Production of phosphatidylcholine containing conjugated linoleic acid mediated by phospholipase A2

Esterification of lysophosphatidylcholine (LPC) with conjugated linoleic acid (CLA) was carried out using porcine pancreatic phospholipase A₂ (PLA₂). PLA₂ only slightly synthesized phosphatidylcholine containing CLA (CLA-PC) at 2.6% by the addition of water. Addition of formamide in place of water markedly increased the yield of CLA-PC. In addition, synthesis of CLA-PC by PLA₂ was affected by the amount of substrate CLA and PLA₂ in the reaction system. Under optimal reaction conditions using 11 mg LPC, 18 mg CLA, 550 mg glycerol, 50 μL formamide, 3.3 × 10⁴ U PLA₂, and 0.3 μmol CaCl₂ at 37 °C for 6 h, the reaction yield of CLA-PC reached 65 mol%. Furthermore, addition of protein such as albumin and casein suppressed the decrease of CLA-PC yield after 6 h. PLA₂ exhibited the highest activity for the 10t,12c-CLA isomer among four CLA isomers (9c,11t-CLA, 9c,11c-CLA, 9t,11t-CLA and 10t,12c-CLA), whereas that for 9c,11c-CLA was the lowest. These results showed that the present esterification system for LPC and CLA by PLA₂ is effective for producing CLA-PC.     

Properties of the Group IV phospholipase A2 family

The Group IV phospholipase A₂ family is comprised of six intracellular enzymes commonly called cytosolic phospholipase A₂ (cPLA₂) , cPLA₂β, cPLA₂γ, cPLA₂δ, cPLA₂ε and cPLA₂ζ. They are most homologous to phospholipase A and phospholipase B/lysophospholipases of filamentous fungi particularly in regions containing conserved residues involved in catalysis. However, a number of other serine acylhydrolases (patatin, Group VI PLA₂s, Pseudomonas aeruginosa ExoU and NTE) contain the Ser/Asp catalytic dyad characteristic of Group IV PLA₂s, and recent structural analysis of patatin has confirmed its structural similarity to cPLA₂. A characteristic of all these serine acylhydrolases is their ability to carry out multiple reactions to varying degrees (PLA₂, PLA₁, lysophospholipase and transacylase activities). cPLA₂, the most extensively studied Group IV PLA₂, is widely expressed in mammalian cells and mediates the production of functionally diverse lipid products in response to extracellular stimuli. It has PLA₂ and lysophospholipase activities and is the only PLA₂ that has specificity for phospholipid substrates containing arachidonic acid. Because of its role in initiating agonist-induced release of arachidonic acid for the production of eicosanoids, cPLA₂ activation is important in regulating normal and pathological processes in a variety of tissues. Current information available about the biochemical properties and tissue distribution of other Group IV PLA₂s suggests they may have distinct mechanisms of regulation and functional roles.     

Ergosterol peroxides as phospholipase A2 inhibitors from the fungus Lactarius hatsudake

Four ergosterol derivatives (1–4) have been isolated for the first time from the fruiting bodies of a basidiomycete fungus, Lactarius hatsudake, through activity-guided fractionation. Their structures were determined, using spectroscopic analysis, as: (22E,24R)-ergosta-5,7,22-dien-3β-ol (ergosterol, 1); 5,8-epidioxy-(22E,24R)-ergosta-6,22-dien-3β-ol (ergosterol peroxide, 2); 5,8-epidioxy-(24S)-ergosta-6-en-3β-ol (3); and (22E,24R)-ergosta-7,22-dien-3β,5,6β-triol (cerevisterol, 4). Compounds 2 and 3 showed selective inhibitory activity against Crotalus adamenteus venom phospholipase A₂ (PLA₂) enzyme, but not against Apis mellifcra bee venom PLA₂. The antiphospholipase A₂ activity of compounds 2 and 3 are reported here for the first time.     

The level of pancreatic PLA2 receptor is closely associated with the proliferative state of rat uterine stromal cells

Rat uterine stromal cells (U_III) express pancreatic type PLA₂ (PLA₂-I) receptor and internalize the enzyme bound to receptors. Here, we investigate the proliferating effect and alterations in binding of PLA₂-I. There is a dramatic decline in PLA₂-I binding in U_III cells as they progress from a nonconfluent proliferating state (40,000 sites/cell) to a confluent state (1300 sites/cell). Intracellular concentration of PLA₂-I changed with the alteration in binding, suggesting that regulation in the PLA₂ binding capacity may have important implications in growth control mechanisms.     

Neo-clerodane diterpenoid, a new metalloprotease snake venom inhibitor from Baccharis trimera (Asteraceae): anti-proteolytic and anti-hemorrhagic properties

Many plants are used in traditional medicine as active agents against various effects induced by snakebite. Few attempts have been made however to identify the nature of plant natural products with anti-ophidian properties. Baccharis trimera (Less) DC (Asteraceae), known in Brazil as carqueja, has been popularly used to treat liver diseases, rheumatism, diabetes, as well as digestive, hepatic and renal disorders. The active component was identified as 7-hydroxy-3,13-clerodadiene-16,15:18,19-diolide, C₂₀H₂₈O₅, (clerodane diterpenoid, Bt-CD). We report now the anti-proteolytic and anti-hemorrhagic properties against snake venoms of a Bt-CD inhibitor from B. trimera. Bt-CD exhibited full inhibition of hemorrhage and proteolytic activity caused by Bothrops snake venoms. The inhibitor was able to neutralize the hemorrhagic, fibrinogenolytic and caseinolytic activities of class P-I and III metalloproteases isolated from B. neuwiedi and B. jararacussu venoms. No inhibition of the coagulant activity was observed. Bt-CD also partially inhibited the edema induced by other crude venoms, metalloproteases, basic and acidic phospholipases A₂. To further elucidate the inhibitory specificity of Bt-CD against metalloproteases isolated from snake venoms, a deeper understanding of its structure and function is necessary. Furthermore, the potential use of these inhibitors to complement anti-venom as an alternative treatment of snakebite envenomations needs to be evaluated in future studies.     

Production of egg yolk lysolecithin with immobilized phospholipase A2

Production of egg yolk lysolecithin was compared using free phospholipase A₂ (PLA₂) and immobilized PLA₂ in alginate-silicate sol-gel matrix. Choice of solvent, water content, calcium, and temperatures changed the activity of the free and immobilized PLA₂ a lot, owing to their effects on the catalytic properties of the enzyme as well as the conformational change of lecithin in ethanol-buffer mixture. Free PLA₂ shows typical microemulsion kinetics in ethanol-buffer system. The effect of the water content on the enzyme reaction was greatly influenced by the presence of calcium ion. In the absence of calcium ion, certain optimal water content for the production of lysolecithin always exists in the free PLA₂ reaction. However, with calcium ion, three distinctive regions were observed with free PLA₂ reactions. Initially, in the micro-aqueous region of the ethanol-buffer system with calcium ion, the hydrolysis activity of PLA₂ was proportional to the water content. Beyond the region, concave type of activity profiles were observed as the water content increases. As the water content increases further, the hydrolysis rate of the PLA₂ abruptly decreased by the phase separation. On the contrary, in case of immobilized enzyme, optimal water content for the production of lysolecithin exists regardless of the presence of calcium ion. The calcium ion was essential for achieving the maximum activity of both free and immobilized PLA₂. The addition of calcium ion not only affected the catalytic activity of the enzyme but also was necessary to improve the enzyme stability. As the immobilization of the enzyme remarkably increased thermal stability of the free enzyme, the immobilized PLA₂ is more desirable to be used in the production of various lysophospholipids. It was successfully reused over 250 h.     

On the purification of notexin. Isolation of a single amino acid variant from the venom of Notechis scutatus scutatus

Venom of the Australian tiger snake, Notechis scutatus scutatus was fractionated by conventional ion-exchange chromatography. The fraction containing notexin, a well-known single-chain toxic phospholipase A2, was further purified by reverse-phase high-performance liquid chromatography. Two main components were isolated and the major one corresponded to notexin. The other component, designated as notechis Ns, was an isoform of notexin. Notechis Ns and notexin possessed similar in vitro esterase activity, in vitro neuromuscular activity and in vivo lethality. Amino acid composition and sequence of the Staphylococcus aureus V8-protease peptides demonstrated that primary structures of notechis Ns and notexin differed from each other by a single substitution amongst 119 amino acids: Lys----Arg at position 16.     

PI 3-kinase and MAP kinase regulate bradykinin induced prostaglandin E(2) release in human pulmonary artery by modulating COX-2 activity

Here we studied the role of phosphoinositide 3-kinase (PI 3-kinase) and mitogen activated protein (MAP) kinase in regulating bradykinin (BK) induced prostaglandin E₂ (PGE₂) production in human pulmonary artery smooth muscle cells (HPASMC). BK increased PGE₂ in a three step process involving phospholipase A₂ (PLA₂), cyclooxygenase (COX) and PGE synthase (PGES). BK stimulated PGE₂ release in cultured HPASMC was inhibited by the PI 3-kinase inhibitor LY294002 and the p38 MAP kinase inhibitor SB202190. The inhibitory mechanism used by LY294002 did not involve cytosolic PLA₂ activation or COX-1, COX-2 and PGES protein expression but rather a novel effect on COX enzymatic activity. SB202190 also inhibited COX activity.     

The membrane effects of 17beta-estradiol on chondrocyte phenotypic expression are mediated by activation of protein kinase C through phospholipase C and G-proteins

Growth plate chondrocytes from both male and female rats have nuclear receptors for 17β-estradiol (E₂); however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the female cell response. E₂ directly affects the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E₂ activates PKC in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E₂-dependent alkaline phosphatase activity in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of this study were: (1) to examine if PKC mediates the effect of E₂ on chondrocyte proliferation, differentiation, and matrix synthesis; and (2) to determine the pathway that mediates the membrane effect of E₂ on PKC. Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10⁻¹⁰ to 10⁻⁷ M E₂ in the presence or absence of the PKC inhibitor chelerythrine, and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [³H]thymidine incorporation were measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E₂ in the presence or absence of genistein (an inhibitor of tyrosine kinases), U73122 or D609 (inhibitors of phospholipase C [PLC]), quinacrine (an inhibitor of phospholipase A₂ [PLA₂]), and melittin (an activator of PLA₂). Alkaline phosphatase specific activity and proteoglycan sulfation were increased and [³H]thymidine incorporation was decreased by E₂. The effects of E₂ on all parameters were blocked by chelerythrine. Treatment of the cultures with E₂ produced a significant dose-dependent increase in PKC. U73122 dose-dependently inhibited the activation of PKC in E₂-stimulated female chondrocyte cultures. However, the classical receptor antagonist ICI 182780 was unable to block the stimulatory effect of E₂ on PKC. Moreover, the classical receptor agonist diethylstilbestrol (DES) had no effect on PKC, nor did it alter the stimulatory effect of E₂. Inhibition of tyrosine kinase and PLA₂ had no effect on the activation of PKC by E₂. The PLA₂ activator also had no effect on PKC activation by E₂. E₂ stimulated PKC activity in membranes isolated from the chondrocytes, demonstrating a direct membrane effect for this steroid hormone. These data indicate that the rapid nongenomic effect of E₂ on PKC activity in chondrocytes from female rats is sex-specific and dependent upon a G-protein-coupled phospholipase C.     

Preparation and characterization of immobilized phospholipase A2 on chitosan beads for lowering serum cholesterol concentration

Phospholipase A₂ (PLA₂) from cobra venom, which can hydrolyze the S_N2 ester bond of 1,2-diacylphosphatides, was immobilized by covalent binding to porous chitosan beads. Immobilization has to be carried out by using the carboxylic groups instead of the amine groups of the enzyme to get reasonable activity retention (higher than 50%). The effects of amount of activating reagent EDC and enzyme loading during the immobilization step were investigated. Since EDC could modify important Asp groups in the enzyme, the EDC/enzyme weight ratio should be less than 10. Although the activity retention of immobilized enzyme increased with enzyme/bead weight ratio, this ratio should be kept to a minimum at 1×10⁻³ to optimize coupling yield of enzyme activity and reduce internal diffusion resistance. The kinetic properties and stability of the immobilized enzyme were determined. The immobilized PLA₂ was packed into a column to hydrolyze phospholipid in a circulating packed-bed reactor. The flow rate of the substrate solution should be set at 37.5 cm/min (superficial velocity) to eliminate external diffusion resistance, under which condition the column reactor could be reused up to 10 times with less than 20% loss of activity. Since enzymatic hydrolysis of phospholipid on low density lipoprotein (LDL) particle surface with PLA₂ could result in faster plasma clearance of the modified LDL particles, an in vitro bioreactor containing immobilized PLA₂ should be able to lower serum cholesterol concentration. A significant decrease in total serum cholesterol concentration in hypercholesterolemic rabbits was observed after 90-min treatment.     

The N-terminal amino group essential for the biological activity of notexin from Notechis scutatus scutatus venom

Notexin from Notechis scutatus scutatus snake venom was modified with trinitrobenzenesulfonic acid, and the major trinitrophenylated (TNP) derivative was separated by high-performance liquid chromatography. Modification resulted in the incorporation of only one TNP group on the N-terminal alpha-amino group. The TNP derivative showed a precipitous decrease in enzymatic activity and lethal toxicity, whereas the antigenicity remained unchanged. However, trinitrophenylation did not significantly affect the secondary structure of the toxin molecule as revealed by the CD spectra. The results, that the modification reaction was accelerated by the Ca2+ and that the TNP derivative retains its affinity for Ca2+, indicate that the N-terminal alpha-amino group did not participate in the Ca2(+)-binding. The TNP derivative could be regenerated with hydrazine hydrochloride. The biological activities of the regenerated notexin are almost the same as those of native notexin. These results suggest that the N-terminal alpha-amino group is essential for the phospholipase A2 activity and lethal toxicity of notexin, and that incorporation of the TNP group on the N-terminal alpha-amino group might give rise to a distortion of the active conformation of notexin.     

Chemical modification of notexin fromNotechis scutatus scutatus (Australian tiger snake) venom with pyridoxal-5′-phosphate

Notexin fromNotechis scutatus scutatus snake venom was subjected to Lys modification with pyridoxal 5′-phosphate (PLP), and one major modified derivative was purified on a cation-exchanger SP-8HR column. The results of amino acid analysis and sequence determination revealed that only 2 Lys residues at positions 82 and 115 out of 11 Lys residues in notexin were modified. The incorporation of PLP into the protein was accompanied by the loss of 53% lethal toxicity, but the modified notexin showed an about 1.2-fold increase in enzymatic activity. However, the secondary structure of the toxin molecule did not significantly change after modification with PLP as revealed by the CD spectra, and the antigenicity of PLP derivative remained unchanged. The modified derivative retained its affinity for Ca²⁺, indicating that the modified Lys residues did not participate in Ca²⁺ binding. These results indicate that modification of Lys residues causes a differential effect on the enzymatic activity and lethal toxicity of notexin, and suggest that notexin might possess two functional sites, one responsible for the catalytic activity and the other associated with its lethal effect.     

Neutralization of lethal potency and inhibition of enzymatic activity of a phospholipase A2 neurotoxin, crotoxin, by non-precipitating antibodies (Fab)

Rabbit antibodies were prepared against both purified catalytic (component-B) and purified non-catalytic (component-A) subunits of crotoxin, the major phospholipase A2 neurotoxin from the South American rattlesnake. They cross-react with crotoxin-like toxins from the venom of several Crotalus species as well as with single-chain phospholipase A2 neurotoxins from Crotalid and Viperid venoms (agkistrodontoxin and ammodytoxin A) but not from Elapid venoms (notexin). Immunological cross-reactions of anti-component-A and anti-component-B sera with crotoxin and with its isolated components A and B showed that component-A exposes determinants of low immunogenicity which are present on component-B, whereas the major antigenic determinants of component-B are not present on component-A. Anti-component-B antibodies, but not anti-component-A antibodies, neutralize the lethal potency of crotoxin and inhibit its enzymatic activity. Furthermore, non-precipitating anti-component-B Fab fragments were as potent as antibodies, indicating that crotoxin neutralization results from the binding of the antibodies to the catalytic subunit, rather than the formation of an immunoprecipitate.     

Diplatinum(III) complexes with bridging 1-methylcytosinate ligands and variable axial ligands, including guanine nucleobases

A series of diplatinum(III) complexes derived from cis-(NH₃)₂Pt^II and the model nucleobase 1-methylcytosine (1-MeC) has been prepared and X-ray structurally characterized, all of which contain two anionic base ligands (1-MeC⁻) in a head–tail (ht) arrangement: ht-cis-[(ONO₂)(NH₃)₂Pt(1-MeC⁻-N3,N4)₂Pt(NH₃)₂(ONO₂)](NO₃)₂·HNO₃·3H₂O (2b), ht-cis-[(NO₂) (NH₃)₂ Pt(1-MeC⁻-N3,N4)₂Pt(NH₃)₂(OH₂)](ClO₄)₃·3.5H₂O (3), ht-cis-[(OH₂)(NH₃)₂Pt(1-MeC⁻-N3,N4)₂Pt(NH₃)₂(OH₂)](ClO₄)₄·H₂O (4b), and ht-cis-[(9-EtGH-N7)(NH₃)₂Pt(1-MeC⁻-N3,N4)₂Pt (NH₃)₂(9-EtGH-N7)](NO₃)₄·9H₂O (7b) (9-EtGH=9-ethylguanine). Several other compounds, differing in the nature of the axial ligands, have been isolated and or observed in solution by ¹H and ¹⁹⁵Pt NMR spectroscopy. The chemistry of these diplatinum(III) compounds is dominated by facile substitution reactions of the axial ligands. Of particular interest in this context is the ready reaction of 2b or 3 with guanine nucleobases. Since similar compounds are not obtained with any of the other common nucleobases, 2b and 3 can be considered guanine-specific chemical probes.     

The stereochemistry of Co(III)-amine complexes. The use of nOe and COSY H NMR spectroscopy to determine structure in solution

It is shown how 1D nOe and 2D COSY ¹H NMR spectroscopy can be used to assign the stereochemistry of Co(III) amine complexes. By using d₆-DMSO as solvent together with a small quantity of DCl all non-equivalent N---H hydrogens can be distinguished at 300 MHz. Through-space (nOe), and through-bond (COSY), associations with other N---H and C---H hydrogens can then be determined. This leads to a complete assignment of structure in solution. The technique is applied to the complexes syn(N), anti(N)-[Co(cyclen) (NH₃)₂] (ClO₄)₃, syn(N), anti(Cl)-[Co(cyclen) (NH₃)Cl] (ClO₄)₂, anti(N), syn(Cl)-[Co(cyclen) (NH₃)Cl](ClO₄)₂, syn(N), anti(O)-[Co(Mecyclen)-(GlyO)](ClO₄)₂ and Δ-cis-[Co(δ-en)₂(NO₂)₂](NO₂).     

Differentiation between mutants of Escherichia coli K12 defective in oxidative phosphorylation

Hybrid membrane particles from two mutants of Escherichia coli K₁₂, B_v4 and K_I1, defective in oxidative phosphorylation, have been prepared, in which ATP-driven membrane energization is restored.

A soluble factor of mutant K_I1 was found to have properties similar to parental crude coupling factor, ATPase (EC 3.6.1.3). Membrane particles of this mutant could not be reconstituted by parental coupling factor. Either parental coupling factor, or the soluble factor of mutant K_I1 could reconstitute both respiration-driven and ATP-driven energization to membrane particles of mutant B_V4 or to parental particles depleted of ATPase. Mutant B_V4 was found to be devoid of coupling factor activity, while retaining the ability to hydrolyze ATP. Both mutants possess an ATPase with an altered binding to the membrane.

Mutant K_I1 is impaired in respiration-driven amino acid transport, in contrast to mutant B_V4.

The three major subunits of parental Escherichia coli ATPase have been isolated and antibodies have been prepared against these subunits. Antibodies against the largestsubunit ( component) or against the intact catalytic subunits ( + β components) inhibit both ATP-P_i exchange in the parent organism as well as ATP hydrolytic activity in parent and mutants. Antibodies against the two other subunits (β or γ components) also inhibit these two reactions, but were found to be less effective. Mutant N_I44, which lacks ATPase activity, shows no precipitin lines with anti-, anti-β, anti-γ, or anti-( + β) preparations. In contrast, mutants B_V4 and K_I1, exhibit cross-reactivity with all of the antisera.     

Evaluation of Active Oxygen Effect on Photosynthesis of Chlorella vulgaris

The relationship between O₂ and an active oxygen scavenging system in Chlorella vulgaris var.vulgaris (IAM C-534) was investigated. When Chlorella vulgaris was exposed to 2% O₂, only traces of active oxygen scavenging enzymes were found. When the Chlorella vulgaris was treated with 20% or 50% O₂, it was shown that the level of enzyme activity increased as the O₂ concentration increased. An increase in enzyme activity was not found in any specific enzyme but in all of the enzymes, but the level of glutathione and ascorbate remained the same in all the cases. In addition, the photosynthetic efficiency also decreased as the concentration of O₂ was increased. These results suggest that an O₂ enriched environment can lead to an increase in the production of active oxygen species such as O_bullet2 and H₂O₂ and to a decrease in the photosynthetic efficiency in Chlorella vulgaris. The hydroxyl radical (^bulletOH) was detected directly in the Chlorella vulgaris suspension with a spin trapping reagent. It was also clear that the increase in the ^bulletOH intensity as the visible light intensity increased was unrelated to the O₂ concentration. It was suggested that the conditions for producing ^bulletOH and the other active oxygen species were different, and that two types of oxygen stress should exist in the Chlorella vulgaris.     

A Monoclonal Antibody to Oestradiol Potentiates the Stimulation of the Specific Activity of the Brain Type Creatine Kinase by Oestrogen in vivo and in vitro

We described previously the in vivo immunoneutralization effects of a high affinity anti-oestradiol antibody clone 15 in blocking ovulation and synaptic remodeling in cycling female rats. In the present study we report the enhancing effects of this antibody. Treatment of ovariectomized female rats or female derived skeletal cell cultures in vitro with anti-E₂ 15 plus oestrogen (E₂) potentiated the specific activity of the brain type creatine kinase (CK) response to E₂ in the rat tissues or skeletal cells. The enhancing CK response of anti E₂ 15 plus E₂ was time- and dose-dependent in the uterus, thymus, epiphysis and diaphysis of ovariectomized female rats. In the pituitary, on the other hand, anti-E₂ 15 blocked the stimulatory CK response to E₂. Two other high affinity anti-E₂ anti-bodies, clones 8D₉ and 11B₆, had no effect in augmenting the response of CK to E₂ in rat tissues. Moreover, the enhancing CK response in rat tissues was specific to anti-E₂ 15 plus E₂ since the intact anti-E₂ in the presence of other oestrogen mimetics, such as oestriol or stilbestrol or tamoxifen did not potentiate the CK response in rat tissues. In this model system the Fab' monomer of anti-E₂ 15 abolished the CK response to E₂ in rat tissues and not to anti-E₂ 15 plus E₂ whereas tamoxifen completely blocked the CK response to anti E₂ plus E₂. Anti E₂ 15 may therefore serve as a specific carrier in delivering E₂ to oestrogen sensitive rat tissues or cells containing functional oestrogen receptors and thereby increasing the magnitude of E₂ effects in vivo and in vitro.