Biomedical subjects

B Luu

Publications and source records attributed to B Luu.

At least 37 records · Page 2Linked to original sources

PubMed1991

Membrane-related oxysterol function: preliminary results on the modification of protein kinase C activity and substrate phosphorylation by 7 beta,25-dihydroxycholesterol.

Oxysterols exhibit a wide variety of biological activities, including potent immunosuppressive effects. 7 beta,25-Dihydroxycholesterol (7,25-OHC), a synthetic oxysterol, has been shown to strongly inhibit the lymphocyte response to different stimuli. This compound has been chosen as a model compound to investigate the mechanisms underlying the immunosuppressive effects of oxysterols. As protein kinase C (PKC) constitutes a key enzyme in the pathways leading to cell activation, we have studied the effect of 7,25-OHC on PKC activity in the cytosolic and particulate fractions of spleen cells. Lymphocytes treated with 7,25-OHC showed a decrease of the relative PKC activity in the particulate fractions compared to control cells. These results are confirmed by the observation that 7,25-OHC also reduces the phosphorylation of the endogenous PKC substrates. Thus oxysterols interfere with two membrane related phenomena, ie the modification of membrane PKC activity and the inhibition of the phosphorylation of the substrates of PKC located in the membrane. Previous results obtained by fluorescence polarisation revealed a modification of the membrane fluidity after oxysterol treatment. Furthermore, it has been demonstrated that oxysterols are incorporated into cell membranes. The alteration of the cell membrane could impair the signal transduction and may explain the immunosuppressive activity of oxysterol. Thus, along with other biological effects previously reported, oxysterols decrease membrane associated PKC activity in immune cells.

Animals↗

PubMed1990

Selectivity of lipid-protein interaction with myelin proteolipids PLP and DM-20. A fluorescence anisotropy study.

The two main myelin proteolipids, PLP (30 kDa) and DM-20 (25 kDa), differ by an internal deletion in DM-20. The deleted fragment, of 35 amino acids (116-150), corresponds to the major hydrophilic domain of PLP. Fluorescence anisotropy experiments using diphenylhexatriene as a fluorescent probe were performed to detect the phase separation induced by these two proteolipids in multilamellar vesicles of binary composition. We found that in vesicles composed of 30% L-alpha-PS and 70% DPPC, the PLP boundary layer contained about 18 motionally restricted phospholipids, almost exclusively L-alpha-PS. On the contrary, the DM-20 boundary layer contained only 14 to 15 phospholipids, with a composition no different from that of the bulk vesicle. In mixtures of DMPG and DPPC, the selectivity of PLP for the acidic phospholipid DMPG was maintained, but was lower than that observed for L-alpha-PS. We assume that this selectivity of PLP stems mainly from electrostatic interactions between the charged residues of the 116-150 fragment, deleted in DM-20, and the acidic phospholipids. These results suggest that fragment 116-150 may play a specific role in the interaction of PLP with the lipid bilayer of the myelin membrane.

Apoproteins↗

PubMed1990

Isolation and amino acid sequence of a novel 6.8-kDa mitochondrial proteolipid from beef heart. Use of FAB-MS for molecular mass determination.

We have isolated a 6.8 kDa proteolipid from an acidic chloroform/methanol extract of bovine cardiac muscle. The molecular mass of the polypeptide was measured by fast atom bombardment-mass spectrometry (FAB-MS) (m/z6834.1). Its amino acid sequence was partly determined by direct sequencing and completed by characterization of cyanogen bromide and tryptic fragments (sequencing, FAB-MS and amino acid analysis). The polypeptide consists of 60 amino acid residues. Polyclonal antibodies raised in rabbit allowed its localization by electroimmunoblotting in mitochondria.

Amino Acid Sequence↗

PubMed1990

Isolation and structure elucidation of a novel 5-kDa peptide from neurohaemal lobes of the corpora cardiaca of Locusta migratoria (Insecta, Orthoptera).

Two predominant peptides have been isolated from neurohaemal lobes of corpora cardiaca of 8000 adults of Locusta migratoria. Both peptides have been unambiguously characterized by automated peptide microsequencing and liquid secondary-ion mass spectrometry as a 50-residue peptide (5K peptide) and a 48-residue isologue (5K' peptide). Computer search of sequence data banks did not reveal any significant similarity with other identified proteins. The 5K peptides are remarkably rich in alanine residues (25%) and contain a stretch of five consecutive alanines. This structure suggests that these molecules could correspond to spacer peptides. This assumption is corroborated in the accompanying paper [Lagueux et al. (1990) Eur. J. Biochem. 187, 249-254] on the molecular cloning of the precursor protein which attributes to the 5K peptides a role analogous to that of the C peptides of insulins.

Amino Acid Sequence↗

PubMed1990

Polyoxygenated sterols and triterpenes: chemical structures and biological activities.

Cholesterol and other sterols are precursors of many hormones of vertebrates (pregnane, androstane, estrane derivatives), invertebrates (ecdysteroids) and even of plants (brassinoids). The normal biosynthetic processes which begin by a series of oxidations lead to a family of compounds, some of which exhibit a wide variety of biological activities. Among the latter, those well-established are their inhibitory effect on the biosynthesis of cholesterol in mammalian cells, their toxic effect on tumor cells and their ability to modify some immunological responses. None of the members of this family has all of the activities just mentioned. The intensity of the effect depends markedly upon the specific structure of each compound.

Animals↗

PubMed1990

Monophosphoric acid diesters of 7 beta-hydroxycholesterol and of pyrimidine nucleosides as potential antitumor agents: synthesis and preliminary evaluation of antitumor activity.

7 beta-Hydroxycholesterol, which has been shown to be selectively cytotoxic toward tumor cells cultured in vitro, was converted into the corresponding water-soluble phosphoric acid ester and linked to a pyrimidine nucleoside such as 5-fluoro-2'-deoxyuridine or 2'-deoxyuridine. 2-Chlorophenyl phosphorodichloridate (3), without activation, was used directly to phosphorylate the protected oxygenated sterol. The intermediate phosphorylated the 5'-OH group of nucleoside selectively, leading to compounds 1a and 1b after deprotection. These compounds were screened for their antiproliferative activity toward EL-4 murine leukemia cells in vitro and for their antitumor activity against the mice bearing Krebs II ascitic carcinoma in vivo.

Animals↗

PubMed1990

Developmental study of proteolipids in bovine brain: a novel proteolipid and DM-20 appear before proteolipid protein (PLP) during myelination.

In a developmental study, we have shown that DM-20 is present before proteolipid protein (PLP) in the fetal bovine cerebral hemispheres. When the white matter appears (27-30 weeks of gestation), the amount of DM-20 drastically increases. DM-20 remains the major proteolipid until birth. PLP is detected only 2-4 weeks after the appearance of white matter, that is, more than 4 weeks after the appearance of DM-20. The early appearance of DM-20 at the beginning of myelination raises the question of its particular function. In the adult bovine cerebral hemispheres, PLP is the major proteolipid but DM-20 remains quantitatively important because the PLP/DM-20 ratio ranges from 1.5 to 1.7. In the same developmental study we have, in the fetal cerebral hemispheres, isolated and characterized a novel proteolipid (apparent Mr 20,000), which appears even before DM-20 and is not detected in the adult brain. It is structurally related to PLP and DM-20 because the first 31 N-terminal amino acid residues are the same. However, in immunoblot, it did not react either with the antitridecapeptide 117-129 antiserum of PLP or with the anti-C-terminal hexapeptide antiserum of PLP.

Animals↗

PubMed1990

Antiproliferative effects of two hydrosoluble derivatives of oxysterols on cultured lymphoma cells.

The antitumor effects of two new hydrosoluble derivatives of oxygenated sterols: JB69 and JC40 have been evaluated in vitro on a panel of lymphoma and leukemia cells from human and murine origins. These compounds result from the combination of a nucleotide with 7 beta-hydroxycholesterol (JB69) or 7 beta,25-dihydroxycholesterol (JC40). Both derivatives exhibit a significant cytotoxic activity against the different tumor cell lines tested, with some degree of difference between them. On the whole, the concentrations needed to inhibit the cell growth were found to be higher than those required for their parent compounds. However, two interesting features appeared in our experiments. (1) In a serum-free culture medium, cell lysis occurred within the first hours of incubation and seemed to result from the detergent-like properties possessed by this type of compounds. (2) In a culture medium supplemented with serum, we noted, that at high concentrations of JB69 (40 microM or 20 microM) and only with this oxysterol derivative, an important increase of incorporation of tritiated thymidine and uridine into DNA and RNA by viable cells. The origin of this effect is as yet unknown, but it strongly suggests a possible action on nucleic acids synthesis and metabolism. Taken together, these results emphasize the diversity and the complexity of the mechanisms involved in the cytotoxicity of these derivatives of oxysterols.

Animals↗

PubMed1990

Studies on the immunological properties of oxysterols: in vivo actions of 7,25-dihydroxycholesterol upon murine peritoneal cells.

Oxygenated derivatives of cholesterol are potent immunosuppressors. It has been reported previously that 7,25-dihydroxycholesterol (7,25-OHC), synthesized in the URA31, strongly inhibits the early steps of T-cell activation. So far, the mechanisms underlying this type of effect have been mainly investigated in vitro, and the activity of these substances on the immune system has been poorly studied. This study describes that a single i.p. injection of 7,25-OHC induces a strong inflammatory response, consisting of a massive influx of macrophages and neutrophils into the abdominal cavity. Macrophages harvested from 7,25-OHC-treated mice express class II antigen to a lesser extent. Moreover, the 7,25-OHC treatment abolishes the class II induction by bacillus Calmette-Guérin (BCG) or interferon-gamma (IFN-gamma). The inflammatory process triggered by the oxysterol is not the consequence of a non-specific effect due, for instance, to the presence of crystals in the abdominal cavity. Moreover, treatments by inhibitors of the acid arachidonic cascade do not affect the peritoneal exudate cell (PEC) influx induced by these substances. This study could be an important contribution to the mechanism determining the oxysterol-induced immunosuppression.

Animals↗

PubMed1989

Isolation and structure of two novel 6-kDa dimeric peptides from the corpora cardiaca of the insect Locusta migratoria. Molecular mass determination by mass spectrometry.

We have isolated two major 6-kDa peptides from extracts of corpora cardiaca of adult females of Locusta migratoria. These peptides have been characterized by peptide sequencing and liquid secondary-ion mass spectrometry. They are structurally related dimers, one (6278.5 Da) being a homodimer (A-A chains), the other (6280.5 Da) being a heterodimer (A-B chains). A 60% similarity exists between the A and B chains. Both peptides have been chemically synthesized and the synthetic compounds appeared to be identical to the native ones. Polyclonal antibodies raised against each of these peptides demonstrated that they were contained within the secretory granules of the intrinsic cells of the glandular lobes of the corpora cardiaca. The physiological significance of these two peptides is unknown but, using the synthetic peptides, we are currently probing their biological role.

Amino Acid Sequence↗

PubMed1989

Toxic effects of 7 beta-hydroxycholesterol on rat liver primary cultures, epithelial lines and co-cultures.

The toxic effects of 7 beta-hydroxycholesterol (7 beta-OHC) on cultures and co-cultures of rat hepatocytes, rat liver epithelial cell lines, and rat liver fibroblast lines were investigated. Hepatocytes in primary culture or co-cultured with proliferative epithelial cells, were not affected by the presence of 7 beta-OHC at a concentration of 400 microM over a period of 72 hours. In contrast, proliferative cultures of liver epithelial cell lines and liver fibroblast lines were killed by 50 microM 7 beta-OHC within the first 24 hours. Established liver epithelial cells (hyperploid) were more sensitive to 7 beta-OHC than the same line at early passages (diploid). When hepatocytes and liver epithelial cells were co-cultured and treated with 100 microM 7 beta-OHC, only epithelial cells were lysed. A concentration of 50 microM 7 beta-OHC was toxic to co-cultures of liver epithelial cell and fibroblasts together. In a serum-free medium, the cytotoxic concentration of 7 beta-OHC was lower than that in the serum-supplemented medium. Thus, liver epithelial cells cultured alone or co-cultured with hepatocytes were killed at 12.5 microM and 50 microM 7 beta-OHC, respectively. Finally, cholesterol concentrations four-fold that of 7 beta-OHC antagonized the lethal effects of 7 beta-OHC in the serum-free medium.

Albumins↗

PubMed1989

Purification of phospholamban from bovine cardiac muscle with organic solvents.

Phospholamban (PLB), an integral membrane protein of cardiac sarcoplasmic reticulum, was extracted from bovine cardiac muscle with an acidic chloroform/methanol mixture. A combination of gel permeation and ion-exchange chromatographies in organic solvents allowed the purification of PLB. The intensive use of organic solvents throughout the isolation yielded a highly purified and intact protein that can be phosphorylated by cAMP protein kinase. The ease of purification and the high yield obtained (2.5 mg/100 g of fresh tissue) show that organic solvents can be very useful in the extraction and purification of hydrophobic membrane proteins.

Amino Acid Sequence↗

PubMed1989

Studies on the immunosuppressive properties of 7,25 dihydroxycholesterol--II. Effects on early steps of T-cell activation.

The effects of an oxysterol, 7,25-dihydroxycholesterol (7,25-OHC), cyclosporin A (CsA) and dexamethasone (Dex) on the blastogenic response of murine lymphocytes to various stimuli were investigated. 7,25-OHC markedly depressed the response to Con A, anti-T3 monoclonal antibodies and to the combination phorbol myristate acetate (PMA) + IL-2 and PMA + ionomycin. Dexamethasone was also active within the same range of concentrations. However, it did not inhibit the stimulation induced by PMA + ionomycin. On the other hand, Cs A failed to depress the lymphocyte response to IL-2 + PMA. Therefore, the mechanisms of action involved in the blockage of lymphocyte activation are different for these three compounds. Moreover, we noted that 7,25-OHC was less active upon purified T-cells. Taken together, these results suggest that 7,25-OHC may act at the level of signal transduction across the membrane.

Animals↗

PubMed1988

Purification and characterization of low-molecular-weight beef heart proteolipids: use of fast atom bombardment-mass spectrometry for identification.

Bovine cardiac muscle was extracted by an acidic chloroform/methanol mixture. A combination of gel permeation and ion-exchange chromatographies in organic solvents and HPLC allowed the purification of subunits VIIIa (Mr 5400) and VIIIb (Mr 4900) of cytochrome c oxidase and of A6L protein (Mr 7900) of ATP synthase. The identification of the proteins was made possible by measurement of their molecular weight by fast atom bombardment-mass spectrometry (FAB-MS) in conjunction with conventional Edman degradation. The determination by FAB-MS of the molecular weight of A6L protein confirmed its supposed formylated N-terminal methionine.

Amino Acid Sequence↗

PubMed1988

6-Nitrocholesterol inhibits 3-hydroxy-3-methylglutaryl coenzyme A reductase and tumor cell growth.

6-Nitrocholesterol has been shown to cause a 50% reduction in the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in animal cells in culture at 1.9 microM and it has relative binding affinity for the cytosolic oxysterol binding protein of 357 nM in cell-free extracts from the same cell line. In addition, significant cytotoxicity was observed when this sterol was incubated with hepatoma and lymphoma cells in culture.

Animals↗

PubMed1988

Effect of 7 beta-hydroxycholesterol on growth and membrane composition of Mycoplasma capricolum.

7 beta-OH cholesterol in a cholesterol rich growth medium (5-10 micrograms/ml) extended the lag period and slowed down the growth rate of Mycoplasma capricolum cells. In a cholesterol poor medium (0.5 micrograms/ml) inadequate to support growth, 7 beta-OH cholesterol exerts a synergistic effect on growth. The 7 beta-OH cholesterol was incorporated unchanged from the growth medium and could be recovered exclusively in the membrane fraction. The incorporation of the 7 beta-OH cholesterol has no effect on the total phospholipid content but the DPG to PG ratio was markedly decreased. Exchange studies with lipid vesicles revealed that whereas most of the cholesterol underwent exchange, only about 20% of the 7 beta-OH cholesterol was exchanged.

Cardiolipins↗

PubMed1988

Metabolism of cytotoxic hydroxysterols in cultured cells. Chemical characterization of metabolites.

The metabolism of labelled 7 alpha- and 7 beta-hydroxycholesterol was investigated in two lymphoma cell lines (YAC-1, RDM-4), in murine splenocytes and in HTC hepatoma cells. The structures of the metabolites in lymphoma cells were determined as 3 beta-esters of C14-C20 fatty acids by nuclear magnetic resonance and mass spectrometric studies. In hepatoma cells, more polar metabolites of 7 alpha- and 7 beta-hydroxycholesterol were detected whereas, in non-dividing lymphocyte cells, no metabolic transformation occurs. Therefore, metabolic transformation of the hydroxycholesterol is not required for the expression of their activity and the question of the physiological role of the metabolic products is raised.

Animals↗

PubMed1988

Studies on the immunosuppressive properties of 7,25-dihydroxycholesterol--I. Reduction of interleukin production by treated lymphocytes.

Several oxysterols are powerful immunosuppressors. Among them 7,25-dihydroxycholesterol (7,25-OHC) seems to be particularly interesting. The effect of this synthetic sterol on murine lymphocytes, stimulated by Con A, or by allogeneic or TNP-modified syngeneic lymphocytes, was investigated. The results showed that the induction of cytotoxic T-lymphocytes (CTL) was strongly inhibited by a treatment with non-toxic concentrations of this sterol. A marked reduction of interleukin 2 (IL-2) production by alloantigen stimulated lymphocytes occurred at the same concentrations. We also showed that receptors to IL-2 did not appear on lymphocytes stimulated by Con A, when 7,25-OHC was added at the beginning of the culture. The addition of exogenous IL-2 to the culture was found to only slightly reverse the inhibition caused by 7,25-OHC. Taken together, these results indicate that an impairment at the early steps of lymphocyte activation, involving IL-2 secretion, could be responsible, at least in part for the various immunosuppressive effects of 7,25-OHC.

Animals↗