycoprotein in lung microsomes.

Lung microsomes are able to synthesize dolichyl-mannosyl-phosphate, which stimulates the biosynthesis of a product (P1), soluble in chloroform/methanol/water (10:10:3, v/v). In presence of pulmonary dolichyl-mannosyl-phosphate, liver microsomes synthesized 3 mannosylated products: X1 product recovered in chloroform/methanol extract, X2 and X3 products soluble in chloroform/methanol/water (10:10:3, v/v). Mild acid hydrolysis of P1 product released one mannosylated oligosaccharide which remained at the origin in our solvent system, since two mannosylated oligosaccharides, released from products synthesized in liver, moved very slowly.

[Biosynthesis of pulmonary glycoconjugates. Mechanism of glucosylation of protein and lipid acceptors].

In sheep lung microsomes, we have shown that glucosyl-transferases catalize the transfer of glucose from UDP-glucose into four different acceptors. The glucosylated products obtained are as follows: - a glucosyl-phosphoryl-polyprenol (product A) extractable by chloroform/methanol (2:1 by volume). - a product B extractable by chloroform/methanol/water (10:10:3 by volume).

[Mannosylation of lipid and protein acceptors in sheep lung microsomes].

In Sheep lungs, we have shown that a mannosyl-transferase activity is involved at microsomal level. This enzymatic system is able to catalyze the incorporation of mannose from GDP-mannose into two different endogenous acceptors: lipids and proteins. The mannolipid has the properties of a mannosyl-phosphoryl-polyprenol.

Inhibition of viral multiplication by homologous methylated ribonucleic acids. V. Inhibition of myxoviruses in mouse.

Mice respond to intravenous injection of homologous methylated RNA by the production of a circulating interferon-like substance. The treatment with modified RNA induces a significant protection against viral infection. This effect is optimum when the treatment occurs a few hours before viral infection.

[Biosynthesis of glycoproteins in pulmonary parenchyma. II. Galactosyltransferase activity in pneumocyte subcellular fractions].

A soluble galactosyltransferase exists in the cell sap of the alveolar cells of the lung. The optimal conditions of galactosyltransferase are: pH 6, 20 DEGREES C, 10 mM Mn2+, 1 mM Mg2+. Michaelis constant for substrate UDP-galactose is 390 nM.