Chiral molecular self-assembly of phospholipid tubules: A circular dichroism study

We report on spectroscopic studies of the chiral structure in phospholipid tubules formed in mixtures of alcohol and water. Synthetic phospholipids containing diacetylenic moieties in the acyl chains self-assemble into hollow, cylindrical tubules in appropriate conditions. Circular dichroism provides a direct measure of chirality of the molecular structure. We find that the CD spectra of tubules formed in mixtures of alcohol and water depends strongly on the alcohol used and the lipid concentration. The relative spectral intensity of different circular dichroism bands correlates with the number of bilayers observed using microscopy. The results provide experimental evidence that tubule formation is based on chiral packing of the lipid molecules and that interbilayer interactions are important to the tubule structure

Synthesis of novel disulfide containing macrocyclic diacylglycerols

New diacylglycerols (2-4) containing intramolecular disulfide linkages between pendant acyl chains were synthesized. Due to the differences in the location of disulfide units, the present method allows synthesis of macrocycles that vary in sizes. Copyright.

Discovery of genes associated with fruit ripening in arica papaya using expressed sequence tags

To identify genes involved in papaya fruit ripening, a total of 1171 expressed sequence tags (ESTs) were generated from randomly selected clones of two independent fruit cDNA libraries derived from yellow and red-fleshed fruit varieties. The most abundant sequences encoded: chitinase, 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase, catalase and methionine synthase, respectively. DNA sequence comparisons identified ESTs with significant similarity to genes associated with fruit softening, aroma and colour biosynthesis. Putative cell wall hydrolases, cell membrane hydrolases, and ethylene synthesis and regulation sequences were identified with predicted roles in fruit softening. Expressed papaya genes associated with fruit aroma included isoprenoid biosynthesis and shikimic acid pathway genes and proteins associated with acyl lipid catabolism. Putative fruit colour genes were identified due to their similarity with carotenoid and chlorophyll biosynthesis genes from other plant species.

Discovery of genes associated with fruit ripening in Carica papaya using expressed sequence tags

To identify genes involved in papaya fruit ripening, a total of 1171 expressed sequence tags (ESTs) were generated from randomly selected clones of two independent fruit cDNA libraries derived from yellow and red-fleshed fruit varieties. The most abundant sequences encoded:chitinase, 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase, catalase and methionine synthase, respectively. DNA sequence comparisons identified ESTs with significant similarity to genes associated with fruit softening, aroma and colour biosynthesis. Putative cell wall hydrolases, cell membrane hydrolases, and ethylene synthesis and regulation sequences were identified with predicted roles in fruit softening. Expressed papaya genes associated with fruit aroma included isoprenoid biosynthesis and shikimic acid pathway genes and proteins associated with acyl lipid catabolism. Putative fruit colour genes were identified due to their similarity with carotenoid and chlorophyll biosynthesis genes from other plant species.

Beta-Ketoacyl-ACP Synthase I/II from Plasmodium falciparum (PfFabB/F)-Is it B or F?

Condensing enzymes play an important and decisive role in terms of fatty acid composition of any organism. They can be classified as condensing enzymes involved in initiating the cycle and enzymes involved in elongating the initiated fatty acyl chain. In E. coli, two isoforms for the elongation condensing enzymes (FabB and FabF) exists whereas Plasmodium genome contains only one isoform. By in vitro complementation studies in E. coli CY244 cells, we show that PfFabB/ functions like E. coli FabF as the growth of the mutant cells could rescued only in the presence of oleic acid. But unlike bacterial enzyme, PfFabB/F does not increase the cis-vaccenic acid content in the mutant cells upon lowering the growth temperature. This study thus highlights the distinct properties of P. falciparum FabF which sets it apart from E. coli and most other enzymes of this family, described so far.

Purification of clofibrate-induced carnitine acetyltransferase from rat liver mitochondria

The enzyme carnitine acetyltransferase (acetyl-CoA:carnitine O-acetyltransferase, EC 2.3.1.7) has been purified to homogeneity from hepatic mitochondria of clofibrate-fed rats. It is a protein of molecular weight 56 000 composed of two non-identical subunits of molecular weight 34 000 and 25 000. The enzyme is inhibited by palmityl-CoA as well as acetyl carnitine. The inhibition by fatty acyl-CoA is competitive with respect to both the substrates, carnitine and acetyl-CoA. The inhibition by acetylcarnitine is reversed by carnitine but not by acetyl-CoA.

Biophysical Characterization of Oxidized Phospholipids : Implications for Altered Membrane Structure and Function

The present study aims to elucidate the modifications in the structure and functionality of the phospholipid matrix of biological membranes brought about by free radical-mediated oxidative damage of its molecular constituents. To this end, the surface properties of two oxidatively modified phospholipids bearing an aldehyde or carboxyl function at the end of truncated sn-2 acyl chain were studied using a Langmuir balance. The results obtained reveal both oxidized species to have a significant impact on the structural dynamics of phospholipid monolayers, as illustrated by the progressive changes in force-area isotherms with increasing mole fraction of the oxidized lipid component. Moreover, surface potential measurements revealed considerable modifications in the electric properties of oxidized phospholipid containing monolayers during film compression, suggesting a packing state-controlled reorientation of the intramolecular electric dipoles of the lipid headgroups and acyl chains. Based on the above findings, a model describing the conformational state of oxidized phospholipid molecules in biological membranes is proposed, involving the protrusion of the acyl chains bearing the polar functional groups out from the hydrocarbon phase to the surrounding aqueous medium. Oxidative modifications alter profoundly the physicochemical properties of unsaturated phospholipids and are therefore readily anticipated to have important implications for their interactions with membrane-associating molecules. Along these lines, the carboxyl group bearing lipid was observed to bind avidly the peripheral membrane protein cytochrome c. The binding was reversed following increase in ionic strength or addition of polyanionic ATP, thus suggesting it to be driven by electrostatic interactions between cationic residues of the protein and the deprotonated lipid carboxyl exposed to the aqueous phase. The presence of aldehyde function bearing oxidized phospholipid was observed to enhance the intercalation of four antimicrobial peptides into phospholipid monolayers and liposomal bilayers. Partitioning of the peptides to monolayers was markedly attenuated by the aldehyde scavenger methoxyamine, revealing it to be mediated by the carbonyl moiety possibly through efficient hydrogen bonding or, alternatively, formation of covalent adduct in form of a Schiff base between the lipid aldehydes and primary amine groups of the peptide molecules. Lastly, both oxidized phospholipid species were observed to bind with high affinity three small membrane-partitioning therapeutic agents, viz. chlorpromazine, haloperidol, and doxorubicin. In conclusion, the results of studies conducted using biomimetic model systems support the notion that oxidative damage influences the molecular architecture as well as the bulk physicochemical properties of phospholipid membranes. Further, common polar functional groups carried by phospholipids subjected to oxidation were observed to act as molecular binding sites at the lipid-water interface. It is thus plausible that oxidized phospholipid species may elicit cellular level effects by modulating integration of various membrane-embedded and surface-associated proteins and peptides, whose conformational state, oligomerization, and functionality is known to be controlled by highly specific lipid-protein interactions and proper physical state of the membrane environment.

Biosynthesis of Long-Chain Alcohols by Developing and Regenerating Rat Sciatic Nerve

Cell-free preparations of rat sciatic nerve were found to catalyze the reduction of fatty acid to alcohol in the presence of NADPH as reducing cofactor. The reductase was membrane-bound and associated primarily with the microsomal fraction. When fatty acid was the substrate, ATP, coenzyme A (CoA), and Mg2+ were required, indicating the formation of acyl CoA prior to reduction. When acyl CoA was used as substrate, the presence of albumin was required to inhibit acyl CoA hydro-lase activity. Fatty acid reductase activity was highest with palmitic and stearic acids, and somewhat lower with lauric and myristic acids. It was inhibited by sulfhydryl reagents, indicating the participation of thiol groups in the reduction. Only traces of long-chain aldehyde could be detected or trapped as semicarbazone. Fatty acid reductase activity in rat sciatic nerve was highest between the second and tenth days after birth and decreased substantially thereafter. Microsomal preparations of sciatic nerve from 10-day-old rats exhibited about four times higher fatty acid reductase activity than brain or spinal cord microsomes from the same animals. Wallerian degeneration and regeneration of adult rat sciatic nerve resulted in enhanced fatty acid reductase activity, which reached a maximum at about 12 days after crush injury.

Degradation of monogalactosyl diglyceride and diagalactosyl diglyceride by sheep pancreatic enzymes

1. Saline extract of sheep pancreas acetone-dried powder was shown to catalyse acyl ester hydrolysis of spinach leaf galactosyl diglycerides and also galactosylglucosyl diglyceride of Lactobacillus casei. 2. Sodium deoxycholate stimulated the enzyme activity. Ca2+ had no effect on the hydrolysis of monogalactosyl diglyceride, but it enhanced that of digalactosyl diglyceride. When added together, there was considerably less activity with both the substrates. 3. Optimal hydrolysis was observed at pH7.2. 4. The initial point of hydrolysis was at position-1, leading to the formation of monogalactosyl monoglyceride and digalactosyl monoglyceride. Further hydrolysis to the corresponding galactosylglycerols and later to galactose and glycerol was also observed, indicating the presence of a- and b-galactosidases in the enzyme preparation. 5. Formation of monogalactosyl diglyceride from digalactosyl diglyceride by the action of a-galactosidase was noted. 6. Monogalactosyl diglyceride was also hydrolysed by b-galactosidase to a limited extent, giving rise to diacylglycerol and galactose. 7. Attempts at purification of monogalactosyl diglyceride acyl hydrolase by using protamine sulphate treatment, Sephadex G-100 filtration and DEAE-cellulose chromatography gave a partially purified enzyme which showed 9- and 81-fold higher specific activity towards monogalactosyl diglyceride and digalactosyl diglyceride respectively. This still showed acyl ester hydrolysis activity towards methyl oleate, phosphatidylcholine and triacylglycerol. 8. When sheep, rat and guinea-pig tissues were compared, guinea-pig tissues showed the highest activity towards both monogalactosyl diglyceride and digalactosyl diglyceride. In all the species pancreas showed higher activity than intestine.

Theoretical studies on penicillins, penicillin sulphones and their 1-oxa-1-dethia and 1-carba-1-dethia analogues: binding specificities of transeptidases and penicillinases

Empirical potential energy calculations have been carried out to determine the preferred conformations of penicillins and penicillin sulphones and their 1-oxa-1-dethia and 1-carba-1-dethia analogues. With the exception of 1-oxa-1-dethia penicillins, all the other compounds favour C2 and the C3 puckered conformations of their five-membered rings. Replacement of C2 methyl groups by hydrogen atoms as in bisnorpenicillin V or oxidation of sulphur in position 1 as in sulphones, makes the C3 puckered form much less favourable. Addition of an amino-acyl group at the C6 atom, however, makes the C3 puckered form more favoured in penicillin G or V and in 1-carba-1-dethia penicillins. Through the replacement of the sulphur atom at position 1 by an oxygen atom or by a -CH2 group increases the non-planarity of the lactam peptide bond, it significantly affects the relative disposition of the C3 carboxyl group with respect to the β-lactam ring. These conformational differences have been correlated with the biological activities of these compounds. The present study suggests that the conformation of the bicyclic ring system may be more important for initial binding with the crosslinking enzyme(s) involved in the biosynthesis of bacterial cell-wall peptidoglycan and that the mode of binding is influenced by the nature of the side-group at the C6 atom. These studies predict, in agreement with experimental results, that the 1-oxa-1-dethia penicillin nulceus is an inhibitor of penicillianses. The study also suggests that the stereospecificities of the crosslinking enzyme(s) and penicillinases are very similar with regard to the nature of the side-group at the 6 atom and the confirmation of the bicyclic ring system. However, the confirmational requirement for the bicyclic ring system appears to be more specific in the former enzyme than in the latter.

Triacylglycerol lipases of yeast and plants: more than just hydrolases

In the yeast, mobilization of triacylglycerols (TAG) is facilitated by TGL3, TGL4 and TGL5 gene products. Interestingly, experiments using [32P] orthophosphate as a precursor for complex glycerophospholipids revealed that tgl mutants had a lower steady-state level of these membrane lipids. To understand a possible link between TAG lipolysis and phospholipid metabolism, we performed overexpression studies with Tgl3p and Tgl5p which clearly demonstrated that these two enzymes enhanced the level of phospholipids. Domains and motifs search analyses indicated that yeast TAG hydrolases posses a GXSXG lipase motif but also a HX4D acyltransferase motif. Purified Tgl3p and Tgl5p did not only exhibit TAG lipase activity but also catalyzed acyl-CoA dependent acylation of lyso-phosphatidylethanolamine and lyso-phosphatidic acid (LPA), respectively. Search for lipase/hydrolase homologues in the Arabidopsis thaliana genome led to the identification of At4g24160 which possess three motifs that are conserved across the plant species such as GXSXG motif, a HX4D motif and a probable lipid binding motif V(X)3HGF. Characterization of At4g24160 expressed in bacteria revealed that the presence of an acyl-CoA dependent LPA acyltransferase activity. In addition, the purified recombinant At4g24160 protein hydrolyzed both TAG and phosphatidylcholine. We hypothesize that the plant enzyme may be involved in membrane repair. In summary, our results indicate that these TAG lipases play a dual role and thereby contribute to both anabolic and catabolic processes in yeast and plants.

The mechanism of intestinal absorption of phosphatidylcholine in rats

1. The mechanism of absorption of phosphatidylcholine was studied in rats by injecting into the intestine phosphatidylcholine specifically labelled either in the fatty acid or in the glycerol moiety or with 32P, when considerable amounts of 1-acyl-lysophosphatidylcholine were found in the intestinal lumen. 2-([14C]Acyl)phosphatidylcholine gave markedly more radioactive unesterified fatty acids in the lumen, compared with the 1-([14C]acyl) derivative. Some of the radioactivity from either the fatty acid or the glycerol moiety of the injected phosphatidylcholine appeared in the mucosal triacylglycerols. 2. Injection of 32P-labelled phosphatidylcholine or 32P-labelled lysophosphatidylcholine led to the appearance of radioactive glycerylphosphorylcholine, glycerophosphate and Pi in the mucosa. 3. Rat mucosa was found to contain a highly active glycerylphosphorylcholine diesterase. 4. It was concluded that the dietary phosphatidylcholine is hydrolysed in the intestinal lumen by the pancreatic phospholipase A to 1-acylglycerylphosphorylcholine, which on entering the mucosal cell is partly reacylated to phosphatidylcholine, and the rest is further hydrolysed to glycerylphosphorylcholine, glycerophosphate, glycerol and Pi. The fatty acids and glycerophosphate are then reassembled to give triacylglycerols via the Kennedy (1961) pathway.

Quorum sensing in the plant pathogen Erwinia carotovora subsp. carotovora

Erwinia carotovora subsp. carotovora (Ecc) is a Gram-negative enterobacterium that causes soft-rot in potato and other crops. The main virulence determinants, the extracellular plant cell wall -degrading enzymes (PCWDEs), lead to plant tissue maceration. In order to establish a successful infection the production of PCWDEs are controlled by a complex regulatory network, including both specific and global activators and repressors. One of the most important virulence regulation systems in Ecc is mediated by quorum sensing (QS), which is a population density -dependent cell-to-cell communication mechanism used by many Gram-negative bacteria. In these bacteria N-acylhomoserine lactones (AHSL), act as diffusible signaling molecules enabling communication between bacterial cells. The AHSLs are structurally diverse and differ in their acyl chain length. This gives the bacteria signaling specificity and enables the recognition and communication within its own species. In order to detect and respond to the AHSLs the bacteria use QS regulators, LuxR-type proteins. The aim of this study was to get a deeper understanding of the Ecc QS system. In the first part of the study we showed that even different strains of Ecc use different dialects and of physiological concentrations, only the cognate AHSL with the correct acyl chain is recognized as a signal that can switch on virulence genes. The molecular basis of the substrate specificity of the AHSL synthase ExpI was investigated in order to recognize the acyl chain length specificity determinants of distinct AHSL synthases. Several critical residues that define the size of the substrate-binding pocket were identified. We demonstrated that in the ExpISCC1 mutations M127T and F69L are sufficient to change the N-3-oxohexanoyl-L-homoserine lactone producing ExpISCC1 to an N-3-oxooctanoyl-L-homoserine lactone (3-oxo-C8-HSL) producing enzyme. In the second study the means of sensing specificity and response to the AHSL signaling molecule were investigated. We demonstrated that the AHSL receptor ExpR1 of Ecc strain SCC3193 has strict specificity for the cognate AHSL 3-oxo-C8-HSL. In addition we identified a second AHSL receptor ExpR2 with a novel property to sense AHSLs with different acyl chain lengths. In the absence of AHSLs ExpR1 and ExpR2 were found to act synergistically to repress the virulence gene expression. This repression was shown to be released by addition of AHSLs and appears to be largely mediated by the global negative regulator RsmA. In the third study random transposon mutagenesis was used to widen the knowledge of the Ecc QS regulon. Two new QS-controlled target genes, encoding a DNA-binding regulator Hor and a plant ferredoxin-like protein FerE, were identified. The QS control of the identified genes was executed by the QS regulators ExpR1 and ExpR2 and as expression of PCWDE genes mediated by the RsmA repressor. Hor was shown to contribute to bacterial virulence at least partly through its control of PCWDE production, while FerE was shown to contribute to oxidative stress tolerance and in planta fitness of the bacteria. In addition our results suggest that QS is central to the control of oxidative stress tolerance in Ecc. In conclusion, these results indicate that Ecc strain SCC3193 is able to react and respond both to the cognate AHSL signal and the signals produced by other bacterial species, in order to control a wide variety of functions in the plant pathogen Ecc.

A fluorescent probe study of the solubilisation of cholesterol by biie salt-phospholipid micelles

The fluorescent probe dansyl cadaverine has been shown to bind strongly to mixed bile salt-phospholipid micelles containing unsaturation in the fatty acyl chains. Incorporation of cholesterol into the mixed micelles reduces the number of molecules of bound dansyl cadaverine without altering the binding affinity. These results suggest a tighter packing of the hydrocarbon matrix of the micelles in the presence of cholesterol.

Design, Development, Synthesis, and Docking Analysis of 2-substituted Triclosan Analogs as Inhibitors for Plasmodium falciparum Enoyl-ACP Reductase

A structure-based approach has been adopted to develop 2'substituted analogs of triclosan. The Cl at position 2' in ring B of triclosan was chemically substituted with other functional groups like NH2, NO2 and their inhibitory potencies against PfENR were determined. The binding energies of the 2' substituted analogs of triclosan for enoyl-acyl carrier protein reductase (ENR) of Plasmodium falciparum were determined using Autodock. Based on the autodock results, we synthesized the potential compounds. The IC50 and inhibition constant (K-i) of 2' substituted analogs of triclosan were determined against purified PfENR. Among them, two compounds,2-(2'-Amino-4'-chloro-phenoxy)-5-chloro-phenol (compound 4) and 5-chloro-2-(4'-chloro-2'-nitro-phenoxy)-phenol) (compound 5) exhibited good potencies. Compound 4 followed uncompetitive inhibition kinetics with crotonoyl CoA and competitive with NADH. It was shown to have an IC50 of 110 nM; inhibition constant was 104 nM with the substrate and 61 nM with the cofactor. IC50 Of compound 5 was determined to be 229 nM. Compounds 4 and 5 showed significant inhibition of the parasite growth in P. falciparum culture. (C) 2009 IUBMB IUBMB Life, 61(11):1083-1091, 2009.