[Interventions for smoking cessation in 2018]. / Les outils du sevrage tabagique en 2018.

Smoking cessation treatments have been proved effective to stop smoking. For pharmacological treatments, nicotine replacement therapies (NRT) as well as bupropion allow to increase 6 month-abstinence rates by more than 80% in comparison with placebo while varenicline prescription doubles success rates in the same conditions. These results mean that for 10 smokers who quit with placebo, 18 are expected to quit with NRT or bupropion and 28 are expected to quit with varenicline. Varenicline is 50% more effective than nicotine patch and 70% more effective than nicotine gum. Nevertheless, a combination including NRT patch and oral nicotine forms is as effective as varenicline, thus leading to encourage the prescription of a combination NRT when NRT are chosen. For these three pharmacological treatments, cardiovascular as well as neuropsychiatric tolerance were not found statistically different from placebo in randomized controlled trials. Yet, bupropion prescription leads to an increasing risk of seizure (1/1000 to 1/1500). For behavioral treatment, motivational interviewing as well as cognitive behavior therapies are been proven to be effective to stop smoking but few smokers have access to this treatment. Smoking cessation mobile application and smartphone application seem to be promising in terms of effectiveness and might be useful to reach more smokers.

First Report of Marasmiellus palmivorus Causing Post-Emergence Damping Off on Coconut Seedlings in Malaysia.

In April and June 2010, coconut seedlings with symptoms of very slow growth, yellowing of leaves, and general abnormal leaf growth were observed in germination beds in Teluk Intan, Perak, Malaysia. The roots were soft, rotten, and brown, extending upward and downward from these lesions. Rhizomorphs and basidiocarps were produced on coconut seeds near the germination eye and identified as Marasmiellus palmivorus according description by Turner (2). Three isolates were obtained by plating surface sterilized symptomatic roots and basidiocarp on malt extract agar (MEA) amended with 85% lactic acid (1 ml added to 11 of the medium). To confirm the identity of the fungus, genomic DNA was extracted from mycelia and basidiocarps of isolates and the large subunit (LSU) region was amplified and sequenced using LR0R/LR7 primers (3). All isolates had identical LSU sequences (GenBank Accession No. JQ654233 to JQ654235). Sequences were identical to each other and 99% similar to a M. palmivorus sequence deposited in the NCBI database (Accession No. AY639434).To confirm pathogenicity, three isolates of M. palmivorus that were obtained from symptomatic plant tissue was inoculated onto seeds of Malaysian Red Dwarf variety. Each isolate was grown in 100 ml of malt extract broth in 250 ml Erlenmeyer flasks and incubated at 27 ± 2°C for 5 days on an orbital shaker (125 rpm). The resulting culture was passed through two layers of sterile cloth. Mycelial suspension was obtained by blending mycelia in 100 ml of sterile water. Seeds were sterilized by soaking in 10% v/v sodium hypochlorite in distilled water for 3 min. The seeds were then rinsed three times over running tap water. The calyx portion of the seed was removed and five holes were made around the germination eye. The seeds were inoculated by injecting 2 ml of suspension into each hole. The control seeds were inoculated with sterile distilled water only. The seeds were transferred to 40-cm diameter plastic pots containing a mixture of sand, soil, and peat in the ratio of 3:2:1, respectively, and steam treated at 100°C for 1.5 h. Pots were placed in the glasshouse with normal exposures to day-night cycles, temperatures of 29 ± 4°C, and high relative humidity (85 to 95%) achieved by spraying water twice daily. After 2 months, 75% of the inoculated seeds failed to germinate. It was speculated that the artificial inoculum was higher than under germination bed conditions. Rhizomorphs and basidiocarps were produced on husk seeds near the germination eye. Seedlings that emerged successfully developed symptoms similar to those observed in the germination bed. No symptoms developed in the noninoculated seeds and seedlings. At 80 days post inoculation, basidiocarps were observed emerging from three diseased seedlings near the germination eye. Three reisolations were made on MEA from root lesions surface sterilized. Pathogenicity tests and LSU sequence analyses indicated that M. palmivorus is the causal agent of the symptoms observed on coconut seedlings. M. palmivorus was first recorded on coconuts and oil palm in the 1920s (1) and attacks the fruit and the petiole on oil palm (2). To our knowledge, this is the first report of M. palmivorus causing post-emergence damping off on coconut seedlings. References: (1) K. G. Singh. A check-list of host and diseases in Malaysia. Ministry of Agriculture and Fisheries, Malaysia, 1973. (2) P. D. Turner. Oil palm diseases and disorders. Oxford University Press. 1981. (3) R. Vilgalys et al. J. Bacteriol. 172:4238, 1990.

Sorption of Cr(VI), Cu(II) and Pb(II) by growing and non-growing cells of a bacterial consortium.

This paper reports the sorption of three metallic ions, namely Cr(VI), Cu(II) and Pb(II) in aqueous solution by a consortium culture (CC) comprising an acclimatised mixed bacterial culture collected from point and non-point sources. Metal sorption capability of growing and non-growing cells at initial pH of between 3 and 8 in the 1-100mg/L concentration range were studied based on Q(max) and K(f) values of the Langmuir and linearised Freundlich isotherm models, respectively. Maximal metal loading was generally observed to be dependent on the initial pH. Growing cells displayed significant maximal loading (Q(max)) for Pb(II) (238.09 mg/g) and Cu(II) (178.87 mg/g) at pH 6 and at pH 7 for Cr(VI) (90.91 mg/g) compared to non-growing cells (p < 0.05). At the pH range of 6-8, growing cells showed higher loading capacity compared to non-growing cells i.e. 38-52% for Cr, 17-28% for Cu and 3-17% for Pb. At lower metal concentrations and at more acidic pH (3-4) however, non-growing cells had higher metal loading capacity than growing cells. The metal sorption capacity for both populations were as follows: Pb(II) > Cu(II) > Cr(VI).

A lipid transfer protein binds to a receptor involved in the control of plant defence responses.

Lipid transfer proteins (LTPs) and elicitins are both able to load and transfer lipidic molecules and share some structural and functional properties. While elicitins are known as elicitors of plant defence mechanisms, the biological function of LTP is still an enigma. We show that a wheat LTP1 binds with high affinity sites. Binding and in vivo competition experiments point out that these binding sites are common to LTP1 and elicitins and confirm that they are the biological receptors of elicitins. A mathematical analysis suggests that these receptors could be represented by an allosteric model corresponding to an oligomeric structure with four identical subunits.

A novel patatin-like gene stimulated by drought stress encodes a galactolipid acyl hydrolase.

A cDNA (Vupat1) encoding a predicted 43 kDa protein was isolated from drought-stressed cowpea (Vigna unguiculata) leaves. It has homology with patatin, a potato tuber storage protein with lipolytic acyl hydrolase activity. The recombinant protein VUPAT1 expressed in the baculovirus system displays preferentially galactolipid acyl hydrolase activity. Phospholipids are very slowly hydrolyzed and apparently triacylglycerols are not deacylated. Vupat1 promoter contains putative drought-inducible sequences. Northern blots showed that gene expression is stimulated by drought stress and is more pronounced in a drought-sensitive cultivar than in a drought-tolerant one. An involvement in drought-induced galactolipid degradation is proposed for VUPAT1.

Lipid transfer proteins are encoded by a small multigene family in Arabidopsis thaliana.

Lipid transfer proteins (LTPs) are small, basic and abundant proteins in higher plants. They are capable of binding fatty acids and of transferring phospholipids between membranes in vitro. LTPs from this family contain a signal peptide and are secreted in the cell wall. Their biological function is presently unknown. LTPs have been suggested to participate to cutin assembly and to the defense of the plants against pathogens. A genetic approach should prove useful to provide clues on their in vivo functions. Here, the characterization of the LTP gene family in Arabidopsis thaliana is described. At least 15 genes were identified, their map position determined and the expression pattern characterized for six of them. All the sequences exhibit the typical features of plant LTPs. The molecular weight is close to 9 kDa, the isoelectric point is near 9 (except for three acidic LTPs), and typical amino acid residues such as cysteines are conserved. Genomic DNA blotting hybridization experiments performed using ltp1 to ltp6 as probes indicate that ltps form distinct 1-3 gene subfamilies which do not cross hybridize. Expression studies indicate that all the genes tested are expressed in flowers and siliques, but not in roots. Ltp1, ltp5 and ltp2 are expressed significantly in leaves, while ltp6 is detected only in 2-4-week-old leaves. In addition, ltp4 and ltp3 are strongly upregulated by abscisic acid (ABA). Tandem repeats can be noted concerning ltp1 and ltp2 on chromosome 2, ltp3 and ltp4 on chromosome 5 and ltp5 and ltp12 on chromosome 3. While ltp7, ltp8 and ltp9 map at the same position on chromosome 2, the other genes are dispersed throughout the genome. The characterization of the Arabidopsis ltp gene family will permit to initiate a genetic approach for determining the in vivo function(s) of these proteins.

Two novel plant cDNAs homologous to animal type-2 phosphatidate phosphatase are expressed in cowpea leaves and are differently regulated by water deficits.

Two cDNAs encoding putative phosphatidate phosphatases (PAPs) designated VuPAP-alpha and VuPAP-beta were cloned in cowpea (Vigna unguiculata L.) leaves. The predicted proteins have six membrane-spanning regions in common with animal type-2 PAPs. Unlike VuPAP-beta, VuPAP-alpha has an N-terminal transit peptide and is targeted in vitro to the chloroplasts. Gene expression of VuPAP-beta is stimulated by rapid air-desiccation of leaves and VuPAP-alpha transcripts increase during rehydration of plants exposed to drought-like conditions.

Comparison of lipid binding and transfer properties of two lipid transfer proteins from plants.

Plant lipid transfer proteins (LTPs) are soluble proteins which are characterized by their in vitro ability to transfer phospholipids between two membranes. We have compared the functional properties of two LTPs purified from maize and wheat seeds knowing that, despite a high degree of sequence identity, the two proteins exhibit structural differences. It was found that wheat LTP had a lower transfer activity than the maize LTP, consistent with a lower kinetics of fatty acid binding. The lower affinity for the fatty acids of the wheat LTP could be explained by a narrowing occurring in the middle part of the binding site, as revealed by comparing the fluorescence spectra of various anthroyloxy-labeled fatty acids associated with the two LTPs. The affinity for some natural fatty acids was studied by competition with fluorescent fatty acids toward binding to the protein. Again, wheat LTP had a lower affinity for those molecules. All together, these observations reveal the complexity of the LTP family in plants, probably reflecting the multiple roles played by these proteins.

Identification of AtPIS, a phosphatidylinositol synthase from Arabidopsis.

Phosphatidylinositol synthase is the enzyme responsible for the synthesis of phosphatidylinositol, a key phospholipid component of all eukaryotic membranes and the precursor of messenger molecules involved in signal transduction pathways for calcium-dependent responses in the cell. Using the amino acid sequence of the yeast enzyme as a probe, we identified an Arabidopsis expressed sequence tag potentially encoding the plant enzyme. Sequencing the entire cDNA confirmed the homology between the two proteins. Functional assays, performed by overexpression of the plant cDNA in Escherichia coli, a bacteria which lacks phosphatidylinositol and phosphatidylinositol synthase activity, showed that the plant protein induced the accumulation of phosphatidylinositol in the bacterial cells. Analysis of the enzymatic activity in vitro showed that synthesis of phosphatidylinositol occurs when CDP-diacylglycerol and myo-inositol only are provided as substrates, that it requires manganese or magnesium ions for activity, and that it is at least in part located to the bacterial membrane fraction. These data allowed us to conclude that the Arabidopsis cDNA codes for a phosphatidylinositol synthase. A single AtPIS genetic locus was found, which we mapped to Arabidopsis chromosome 1.

A plastidial lysophosphatidic acid acyltransferase from oilseed rape.

The biosynthesis of phosphatidic acid, a key intermediate in the biosynthesis of lipids, is controlled by lysophosphatidic acid (LPA, or 1-acyl-glycerol-3-P) acyltransferase (LPAAT, EC 2.3.1.51). We have isolated a cDNA encoding a novel LPAAT by functional complementation of the Escherichia coli mutant plsC with an immature embryo cDNA library of oilseed rape (Brassica napus). Transformation of the acyltransferase-deficient E. coli strain JC201 with the cDNA sequence BAT2 alleviated the temperature-sensitive phenotype of the plsC mutant and conferred a palmitoyl-coenzyme A-preferring acyltransferase activity to membrane fractions. The BAT2 cDNA encoded a protein of 351 amino acids with a predicted molecular mass of 38 kD and an isoelectric point of 9.7. Chloroplast-import experiments showed processing of a BAT2 precursor protein to a mature protein of approximately 32 kD, which was localized in the membrane fraction. BAT2 is encoded by a minimum of two genes that may be expressed ubiquitously. These data are consistent with the identity of BAT2 as the plastidial enzyme of the prokaryotic glycerol-3-P pathway that uses a palmitoyl-ACP to produce phosphatidic acid with a prokaryotic-type acyl composition. The homologies between the deduced protein sequence of BAT2 with prokaryotic and eukaryotic microsomal LAP acytransferases suggest that seed microsomal forms may have evolved from the plastidial enzyme.

Lipid-transfer proteins from plants: structure and binding properties.

Plant cells contain lipid-transfer proteins (LTPs) able to transfer phospholipids between membranes in vitro. Plant LTPs share in common structural and functional features. Recent structural studies carried out by NMR and X-ray crystallography on an LTP isolated from maize seeds have showed that this protein involves four helices packed against a C-terminal region and stabilized by four disulfide bridges. A most striking feature of this structure is the existence of an internal hydrophobic cavity running through the whole molecule and able to accommodate acyl chains. It was thus of interest to study the ability of maize LTP to bind hydrophobic ligands such as acyl chains or lysophosphatidylcholine and to determine the effect of this binding on phospholipid transfer. The binding abilities of maize LTP, presented in this paper, are discussed and compared to those of lipid-binding proteins from animal tissues.

Characterisation of acyl binding by a plant lipid-transfer protein.

Maize lipid-transfer protein (LTP) is a small soluble protein which is able to transfer in vitro phospholipids between membranes and to bind fatty acids or lysoderivatives. In the studies reported here, fluorescent-labelled fatty acids were used to characterise the nature of the binding site on LTP. A fluorescent analogue of 12 carbons with a pyrene moiety attached at the end, alone or in conjunction with an anthroyloxy analogue, indicated that LTP could bind two fatty acids although with a marked difference in affinity. The binding capacity was strongly affected after reduction of the protein by dithiothreitol, showing that the four S-S bonds of LTP are essential for its lipid binding property. Other analogues used were 16-carbon or 18-carbon fatty acids with an anthracene moiety attached at different points of the hydrocarbon chain. Emission maxima of these molecules varied with the analogue and suggested a motional constraint for the bound fatty acid which is more important around the middle of the chain than at its extremities. Binding displacement studies were carried out with a wide range of fatty acids or fatty acyl derivatives. Fatty acids of 16 to 19 carbons were found to be the preferred ligands. The presence of one double bond did not change appreciably the affinity of LTP, although the presence of two or three double bonds or of a hydroxyl moiety significantly reduced the affinity. Fatty acyl-CoA or lysoderivatives bound as well as the corresponding fatty acid.

Isolation of a cDNA from Arabidopsis thaliana that complements the sec14 mutant of yeast.

The SEC14 gene of Saccharomyces cerevisiae codes for a phosphatidylinositol-transfer protein (Sec14p(sc)) which is capable of transferring both phosphatidylinositol and phosphatidylcholine between membranes in vitro. Genetic and biochemical studies conducted in S. cerevisiae have shown that this protein acts as an inhibitor of phosphatidylcholine biosynthesis via the so-called Kennedy pathway only. This inhibition is controlled by the binding of phospholipids to the Sec14p(sc) protein. Here we describe the isolation of a cDNA from Arabidopsis thaliana by functional complementation of a sec14(ts) mutant of S. cerevisiae. This cDNA, designated AtSEC14, is capable of restoring the growth of the sec14(ts) mutant at the restrictive temperature of 37 degrees C. Extracellular invertase measurements indicated that the cDNA can partly restore protein secretion. In addition, the phosphatidylinositol-transfer activity measured in protein extracts is greatly enhanced in the complemented mutant strain when compared with the sec14(ts) mutant. The best sequence similarity at the amino acid level is found with the Sec14p protein of S. cerevisiae (36.5% similarity), and most of the amino acids that are thought to be involved in the binding of phospholipids in the yeast protein are conserved in the AtSEC14 gene product. Southern analysis suggests the presence of a single gene in the Arabidopsis genome, although the existence of distantly related sequences cannot be excluded. This gene is expressed in roots, leaves, flowers and siliques of Arabidopsis.

Rice lipid transfer protein (LTP) genes belong to a complex multigene family and are differentially regulated.

Several cDNA clones encoding three different lipid transfer proteins (LTPs) have been isolated from rice (Oryza sativa L.) in order to analyse the complexity, the evolution and the expression of the LTP gene family. The mature proteins deduced from three clones exhibited a molecular mass of 9 kDa, in agreement with the molecular mass of other LTPs from plants. The clones were shown to be homologous in the coding region, while the 3' non-coding regions diverged strongly between the clones. The occurrence of at least three small multigene families encoding these proteins in rice was confirmed by Southern blot analysis. When compared with each other and with LTPs from other plants, the cluster including rice LTPs and other cereal LTPs indicated that these genes duplicated rather recently and independently in the different plant phyla. The expression pattern of each gene family was also investigated. Northern blot experiments demonstrated that they are differentially regulated in the different tissues analysed. Components such as salt, salicylic acid and abscisic acid were shown to modulate Ltp gene expression, depending on tissues and gene classes, suggesting a complex regulation of these genes.

Solution structure and lipid binding of a nonspecific lipid transfer protein extracted from maize seeds.

The three-dimensional solution structure of a nonspecific lipid transfer protein extracted from maize seeds determined by 1H NMR spectroscopy is described. This cationic protein consists of 93 amino acid residues. Its structure was determined from 1,091 NOE-derived distance restraints, including 929 interresidue connectivities and 197 dihedral restraints (phi, psi, chi 1) derived from NOEs and 3J coupling constants. The global fold involving four helical fragments connected by three loops and a C-terminal tail without regular secondary structures is stabilized by four disulfide bridges. The most striking feature of this structure is the existence of an internal hydrophobic cavity running through the whole molecule. The global fold of this protein, very similar to that of a previously described lipid transfer protein extracted from wheat seeds (Gincel E et al., 1994, Eur J Biochem 226:413-422) constitutes a new architecture for alpha-class proteins. 1H NMR and fluorescence studies show that this protein forms well-defined complexes in aqueous solution with lysophosphatidylcholine. Dissociation constants, Kd, of 1.9 +/- 0.6 x 10(-6) M and > 10(-3) M were obtained with lyso-C16 and -C12, respectively. A structure model for a lipid-protein complex is proposed in which the aliphatic chain of the phospholipid is inserted in the internal cavity and the polar head interacts with the charged side chains located at one end of this cavity. Our model for the lipid-protein complex is qualitatively very similar to the recently published crystal structure (Shin DH et al., 1995, Structure 3:189-199).

Germination-specific lipid transfer protein cDNAs in Brassica napus L.

Three cDNA clones encoding lipid transfer proteins (LTPs) were isolated by applying the rapid amplification of cDNA ends (RACE) protocol to imbibed seeds and germinating seedlings Brassica napus. The deduced amino-acid sequences show a great degree of homology and they exhibit the common features shared by all LTPs. Their expression pattern indicates a strong developmental, hormonal, and environmental regulation. They are expressed only in cotyledons and hypocotyls of germinating seedlings and their levels of expression increase upon treatment with cis-abscisic acid and NaCl. Their distribution in the cotyledons of young seedlings is suggestive of a role related to the mobilization of lipid reserves.

A potent antimicrobial protein from onion seeds showing sequence homology to plant lipid transfer proteins.

An antimicrobial protein of about 10 kD, called Ace-AMP1, was isolated from onion (Allium cepa L.) seeds. Based on the near-complete amino acid sequence of this protein, oligonucleotides were designed for polymerase chain reaction-based cloning of the corresponding cDNA. The mature protein is homologous to plant nonspecific lipid transfer proteins (nsLTPs), but it shares only 76% of the residues that are conserved among all known plant nsLTPs and is unusually rich in arginine. Ace-AMP1 inhibits all 12 tested plant pathogenic fungi at concentrations below 10 micrograms mL-1. Its antifungal activity is either not at all or is weakly affected by the presence of different cations at concentrations approximating physiological ionic strength conditions. Ace-AMP1 is also active on two Gram-positive bacteria but is apparently not toxic for Gram-negative bacteria and cultured human cells. In contrast to nsLTPs such as those isolated from radish or maize seeds, Ace-AMP1 was unable to transfer phospholipids from liposomes to mitochondria. On the other hand, lipid transfer proteins from wheat and maize seeds showed little or no antimicrobial activity, whereas the radish lipid transfer protein displayed antifungal activity only in media with low cation concentrations. The relevance of these findings with regard to the function of nsLTPs is discussed.

Selective modifications of the phospholipid fatty acid composition in human platelet membranes using nonspecific and specific lipid transfer proteins.

In order to specifically modify the fatty acid composition of cell membrane phospholipids, we have developed an original method based on the transfer of pure phospholipid molecular species to membranes. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) subclasses containing 18:2n-6 and 22:6n-3 at the sn-2 position were incorporated into human platelet membranes using the endogenous phosphatidylinositol/PC transfer protein (PI/PC-TP) and the phospholipid transfer protein from maize (L-TP), respectively. PI/PC-TP was shown to catalyze a strict exchange of phospholipids between platelet membranes and unilamellar vesicles containing 1,2-diacylglycerophosphocholine (diacyl-GPC; 16:0/18:2-GPC, or 16:0/22:6-GPC). The proportions of 18:2n-6 and 22:6n-3 in diacyl-GPC of platelet membranes were gradually increased from 10.7 to 16.9% and from 0.8 to 10.1%, respectively, whereas the PE and PI fatty acid compositions were not changed. The diacyl-GPC enrichment in 22:6n-3 and 18:2n-6 did not induce changes in membrane fluidity parameters measured by electron-spin resonance of 5- and 16-nitroxy stearic acids. Similarly, 18:2n-6 and 22:6n-3 esterified in 1,2-diacylglycerophosphoethanolamine (diacyl-GPE) have been incorporated in platelet membranes by an apparent exchange process under conditions where donor vesicles had a phospholipid composition equivalent to that of platelet membranes. The proportions of 18:2n-6 and 22:6n-3 were selectively and progressively increased from 6.0 to 21.2% and from 2.2 to 17.2%, respectively, in diacyl-GPE of platelet membranes. Thus, the L-TP- and PI/PC-TP-catalyzed enrichment can be used for studying the modulation of membrane biological activities by defined changes of fatty acid composition of specific phospholipid classes or subclasses.

Compared selectivities of the phosphatidylinositol-synthase from maize coleoptiles either in microsomal membranes or after solubilization.

PI-synthase selectivity from etiolated maize coleptiles was studied either associated with the microsomal membranes or after solubilization by CHAPS and prepurification on a DEAE-trisacryl M column. When maize microsomes were incubated with [3H]inositol without any exogenous CPM-PA, the most heavily labelled molecular species were 16:0/18:2-PI (77%), 16:0/18:3-plus 18:2/18:2-PI (15%), 16:0/18:1-PI (4%) and 18:0/18:2-PI (4%). Addition to the incubation medium of up to 300 microM 16:0/16:0-CMP-PA unexpectedly resulted in the formation of very little labelled 16:0/16:0-PI. When the solubilized fraction from microsomes was incubated with [3H]inositol in absence of 16:0/16:0-CPM-PA, the same PI molecular species as above were synthesized. However, with increasing concentrations of 16:0/16:0-CMP-PA in the medium, increasing amounts of labelled 16:0/16:0-PI appeared as well. With prepurified PI-synthase eluted from a DEAE column, endogenous CMP-PA was poorly utilized for PI biosynthesis whereas the exogenous 16:0/16:0-CPM-PA was used actively. With time, the endogenous CMP-PA was utilized first and the exogenous substrate was utilized, albeit, much more slowly. The results demonstrate that the selectivity displayed by PI-synthase towards various molecular species of CMP-PA depends on the integration of the enzyme in the membrane structure. Solubilization of the enzyme, i.e., inclusion of the protein in micelles with detergents and lipids, results in an apparent loss of the selectivity for CMP-PA.