Biomedical subjects

B Luu

Publications and source records attributed to B Luu.

At least 19 recordsLinked to original sources

PubMed1996

Solution structure of PMP-C: a new fold in the group of small serine proteinase inhibitors.

The solution structure and the disulfide pairings of a 36-residue proteinase inhibitor isolated from the insect Locusta migratoria have been determined using NMR spectroscopy and simulated annealing calculations. The peptide, termed PMP-C, was previously shown to inhibit bovine alpha-chymotrypsin as well as human leukocyte elastase, and was also found to block high-voltage-activated Ca2+ currents in rat sensory neurones. PMP-C has a prolate ellipsoid shape and adopts a tertiary fold hitherto unobserved in the large group of small "canonical" proteinase inhibitors. The over-all fold consists mainly of three strands arranged in a right-handed twisted, antiparallel, beta-sheet that demarcates a cavity, together with a linear amino-terminal segment oriented almost perpendicular to the three strands of the beta-sheet. Inside the cavity a phenyl ring constitutes the centre of a hydrophobic core. The proteinase binding loop is located in the carboxy-terminal part of the molecule, between two cysteine residues involved in disulfide bridges. Its conformation resembles that found in other small canonical proteinase inhibitors. A comparison of PMP-C structure with the recently published solution structure of the related peptide PMP-D2 shows that the most significant differences are complementary changes involved in the stabilization of similar folds. This comparison led us to review the structure of PMP-D2 and to identify two salt bridges in PMP-D2.

Amino Acid Sequence↗

PubMed1995

Serine protease inhibition by insect peptides containing a cysteine knot and a triple-stranded beta-sheet.

Three insect peptides showing high sequence similarity and belonging to the same structural family incorporating a cysteine knot and a short three-stranded antiparalled beta-sheet were studied. Their inhibitory effect on two serine proteases (bovine alpha-chymotrypsin and human leukocyte elastase) is reported. One of them, PMP-C, is a strong alpha-chymotrypsin inhibitor (Ki = 0.2 nM) and interacts with leukocyte elastase with a Ki of 0.12 microM. The other two peptides, PMP-D2 and HI, interact only weakly with alpha-chymotrypsin and do not inhibit leukocyte elastase. Synthetic variants of these peptides were prepared by solid-phase synthesis, and their action toward serine proteases was evaluated. This enabled us to locate the P1 residues within the reactive sites (Leu-30 for PMP-C and Arg-29 for PMP-D2 and HI), and, interestingly, variants of PMP-D2 and HI were converted into powerful inhibitors of both alpha-chymotrypsin and leukocyte elastase, the most potent elastase inhibitor obtained in this study having a Ki of 3 nM.

Amino Acid Sequence↗

PubMed1995

[From cholesterol to oxysterols. Current data].

Oxysterols, a class of cholesterol oxidation products exhibit several important biological activities. Some of these natural compounds are potent inhibitors of the enzyme HMG-CoA reductase, a key enzyme in the cholesterol biosynthetic pathway. Many studies have been directed towards to the verification of the hypothesis that some oxysterols are endogenous intracellular regulators of cholesterol homeostasis. In adition to oxysterols derived directly from oxidation of cholesterol, several others are formed from squalene dioxide. It is presently well established that, in addition to the classical cholesterol biosynthetic pathway, there exists an alternate bifurcation from squalene oxide. The cyclisation of squalene dioxide leads to a series of new oxysterols. Thus, several types of oxysterols and several molecular targets are involved in the regulation of steroid biosynthesis. Many oxysterols, particularly those obtained from the oxidations of phytosterols and tetracyclic triterpenes are potent cytotoxic agents. They are selectively cytotoxic against tumorous cells. This cytotoxicity depends markedly on the specific structure of each oxysterol. Some structures are very cytotoxic, while their stereoisomers are inactive. The activity depends on the tumor cells which are used in the assay system: some compounds display inhibitory activity towards hepatoma cells but are inactive against lymphoma cells while others act in the opposite manner. Free oxysterols do not depress tumor growth in living animals. However, several water soluble prodrugs of oxysterols are able to depress different type of tumors in vivo. Clinical trial studies are presently conducted in order to learn the therapeutic values of these oxysterols.

Adjuvants, Immunologic↗

PubMed1994

Solution structure of PMP-D2, a 35-residue peptide isolated from the insect Locusta migratoria.

The three-dimensional solution structure of PMP-D2, a 35 amino acid peptide isolated from the insect Locusta migratoria, has been determined from two-dimensional 1H NMR spectroscopy data. The structure calculations were performed from 222 NOE-derived interproton distances and 11 dihedral angles calculated from the JHN-H alpha coupling constants, using either a combination of distance geometry and restrained simulated annealing or by restrained simulated annealing alone. PMP-D2 contains three disulfide bridges that have been assigned from NMR data and structure calculations and independently confirmed using chemical and enzymatic methods. The core region of PMP-D2 adopts a compact globular fold, stabilized by hydrophobic interactions, which consists of a short three-stranded antiparallel beta-sheet involving residues 8-11, 15-19, and 25-29. Back-calculation of the NOESY spectra was used to validate the final structures. Analysis of the CD spectra of PMP-D2 under various conditions of ionic strength and in the presence of organic solvents demonstrates the high stability of this molecule. PMP-D2 was recently shown to inhibit Ca2+ currents. This activity is discussed based on the comparison of PMP-D2 three-dimensional structure with the recently established three-dimensional structure of the Ca2+ channel blocker omega-conotoxin GVIA.

Amino Acid Sequence↗

PubMed1993

Apoptosis induced by oxysterols in murine lymphoma cells and in normal thymocytes.

Oxygenated derivatives of cholesterol (oxysterols), a family of naturally occurring compounds, possess marked anti-proliferative and immunosuppressive activities, in particular they have been shown to inhibit T-cell responses to different stimuli. 25-Hydroxycholesterol (25-OHC) and 7 beta,25-dihydroxycholesterol (7.25-OHC) are able to kill not only RDM4 murine lymphoma in vitro, but also, surprisingly, mouse thymocytes after several hours of incubation. In this study, we report that the death of RDM4 and thymocytes induced by oxysterols exhibits the features of apoptosis. This phenomenon was identified by agarose gel electrophoresis of DNA fragments extracted from the cells and quantified by flow cytometric analysis of the DNA fluorescence of propidium iodide-stained cells. Cycloheximide and actinomycin D were found to decrease the number of apoptotic cells and to increase cell viability, indicating a requirement for the synthesis of macromolecules in oxysterol-induced programmed cell death. The pathway by which 25-OHC and 7.25-OHC are able to induce apoptosis in this type of cell and the possible contribution of these compounds to thymus involution during development are discussed.

Animals↗

PubMed1992

Isolation and structural determination of three peptides from the insect Locusta migratoria. Identification of a deoxyhexose-linked peptide.

We have isolated three novel peptides from the aqueous extract of the pars intercerebralis of male and female adults of the insect Locusta migratoria. After extensive HPLC purification, the peptides were characterised by automated Edman degradation and electrospray mass spectrometry: one is a 35-residue peptide (3752.3 +/- 1.1 Da) containing six cysteines involved in three intramolecular disulfide bridges, the second is a 36-residue peptide (3919.2 +/- 0.9 Da), also cross-linked by three intramolecular disulfide bridges. This second peptide is post-translationally modified by a single fucose moiety, which was identified by gas chromatography coupled to mass spectrometry. These two peptides show a strong sequence similarity. Additionally, they were also found in larger amounts in the fat body of Locusta; this finding raises the question of their exact site of synthesis. The third peptide (5776.3 +/- 0.9 Da), a 54-residue peptide cross-linked by six intramolecular disulfide bridges, is structurally related to the two other peptides, but to a lesser extent. Mass spectrometry has shown that all the cysteines within these three peptides are involved in intramolecular disulfide bridges; however, the location of these bridges is not yet established and is currently being investigated. A computer search of sequence data banks did not reveal any significant similarity of these three peptides with other known proteins.

Amino Acid Sequence↗

PubMed1992

Studies on the immunological effects of fatty alcohols--I. Effects of n-hexacosanol on murine macrophages in culture.

n-Hexacosanol (hexa), a long chain fatty alcohol extracted from Hygrophila erecta, has proved to possess neurotrophic activities on cultured neurons, and to attenuate the degeneration of cholinergic neurons after injury. In the present study, we show that hexa has also interesting properties on macrophages, a cell type largely represented in the brain: when added to mice resident peritoneal macrophages, it provokes significant morphological changes, and increases their phagocytosis capacity. These results may indicate that some membrane properties involved in these different effects and in macrophage functions are affected by n-hexacosanol, but other sites of action could also be considered.

Animals↗

PubMed1991

Electrophysiological activity of the C-peptide of the Locusta insulin-related peptide. Effect on the membrane conductance of Locusta neurones in vitro.

The C-peptide of Locusta insulin-related peptide, which is a 50 residue peptide originally isolated from the corpora cardiaca of the insect Locusta migratoria and to which we refer as 5-kDa peptide, has been synthesised chemically by the solid-phase method, using a BOC strategy. Since this peptide contains in its sequence a potential monobasic cleavage site, we also synthesised its 1-38 residue-related fragment, named 4-kDa peptide, although we have no hints of its natural occurrence in the corpora cardiaca. Electrophysiological studies have shown that both the 5-kDa and 4-kDa peptides depolarise the membrane and increase the membrane conductance of neurones freshly isolated from the thoracic ganglia of Locusta. Under voltage-clamp conditions, the current underlying these effects was inwardly directed and could be resolved into 2 components. One component, I(5-kDa)1, activated at potentials more hyperpolarised than -50 mV, peaked at about -75 mV and was blocked by the potassium channel blockers cesium and rubidium. The second component, I(5-kDa)2 was activated at potentials more depolarised than -50 mV, increased with depolarisation and was not blocked by cesium and rubidium. The effects of the 5-kDa and 4-kDa peptides on the membrane potential and membrane conductance of Locusta neurones suggest that these peptides may have a physiological role in the central nervous system of insects.

Animals↗

PubMed1991

Differential sensitivity of astrocyte primary cultures and derived spontaneous transformed cell lines to 7 beta-hydroxycholesterol: effect on plasma membrane lipid composition and fluidity, and on cell surface protein expression.

The cytotoxicity of 7 beta-hydroxycholesterol (7 beta-OHC) was investigated on rat astrocyte primary cultures and spontaneously transformed cell lines derived from them. Confluent astrocyte primary cultures (normal cells) were unaffected by 20 microM 7 beta-OHC over a period of 72 h whereas 30 microM markedly affected the viability of the transformed cells within the first 72 h. Both cell types incorporated 18% of the total amount of 7 beta-OHC added to the cultures at concentrations of 20 microM or 30 microM. Cellular fractionation after incubation with 20 microM or 30 microM 7 beta-OHC indicated that the plasma membrane incorporated 2 or 6 fold more 7 beta-OHC than the intracellular one's respectively. Plasma membrane cholesterol (CH) and phospholipid (PL) analysis showed that 20 microM 7 beta-OHC did not affect CH/PL in normal cells; in contrast, plasma membranes of transformed cells displayed a significant CH/PL decrease, which was more pronounced with 30 microM 7 beta-OHC treatment. Fluorescence anisotropy measurements indicated that 20 microM 7 beta-OHC slightly fluidified the plasma membrane of normal cells whereas it has not effect on that of the transformed cells one; however, an increase in plasma membrane fluidity was observed when the transformed cells were treated with 30 microM 7 beta-OHC. Lactoperoxidase catalyzed radioiodination of cell surface proteins and subsequent autoradioelectrophoretic analysis demonstrated that the labelled protein pattern was unchanged when both cell types were incubated with 30 microM 7 beta-OHC. These findings demonstrate that 7 beta-OHC is lethal to highly proliferating cultured glial cells. The high accumulation of 7 beta-OHC in the plasma membrane and its decrease in fluidity, by themselves, do not seem to be involved in the processes leading to cellular death. However, increase of plasma membrane fragility associated with the decrease of CH/PL, which occurs exclusively in plasma membranes isolated from 7 beta-OHC treated transformed cells together with high 7 beta-OHC uptake, are probably implicated in 7 beta-OHC cytotoxicity. The possibility of an additional action mechanism is discussed.

Animals↗

PubMed1991

Revision of the amino acid sequence of the smallest bc1 complex subunit: use of fast atom bombardment mass spectrometry and mass-analysed ion kinetic energy spectrum analysis.

We have isolated the smallest bc1 complex subunit from an acidic chloroform/methanol extract of bovine cardiac muscle. The identification of the polypeptide was made possible by classical Edman degradation and amino acid analysis. The measurement of its exact molecular weight by fast atom bombardment mass spectrometry (m/z 6519.8), the characterization of a tryptophan cleavage peptide and pepsic peptides by mass measurements and by mass-analysed ion kinetic energy spectrum analysis allow rectification of the amino acid sequence of the smallest bc1 complex subunit. We found a serine residue instead of Gln 22 and tryptophan residues in place of Ser 34 and Ser 38.

Amino Acid Sequence↗

PubMed1991

Oxysterols: biological activities and physicochemical studies.

To improve the understanding of the various biological activities of oxysterols (oxygenated derivatives of cholesterol), studies of their physicochemical properties have been undertaken. Oxysterols modify membrane dynamic properties which consequently trigger several biological effects. Despite the presence of at least one oxygenated group in addition to the C3 beta-hydroxyl, oxysterols insert perfectly into the lipidic bilayer of the membrane inducing a condensing effect similar to, but less potent than, that of cholesterol. In biological membranes oxysterols probably interact with membrane components as they are not easily exchanged after their incorporation into the cell membrane. These lipid-protein interactions are probably crucial for the expression of the biological activities of the oxysterols.

Cell Membrane↗

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↗