Involvement of sulfhydryl oxidase QSOX1 in the protection of cells against oxidative stress-induced apoptosis.

The QSOX1 protein, belonging to a new class of FAD-linked Quiescin/Sulfhydryl oxidase, catalyzes disulfide bond formation. To give new insight into the biological function of QSOX1, we studied its involvement in oxidative stress-induced apoptosis and cell recovery of PC12 cells. By real time RT-PCR and flow cytometric analysis, we show that the QSOX1 mRNA and protein levels increased late after the beginning of oxidative treatment and were sustained for 72 h.

Identification and expression of a new splicing variant of FAD-sulfhydryl oxidase in adult rat brain.

Flavoproteins of the quiescin/sulfhydryl oxidase (QSOX) family catalyze oxidation of peptide and protein thiols to disulfides with the reduction of oxygen to hydrogen peroxide. We report here the molecular cloning of a new putative sulfhydryl oxidase cDNA, rQSOX-L (GenBank Accession no ), from adult rat brain and its expression studied by RT-PCR, Northern and Western blots in rat tissues. DNA-sequencing demonstrated the existence of two cDNAs in rat cortex, corresponding to a long transcript (rQSOX-L) and a short transcript (rQSOX-S) which differed by 851 nucleotides due to alternative splicing.

A novel early estrogen-regulated gene gec1 encodes a protein related to GABARAP.

We have isolated, in guinea-pig endometrial cells, an estrogen-induced 1.8 kb RNA called gec1. Screening of a guinea-pig genomic library led to identification of gec1 gene consisting of 4 exons and 3 introns.

Cytotoxic activity of a recombinant GnRH-PAP fusion toxin on human tumor cell lines.

Pokeweed antiviral protein (PAP), a ribosome-inactivating protein isolated from the leaves of Phytolacca americana, reveals potent antiviral activity against viruses or cytotoxic action against cells once inside the cytoplasm. Therefore PAP is a good candidate to be used as an immunotoxin. We constructed a bacterial expression plasmid encoding PAP as a fusion protein with gonadotropin-releasing hormone (GnRH), a neuropeptide with receptor sites on several gynaecologic tumors.

Isolation and characterization of a cDNA clone encoding the pokeweed antiviral protein II from Phytolacca americana and its expression in E. coli.

Three distinct ribosome-inactivating proteins (RIPs) were isolated from pokeweed (Phytolacca americana). We identified and sequenced for the first time a complete cDNA encoding the pokeweed antiviral protein II (PAP II), which is expressed in the late summer leaves of pokeweed. The cDNA of PAP II consists of 1,187 nucleotides and encodes a mature protein of 285 amino acids.

Influence of chain length of pyrene fatty acids on their uptake and metabolism by Epstein-Barr-virus-transformed lymphoid cell lines from a patient with multisystemic lipid storage myopathy and from control subjects.

The uptake and intracellular metabolism of 4-(1-pyrene)butanoic acid (P4), 10-(1-pyrene)decanoic acid (P10) and 12-(1-pyrene)dodecanoic acid (P12) were investigated in cultured lymphoid cell lines from normal individuals and from a patient with multisystemic lipid storage myopathy (MLSM). The cellular uptake was shown to be dependent on the fatty-acid chain length, but no significant difference in the uptake of pyrene fatty acids was observed between MLSM and control lymphoid cells. After incubation for 1 h the distribution of fluorescent fatty acids taken up by the lymphoid cell lines also differed with the chain length, most of the fluorescence being associated with phospholipid and triacylglycerols.

Metabolism of pyrenedecanoic acid in Epstein-Barr virus-transformed lymphoid cell lines from normal subjects and from a patient with multisystemic lipid storage myopathy.

A lymphoid cell line has been established from a patient with multisystemic lipid storage myopathy and showed a major triacylglycerol storage, whereas the content of other neutral lipids and phospholipids was in the normal range. The metabolism of the triacylglycerols has been investigated in this lymphoid cell line from multisystemic lipid storage myopathy as well as in control cells through pulse-chase experiments using 10-(1-pyrene)decanoic acid (P10), a fluorescent fatty acid derivative, as precursor. After 1 h incubation, the uptake of P10 was not significantly different in multisystemic lipid storage myopathy and control lymphoid cells.

Independence of triacylglycerol-containing compartments in cultured fibroblasts from Wolman disease and multisystemic lipid storage myopathy.

The functional relationship between the two subcellular compartments involved in catabolism of triglycerides, i.e. lysosomes and lipid-containing cytoplasmic vacuoles, has been investigated using cultured fibroblasts from patients affected with two different genetic lipid (triacylglycerol) storage disorders: Wolman disease and multisystemic lipid storage myopathy.

Biochemical and ultrastructural features of human fibroblasts cultured from a new variant of type 3 lipid storage myopathy.

A new variant of multisystemic lipid storage myopathy (type 3) has been identified. Human cultured fibroblasts present a major triacylglycerol storage whereas other neutral lipids and phospholipids are in the normal range. When feeding the cells in the presence of radiolabelled oleic acid we observed an accumulation of radiolabelled triacylglycerols demonstrating the endogenous biosynthesis of the stored triacylglycerols.

Extracellular origin of the lipid lysosomal storage in cultured fibroblasts from Wolman's disease.

The experiments reported here allowed us to compare the metabolism of neutral lipids from extracellular origin (lipoproteins) and endogenous origin (triacylglycerol biosynthesis induced by feeding cells with high levels of free fatty acid) in normal and acid-lipase-deficient fibroblasts (Wolman's disease). When the cells were grown in hyperlipemic-rich medium, a major neutral lipid storage appeared in normal as well as in acid-lipase-deficient cells; this storage disappeared rapidly in normal cells during the 'chase', whereas in Wolman cells, the storage of cholesteryl esters and triacylglycerols remained unchanged, or only decreased very slowly. When the cells were fed with high levels of radiolabelled oleic acid, a major accumulation of radiolabelled triacylglycerols was observed.

Metabolism of 1-pyrenedecanoic acid and accumulation of neutral fluorescent lipids in cultured fibroblasts of multisystemic lipid storage myopathy.

The lipid metabolism in cultured fibroblasts from multisystemic (type 3) lipid storage myopathy and controls has been studied through pulse-chase experiments using 1-pyrenedecanoic acid as precursor. The uptake of 1-pyrenedecanoic acid was not significantly different in multisystemic lipid storage myopathy and control fibroblasts. The amount of fluorescent lipids synthesized by the cells was proportionally increasing with rising 1-pyrenedecanoic acid concentration in the culture medium.

Metabolism of neutral lipids in cultured fibroblasts from multisystemic (or type 3) lipid storage myopathy.

The lipid metabolism in cultured fibroblasts from multisystemic (type 3) lipid storage myopathy (MLSM) and controls has been studied through pulse-chase experiments using radiolabelled oleic acid and acetate precursors. The uptake of radiolabelled oleic acid by MLSM fibroblasts was slightly higher than in controls but did not seem to be the primary defect of the multisystemic lipid storage myopathy. The uptake of radiolabelled acetate was quite similar in MLSM and in control cells.

Fluorometric assay for pancreatic lipase.

We report a new fluorometric assay in which a fluorescent triglyceride is used for determining lipase activity in serum, and we compare it with turbidimetric and radiometric methods. Because this fluorometric method is at least 50-fold more sensitive than the turbidimetric method, we have been able to develop a micromethod that requires only very small amounts of substrate reagent and serum. The use of fluorescent-labeled fatty acids allows direct determination of the product of lipase action and obviates the use of a standard for calibrating the method.

[Wolman disease and cholesterol ester storage disease in adults. New means of study and diagnosis].

Two clinical forms of lysosomal storage of neutral lipids with deficiency of acid lipase are known: the severe infantile form is called Wolman disease, whereas the more benign adult form is called "polycorie cholesterolique de l'adulte" or cholesteryl ester storage disease (CESD). We have developed several new tools for the study of hereditary enzymopathies and we report here their use in the study of genetic defects in lysosomal acid lipase. Lymphoid cell lines established by Epstein-Barr Virus transformation represent a new experimental model system in culture.