Diacylglycerol Kinase Is Stimulated by Arachidonic Acid in Neural Membranes.

The effect of arachidonic acid (AA) on the activity of diacylglycerol (DG) kinase in neural membranes was investigated. When rat brain cortical membranes were incubated with 0.5 mM dipalmitin and [gamma-P-32]ATP, formation of phosphatidic acid (PA) was observed. It was linear up to 5 min, and the initial rate was similar to 1.0 nmol/min/mg of protein. The DG kinase activity was stimulated twofold by 0.25 mM AA. The stimulation was apparent at the earliest time point measured (1 min) and with the lowest concentration of AA tested (62.5 mu M). The stimulation was proportional to the concentration of AA up to 250 mu M. AA was the most potent stimulator of DG kinase, and linolenic acid showed similar to 40% stimulation. Oleic acid showed no effect, whereas linoleic and the saturated fatty acids tested were inhibitory. AA stimulation of DG kinase was observed only with membranes of cerebrum, cerebellum, and myelin and not with brain cytosol or liver membranes. AA also stimulated the formation of PA in the absence of added dipalmitin (endogenous activity) with membranes prepared from whole brain. DG kinase of neural membranes was extracted with 2 M NaCl, which on dialysis yielded a precipitate. Both the precipitate and the supernatant showed DG kinase activity, but only the enzyme in the precipitate was stimulated by AA at concentrations as low as 25 mu M. It is suggested that AA, through its effect on DG kinase, regulates the level of DG in neural membranes, which in turn regulates protein kinase C activity.

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.

Lipids of some thermophilic fungi

Total lipid content in the thermophilic fungi—Thermoascus aurantiacus, Humicola lanuginosa, Malbranchea pulchella var.sulfurea, andAbsidia ramosa—varied from 5.3 to 19.1% of mycelial dry weight. The neutral and polar lipid fractions accounted for 56.4 to 80.2% and 19.8 to 43.6%, respectively. All the fungi contained monoglycerides, diglycerides, triglycerides, free fatty acids, and sterols in variable amounts. Sterol ester was detected only inA. ramosa. Phosphatide composition was: phosphatidyl choline (15.9–47%), phosphatidyl ethanolamine (23.4–67%), phosphatidyl serine (9.3–17.6%), and phosphatidyl inositol (1.9–11.9%). Diphosphatidyl glycerol occurred in considerable quantity only inH. lanuginosa andM. pulchella var.sulfurea. Phosphatidic acid, detected as a minor component only inM. pulchella var.sulfurea andA. ramosa, does not appear to be a characteristic phosphatide of thermophilic fungi as suggested earlier. The 16∶0, 16∶1, 18∶0, 18∶1, and 18∶2 acids were the main fatty acid components. In addition,A. ramosa contained 18∶3 acid. Total lipids contained an average of 0.93 double bonds per mole of fatty acids, and neutral lipids tend to be more unsaturated than phospholipids.

Specificity for the Exchange of Phospholipids Through Polymyxin B Mediated Intermembrane Molecular Contacts

Structural specificity for the direct vesicle−vesicle exchange of phospholipids through stable molecular contacts formed by the antibiotic polymyxin B (PxB) is characterized by kinetic and spectroscopic methods. As shown elsewhere [Cajal, Y., Rogers, J., Berg, O. G., & Jain, M. K. (1996) Biochemistry 35, 299−308], intermembrane molecular contacts between anionic vesicles are formed by a small number of PxB molecules, which suggests that a stoichiometric complex may be responsible for the exchange of phospholipids. Larger clusters containing several vesicles are formed where each vesicle can make multiple contacts if sterically allowed. In this paper we show that the overall process can be dissected into three functional steps: binding of PxB to vesicles, formation of stable vesicle−vesicle contacts, and exchange of phospholipids. Polycationic PxB binds to anionic vesicles. Formation of molecular contacts and exchange of monoanionic phospholipids through PxB contacts does not depend on the chain length of the phospholipid. Only monoanionic phospholipids (with methanol, serine, glycol, butanol, or phosphatidylglycerol as the second phosphodiester substituent in the head group) exchange through these contacts, whereas dianionic phosphatidic acid does not. Selectivity for the exchange was also determined with covesicles of phosphatidylmethanol and other phospholipids. PxB does not bind to vesicles of zwitterionic phosphatidylcholine, and its exchange in covesicles is not mediated by PxB. Vesicles of dianionic phospholipids, like phosphatidic acid, bind PxB; however, this phospholipid does not exchange. The structural features of the contacts are characterized by the spectroscopic and chemical properties of PxB at the interface. PxB in intermembrane contacts is readily accessible from the aqueous phase to quenchers and reagents that modify amino groups. Results show that PxB at the interface can exist in two forms depending on the lipid/PxB ratio. Additional studies show that stable PxB-mediated vesicle−vesicle contacts may be structurally and functionally distinct from “stalks”, the putative transient intermediate for membrane fusion. The phenomenon of selective exchange of phospholipids through peptide-mediated contacts could serve as a prototype for intermembrane targeting and sorting of phospholipids during their biosynthesis and trafficking in different compartments of a cell. The protocols and results described here also extend the syllogistic foundations of interfacial equilibria and catalysis.

Effect of phenoxy acids and their derivatives on lipid metabolism in groundnut (Arachis hypogaea) leaves

The effect of four phenoxy compounds [2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid, 4-chlorophenoxyacetic acid 2-(dimethylamino)ethyl ester (centrophenoxine), and 4-chlorophenoxy ethyl 2-(dimethylamino) ethyl ether (neophenoxine)] on lipid metabolism in groundnut (Arachis hypogaea) leaves was investigated under nonphotosynthetic conditions. In experiments with leaf disks, the uptake of [1-14C]acetate, [32P]orthophosphate, [35S]sulfate and [methyl-14C]choline was substantially inhibited by all the phenoxy compounds except neophenoxine. When the incorporation of these precursors into lipids was measured and expressed as percentage of total uptake, there was significant inhibition of incorporation of [1-14C]acetate and [32P]orthophosphate into lipids by all the compounds except neophenoxine. The incorporation of [methyl-14C]choline was unaffected by all except centrophenoxine which showed stastically significant stimulation. [35S]Sulfate incorporation into lipids was markedly inhibited only by centrophenoxine. The fatty acid synthetase of isolated chloroplasts assayed in the absence of light was inhibited 20–50% by the phenoxy compounds at 0.5 mM concentration. This inhibition showed a dependence on time of preincubation with the herbicide suggesting an interaction with the enzyme. It was, however, reversible and excess substrate did not prevent the inhibition, suggesting that the herbicide interaction may not be at the active site. sn-Glycerol-3-phosphate acyltransferase in the chloroplast and microsomal fractions was inhibited by 2,4-D while the phosphatidic acid phosphatase was insensitive to all the phenoxy compounds. It is concluded that phenoxy compounds affect precursor uptake, their incorporation into lipids, and the chloroplast fatty acid synthetase. The free acids were the most potent compounds while the ester (centrophenoxine) was less effective and the ether (neophenoxine) was completely ineffective in their influence on lipid metabolism.

Studies on neurotransmitter-stimulated phospholipid metabolism with cerebral tissue suspensions: A possible biochemical correlate of synaptogenesis in normal and undernourished rats

The phenomenon of neurotransmitter-stimulated incorporation of32Pi into phosphatidic acid and inositol phosphatides (neurotransmitter effect) in developing brain was studied in vitro as a possible measure of synaptogenesis. While the neurotransmitter effect was not observed with brain homogenates, highly consistent and significant effects were noted with brain tissue suspensions obtained by passing the tissue through nylon bolting cloth. The magnitude of the effect decreased with the increase in mesh number. Maximum stimulations obtained with the 33 mesh adult brain cortex preparations (mean±S.E.M. of6experiments) were203 ± 8%, 316 ± 11 % and150 ± 8% with 10−3 M acetylcholine (ACh) + 10−3 M eserine; 10−2 M norepinephrine (NE) and 10−2 M serotonin (5-HT), respectively. Experiments with developing rat brain at 7, 14 and 21 days of age showed that the neurotransmitter effects due to ACh, NE and 5-HT increase progressively in different regions of the brain but that there are marked regional differences. It is suggested that the neurotransmitter effect is a valid biochemical correlate of synaptogenesis. In rats undernourished from birth t0 21 days of age, by increasing the litter size, the neurotransmitter effect with ACh, NE or 5-HT was not altered in the cortex but was significantly reduced in the brain stem. In cerebellum the effects due to ACh and NE were significantly altered, while that with 5-HT was unaffected. It is concluded that cholinergic, adrenergic and serotonergic synapses are relatively unaffected in the cortex but are significantly affected in the brain stem by undernutrition. In the cerebellum of undernourished rats the adrenergic and cholinergic, but not serotonergic systems, are altered.

Effect of thiocarbamate, urea, and uracil herbicides on lipid metabolism in groundnut (Arachis hypogaea) leaves

The effect of thiocarbamates (S-ethyldipropylthiocarbamate and diallate), substituted ureas (monuron and diuron), and uracils (bromacil and terbacil) on lipid metabolism in groundnut (Arachis hypogaea) leaves was investigated under nonphotosynthetic conditions. The uptake of [1-14C]acetate by leaf disks was inhibited by the thiocarbamates and marginally by the substituted ureas, but not by the uracil herbicides. The uptake of [methyl-14C]choline was inhibited to a lesser extent by thiocarbamates, while the other herbicides showed a slight stimulation. The thiocarbamates almost completely inhibited uptake of [32P]orthophosphate at 1.0 mM concentration, while diuron and terbacil showed significant inhibition. [1-14C]Acetate incorporation into lipids was inhibited only by diallate. [methyl-14C]Choline incorporation into the choline phosphoglycerides was inhibited by diallate, diuron, and bromacil. The incorporation of [32P]orthophosphate into phospholipids was substantially inhibited (over 90% at 1.0 mM) by the thiocarbamates, but not by the other herbicides. [35S]Sulfate incorporation into sulfoquinovosyl diglycerides was markedly inhibited only by the thiocarbamates. Fatty acid synthesis by isolated chloroplasts was inhibited 40–85% by thiocarbamates, substituted ureas, and bromacil, but not by terbacil. The inhibitory effect of the urea derivatives was reversible, but that of thiocarbamates was irreversible. sn-Glycerol-3-phosphate acyltransferase(s) of the chloroplast and microsomal fractions were profoundly inhibited by thiocarbamates, but not by the other two groups of herbicides. Phosphatidic acid phosphatase was insensitive to all the herbicides tested.

Translational Up-Regulation and High-Level Protein Expression from Plasmid Vectors by mTOR Activation via Different Pathways in PC3 and 293T Cells

Background: Though 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. We recently identified the prostate carcinoma cell line PC3 to be as efficient as 293T in protein expression. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines. Methodology/Principal Findings: In a survey of different cell lines for efficient expression of platelet-derived growth factor-B (PDGF-B), beta-galactosidase (beta-gal) and green fluorescent protein (GFP) from plasmid vectors, PC3 was found to express at 5-50-fold higher levels compared to the bone metastatic prostate carcinoma cell line PC3BM and many other cell lines. Further, the efficiency of transfection and level of expression of the reporters in PC3 were comparable to that in 293T. Comparative analyses revealed that the high level expression of the reporters in the two cell lines was due to increased translational efficiency. While phosphatidic acid (PA)-mediated activation of mTOR, as revealed by drastic reduction in reporter expression by n-butanol, primarily contributed to the high level expression in PC3, multiple pathways involving PA, PI3K/Akt and ERK1/2 appear to contribute to the abundant reporter expression in 293T. Thus the extent of translational upregulation attained through the concerted activation of mTOR by multiple pathways in 293T could be achieved through its activation primarily by the PA pathway in PC3. Conclusions/Significance: Our studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mTOR activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression. Further, PC3 offers an advantage in that the level of expression of the protein can be regulated by simple addition of n-butanol to the culture medium.

Phospholipid Mediated Activation of Calcium Dependent Protein Kinase 1 (CaCDPK1) from Chickpea: A New Paradigm of Regulation

Phospholipids, the major structural components of membranes, can also have functions in regulating signaling pathways in plants under biotic and abiotic stress. The effects of adding phospholipids on the activity of stress-induced calcium dependent protein kinase (CaCDPK1) from chickpea are reported here. Both autophosphorylation as well as phosphorylation of the added substrate were enhanced specifically by phosphatidylcholine and to a lesser extent by phosphatidic acid, but not by phosphatidylethanolamine. Diacylgylerol, the neutral lipid known to activate mammalian PKC, stimulated CaCDPK1 but at higher concentrations. Increase in V-max of the enzyme activity by these phospholipids significantly decreased the K-m indicating that phospholipids enhance the affinity towards its substrate. In the absence of calcium, addition of phospholipids had no effect on the negligible activity of the enzyme. Intrinsic fluorescence intensity of the CaCDPK1 protein was quenched on adding PA and PC. Higher binding affinity was found with PC (K-1/2 = 114 nM) compared to PA (K-1/2 = 335 nM). We also found that the concentration of PA increased in chickpea plants under salt stress. The stimulation by PA and PC suggests regulation of CaCDPK1 by these phospholipids during stress response.

X-ray studies on crystalline complexes involving amino acids and peptides. XXXIII. Crystal structures of L- and DL-arginine complexed with oxalic acid and a comparative study of amino acid oxalic acid complexes

The DL- and L-arginine complexes of oxalic acid are made up of zwitterionic positively charged amino acid molecules and semi-oxalate ions. The dissimilar molecules aggregate into separate alternating layers in the former. The basic unit in the arginine layer is a centrosymmetric dimer, while the semi-oxalate ions form hydrogen-bonded strings in their layer. In the L-arginine complex each semi-oxalate ion is surrounded by arginine molecules and the complex can be described as an inclusion compound. The oxalic acid complexes of basic amino acids exhibit a variety of ionization states and stoichiometry. They illustrate the effect of aggregation and chirality on ionization state and stoichiometry, and that of molecular properties on aggregation. The semi-oxalate/oxalate ions tend to be planar, but large departures from planarity are possible. The amino acid aggregation in the different oxalic acid complexes do not resemble one another significantly, but the aggregation of a particular amino acid in its oxalic acid complex tends to have similarities with its aggregation in other structures. Also, semi-oxalate ions aggregate into similar strings in four of the six oxalic acid complexes. Thus, the intrinsic aggregation propensities of individual molecules tend to be retained in the complexes.

Adenosine-5'-carboxylic Acid

The two molecules in the asymmetric unit of adenosine-5'-carboxylic acid, C10H11N5O5, exist as zwitterions with N1 protonated and the carboxyl groups ionized. Both molecules are in an anti conformation with glycosyl torsion angles of -161.4(3) and -155.5(3)degrees. The ribose moieties adopt a C3-endo-C2-exo twist conformation. The pseudo-rotation parameters are P = 0.01(1) and 6.58(1)degrees, and tau(m) = 36.2(2) and 34.6(2)degrees, for molecules A and B, respectively. The carboxyl groups of A and B are not in the standard g(+), g(-) or t conformations. Both Watson-Crick sites, N1 and N6, of the adenine bases are involved in a pair of hydrogen bonds with the dissociated carboxyl groups, forming a cyclic tetramer. The adenine base of molecule A stacks on the ribose O4' atom of a symmetry-related B molecule at a distance of 2.88 Angstrom; the adenine base of B stacks in an analogous way at a distance of 2.91 Angstrom.

Ultrasonic Degradation of Poly(acrylic acid)

The ultrasonic degradation of poly(acrylic acid), a water-soluble polymer, was studied in the presence of persulfates at different temperatures in binary solvent Mixtures of methanol and water. The degraded samples were analyzed by gel permeation chromatography for the time evolution of the molecular weight distributions. A continuous distribution kinetics model based on midpoint chain scission was developed, and the degradation rate coefficients were determined. The decline in the rate of degradation of poly(acrylic acid) with increasing temperature and with an increment in the methanol content in the binary solvent mixture of methanol and water was attributed to the increased vapor pressure of the solutions. The experimental data showed an augmentation of the degradation rate of the polymer with increasing oxidizing agent (persulfate) concentrations. Different concentrations of three persulfates-potassium persulfate, ammonium persulfate, and sodium persulfate-were used. It was found that the ratio of the polymer degradation rate coefficient to the dissociation rate constant of the persulfate was constant. This implies that the ultrasonic degradation rate of poly(acrylic acid) can be determined a priori in the presence of any initiator.

Ground and Excited State Intramolecular Proton Transfer in Salicylic Acid: an Ab Initio Electronic Structure Investigation

Energetics of the ground and excited state intramolecular proton transfer in salicylic acid have been studied by ab initio molecular orbital calculations using the 6-31G** basis set at the restricted Hartree-Fock (RHF) and configuration interaction-single excitation (CIS) levels and also using the semiempirical method AM1 at the RHF level as well as with single and pair doubles excitation configuration interaction spanning eight frontier orbitals (PECI = 8). The ab initio potential energy profile for intramolecular proton transfer in the ground state reveals a single minimum corresponding to the primary form, in the first excited singlet state, however, there are two minima corresponding to the primary and tautomeric forms, separated by a barrier of similar to 6 kcal/mol, thus accounting for dual emission in salicylic acid. Electron density changes with electronic excitation and tautomerism indicate no zwitterion formation. Changes in spectral characteristics with change in pH, due to protonation and deprotonation of salicylic acid, are also accounted for, qualitatively. Although the AM1 calculations suggest a substantial barrier for proton transfer in the ground as well as the first excited state of SA, it predicts the transition wavelength in near quantitative accord with the experimental results for salicylic acid and its protonated and deprotonated forms.

Synthesis of 2-(5-((5-(4-chlorophenyl)furan 2-yl)methylene)-4-oxo-2-thioxo-thiazolidi n-3-yl)acetic acid derivatives and evaluation of their cytotoxicity and induction of apoptosis in human leukemia cells

In order to explore the anticancer effect associated with the thiazolidinone framework, several 2-(5-((5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid derivatives 5(a-1) were synthesized. Variation in the functional group at C-terminal of the thiazolidinone led to set of compounds bearing amide moiety. Their chemical structures were confirmed by H-1 NMR, IR and Mass Spectra analysis. These thiazolidinone compounds containing furan moiety exhibits moderate to strong antiproliferative activity in a cell cycle stage-dependent and dose dependent manner in two different human leukemia cell lines. The importance of the electron donating groups on thiazolidinone moiety was confirmed by MTT and Trypan blue assays and it was concluded that the 4th position of the substituted aryl ring plays a dominant role for its anticancer property. Among the synthesized compounds, 5e and 5f have shown potent anticancer activity on both the cell lines tested. To rationalize the role of electron donating group in the induction of cytotoxicity we have chosen two molecules (5e and 5k) having different electron donating group at different positions. LDH assay, Flow cytometric analysis and DNA fragmentation suggest that 5e is more cytotoxic and able to induce the apoptosis. (C) 2009 Elsevier Ltd. All rights reserved.