Low Phospholipase A2 Activity Research Articles

Antimüllerian hormone and F2-isoprostanes in the Coronary Artery Risk Development in Young Adults (CARDIA) Study

To examine whether F2-isoprostanes, a marker of systematic oxidative stress, are associated with antimüllerian hormone (AMH), an indicator of ovarian reserve, in a population-based cohort of women of black and white ethnicities. Cross-sectional analysis. Not applicable. The CARDIA Women's Study, a population-based cohort. Black (n = 398) and white (n = 432) late reproductive-aged women (mean age 40 ± 3.6 years) without histories of gynecologic surgery. Log-transformed serum AMH concentrations. Linear regression models evaluated whether plasma F2-isoprostanes were associated with log-transformed AMH after adjustment for age, race, smoking, body mass index, and oral contraceptive pill use. Higher levels of F2-isoprostanes were associated with lower AMH levels (β -0.048 per standard deviation, 95% confidence interval -0.087, -0.01). The observed associations were stronger at younger ages (P=.04 for interaction between levels of age and F2-isoprostanes). Indicators of other steps in the oxidative stress pathway (superoxide dismutase, paraoxonase activity, oxidized low-density lipoprotein cholesterol, and carotenoids) were not associated with AMH, although lower phospholipase A2 activity (β 0.036 per standard deviation, 95% confidence interval 0.001, 0.071) was associated with lower AMH across all ages. In a population-based cohort, higher levels of F2-isoprostanes were associated with lower ovarian reserve, particularly at younger ages.

Biological and molecular properties of yellow venom of the Amazonian coral snake Micrurus surinamensis.

The coral snake Micrurus surinamensis, which is widely distributed throughout Amazonia, has a neurotoxic venom. It is important to characterize the biological and molecular properties of this venom in order to develop effective antitoxins. Toxins from the venom of M. surinamensis were analyzed by two-dimensional polyacrylamide gel electrophoresis and their neurotoxic effects in vivo were evaluated. Most proteins in the venom had masses < 14kDa, low phospholipase A2 activity, and no proteolytic activity. The toxins inhibited the coagulation cascade. The venom had neurotoxic effects in mice, with a median lethal dose upon intravenous administration of 700 µg/kg. Immunogenic studies revealed abundant cross-reactivity of antielapidic serum with 14kDa toxins and limited cross-reactivity with toxins < 10kDa. These results indicate that antielapidic serum against M. surinamensis venom has weak potency (0.35mg/ml) in mice.

Biological activities of Leptodeira annulata (banded cat-eyed snake) venom on vertebrate neuromuscular preparations

The physiological properties of colubrid snake venoms are largely unknown and less frequently investigated. In this study, we assessed the enzymatic properties and biological activities of Leptodeira annulata (banded cat-eyed snake) venom, an opistoglyphous snake from Colombia. The proteolytic, phospholipase A2 and amidolytic activities are assessed using colorimetric assays and the biological activities were analyzed in avian and mammalian neuromuscular preparations. L. annulata venom caused neuromuscular blockade in chick biventer cervicis (BC) preparations (40± 15% and 50± 3% of twitch reduction for 30 and 100 μg/ml, respectively; p < 0.05) following 120 incubation; 10 μg/ml of venom did not induce blockade. There was a mild reduction in contracture response to exogenous acetylcholine (110 μM) in BC preparations exposed to 10 and 30 μg of venom/ml (∼4% and ∼32% of reduction, respectively, p > 0.05, n = 4) compared to basal values whereas the highest concentration (100 μg/ml) abolished it after 120 min. The venom caused a significant reduction in contracture response elicited by KCl (∼58 and ∼90 of reduction for 30 and 100 μg/ml, respectively, p < 0.05, n = 4). In mouse phrenic nerve-diaphragm (PND) preparations, L. annulata venom induced a progressive muscle membrane depolarization [from −85.9 ± 1.6 mV (t0) to −72.2 ± 2.9 mV (t120), p < 0.05, n = 4); the postsynaptic receptors remained functional as shown by carbachol-induced depolarization. The morphological analyses showed a concentration-dependent number of pathological states in muscle fibers from both BC and PND preparations pre-exposed to venom. The venom showed high proteolytic activity and low phospholipase A2 activity; there was no evidence for serine protease activity. These results indicate that the neuromuscular effect induced by L. annulata venom resulted from damaged muscle fibers that lead to the blockade of twitches response. The findings suggest that the myotoxicity might be related to the presence of metalloproteases in this venom.

Decreased platelet phospholipase A2 activity in Alzheimer's disease and mild cognitive Impairment

We investigated the activity of phospholipase A2 (PLA2) in platelets from patients with Alzheimer’s disease (AD), in healthy elderly controls and in individuals with mild cognitive impairment (MCI). PLA2 activity was significantly lower in AD than in MCI patients and controls. MCI showed mean PLA2 values between AD and controls. Lower PLA2 activity was significantly correlated with a worse cognitive performance as rated by the MMSE and CAMDEX. Our data replicate previous findings of reduced PLA2 activity in AD and further suggests that PLA2 activity may also be decreased in non-demented memory impaired subjects. PLA2 activity in platelets may reflect its activity in the brain, considering previous evidence of reduced PLA2 in post-mortem AD brains, and in vivo spectroscopy data suggesting reduced cortical phospholipid turnover in AD.

Isolation and enzymatic characterization of a basic phospholipase A2 from Bothrops jararacussu snake venom.

A novel basic phospholipase A2 (PLA2) isoform was isolated from Bothrops jararacussu snake venom and partially characterized. The venom was fractionated by HPLC ion-exchange chromatography in ammonium bicarbonate buffer, followed by reverse-phase HPLC to yield the protein Bj IV. Tricine SDS-PAGE in the presence or absence of dithiothreitol showed that Bj IV had a molecular mass of 15 and 30 kDa, respectively. This enzyme was able to form multimeric complexes (30, 45, and 60 kDa). Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The N-terminal sequence (DLWSWGQMIQETGLLPSYTTY...) showed a high degree of homology with basic D49 PLA2 myotoxins from other Bothrops venoms. Bj IV had high PLA2 activity and produced moderate myonecrosis in skeletal muscle, but showed no neuromuscular activity in mouse phrenic nerve-diaphragm preparations. Bj IV showed allosteric enzymatic behavior, with maximal activity at pH 8.2 and 35-45 degrees C. Full PLA2 activity required Ca2+ but was inhibited by Cu2+ and Zn2+, and by Cu2+ and Mg2+ in the presence and absence of Ca2+, respectively. Crotapotins from Crotalus durissus terrificus rattlesnake venom significantly inhibited the enzymatic activity of Bj IV. The latter observation suggested that the binding site for crotapotin in this PLA2 was similar to that in the basic PLA2 of the crotoxin complex from C. d. terrificus venom. The presence of crotapotin-like proteins capable of inhibiting the catalytic activity of D49 PLA2 could partly explain the low PLA2 activity of Bothrops venoms.

The amino acid sequence of bothropstoxin-II, an Asp-49 myotoxin from Bothrops jararacussu (Jararacucu) venom with low phospholipase A2 activity.

The complete amino acid sequence of bothropstoxin-II (BthTX-II), a myotoxin isolated from Bothrops jararacussu snake venom, is reported. The results show that BthTX-II is an Asp-49 phospholipase A2 (PLA2)-like protein composed of a single polypeptide chain of 120 amino acid residues (Mr = 13,976), containing one methionine and 14 half-cystines. Despite a high degree of homology with other PLA2's and the presence of the strategic residues known to compose the Ca2+-binding loop, namely Tyr-28, Gly-30, Gly-32, and especially Asp-49, besides His-48, Tyr-52, and Asp-99, all of them directly or indirectly involved in catalysis, BthTX-II revealed a very low PLA2 activity when assayed on egg yolk phosphatidylcholine. We attribute this low catalytic activity to the existence of extra mutations, e.g., Trp-5 for Phe-5, which points to the need of considering other strategic positions, since only Lys-49 PLA2's have been considered to be devoid of this enzymatic activity.

Isolation and Characterization of a Myotoxic Phospholipase A2from the Venom of the Arboreal SnakeBothriechis(Bothrops)schlegeliifrom Costa Rica

A new myotoxic phospholipase A2was isolated from the venom of the arboreal snakeBothriechis schlegelii(formerlyBothrops schlegelii) from Costa Rica, by ion-exchange chromatography on CM-Sephadex.B. schlegeliimyotoxin I is a basic protein (pI> 9.3) with a subunit molecular weight of 15 kDa, which migrates as a dimer in sodium dodecyl sulfate–polyacrylamide gel electrophoresis under nonreducing conditions. This myotoxin is recognized by antibodies generated againstBothrops aspermyotoxin II (a lysine-49 phospholipase A2), by both enzyme-immunoassay and gel immunodiffusion, in the latter case with a pattern of partial identity. The toxin induces rapid myonecrosis upon intramuscular injection in mice, as evidenced by the early increase in plasma creatine kinase activity and by direct intravital microscopic observation.B. schlegeliimyotoxin I also induces edema in the mouse footpad assay and exerts lethal activity (LD50∼2.5 μg/g) upon intravenous injection. The toxin has a low phospholipase A2activity (4.2 μEq·mg−1·min−1) using egg yolk phospholipids as substrate. It also shows a weak anticoagulant effectin vitro.Its N-terminal sequence, SMYELGKMILLETGKNAATSYIAYG, shows 93% homology with bothBothrops aspermyotoxin II andB. jararacussubothropstoxin I, suggesting thatB. schlegeliimyotoxin I may be a new lysine-49 variant of this family of myotoxic phospholipases A2.

Decreased phospholipase A2 activity in the brain and in platelets of patients with Alzheimer's disease.

Phospholipase A2 (PLA2) is a key enzyme in the metabolism of membrane phospholipids. PLA2 influences the processing and secretion of the amyloid precursor protein, which give rise to the beta-amyloid peptide, the major component of the amyloid plaque in Alzheimer's disease (AD). We investigated the PLA2 activity in two samples: in post-mortem brains from 23 patients with AD and 20 non-demented elderly controls, and platelets from 16 patients with a diagnosis of probable AD, 13 healthy controls and 14 elderly patients with a major depression. In AD brains PLA2 activity was significantly decreased in the parietal, and to a lesser degree in the frontal, cortex. Lower PLA2 activity correlated significantly with an earlier onset of the disease, an earlier age at death and higher counts of neurofibrillary tangles and senile plaques. In platelets PLA2 activity was also significantly reduced in the AD group as compared with healthy and depressed controls. The reduction of the enzyme activity in platelets correlated with an early disease onset and with the severity of cognitive impairment, indicating a relationship between abnormally low PLA2 activity and a more severe form of the illness. The present results provide new evidence for a disordered phospholipid metabolism in AD brains and suggest that reduced PLA2 activity may contribute to the production of amyloidogenic peptides in the disease. Further studies are needed to examine whether PLA2 activity in platelets may be useful as a peripheral marker for a subgroup of patients with AD.

Relevance of metabolism of membrane phospholipids for Alzheimer dementia

We found a decreased activity of the enzyme phospholipase A2 (PLA2) in brain tissue from 23 patients with Alzheimer's disease (AD) compared to 20 non-demented controls. The decrement was more pronounced in patients with an early onset of disease and correlated significantly with higher counts of senile plaques and neurofibrillary tangles. Decreased PLA2 activity may inhibit the secretion of the amyloid precursor protein (APP) and thus contribute to the formation of the beta-amyloid peptide, the major component of the amyloid plaque in AD. Because PLA2 activity is under genetic control, it is conceivable that the enzyme activity in the brain is related to the activity in blood cells. To test this assumption we investigated PLA2 in platelet membranes from AD patients compared to healthy and psychiatric controls. Platelets are interesting peripheral models in AD research, because they contain and secrete APP. We determined the platelet PLA2 activity in 16 patients with a "probable" AD (NINCDS-ADRDA criteria) as compared to 13 healthy controls and to 14 psychiatric patients with a major depression. There were no significant differences between the three groups regarding age and sex distribution. In the AD patients the cognitive performance was assessed with the CAMCOG and the Mini Mental State Exam (MMSE). The radioenzymatic assay for the determination of PLA2 activity is described elsewhere. Platelet PLA2 activity was significantly reduced in AD patients as compared to healthy (p < 0.03) and to psychiatric controls (p < 0.002). The reduction of the enzyme activity correlated with an early onset of the disease (rs = .43, p < 0.10) and with the cognitive impairment in the CAMCOG (rs = .55, p < 0.05). AD patients with a MMSE-score lower than 10 (median) showed significantly lower PLA2 activity (11.8 +/- 3.1) than patients with MMSE-score higher than 10 (16.2 +/- 4.6, p < 0.05). These findings in platelets are in line with our previous results in brain tissue. In both studies decreased PLA2 activity was related to a more severe form of AD (early onset, more cognitive impairment and higher number of plaques and tangles). Moreover, reduced platelet PLA2 activity was specific for AD as compared to age-matched psychiatric controls. Further studies should clarify whether PLA2 activity in platelets could be useful as a peripheral marker for a subgroup of AD.

Decreased phospholipase A 2 activity in Alzheimer brains

Phospholipase A2 (PLA2) is a key-enzyme in the metabolism of membrane phospholipids. In cholinergic neurons PLA2 controls the physico-chemical properties of neuronal membranes as well as the breakdown of phosphatidylcholine to produce choline for acetylcholine synthesis. Moreover PLA2 influences the processing and secretion of the amyloid precursor protein, which gives rise to the beta-amyloid peptide, the major component of the amyloid plaque in Alzheimer's disease (AD). In the present study PLA2 activity was investigated in post-mortem brains from 23 patients with AD and 20 nondemented elderly controls. In AD brains PLA2 activity was significantly decreased in the parietal and to a lesser degree in the frontal cortex. Lower PLA2 activity correlated significantly with an earlier onset of the disease, higher counts of neurofibrillary tangles and senile plaques and an earlier age at death, indicating a relationship between abnormally low PLA2 activity and a more severe form of the illness. The present results provide new evidence for a disordered phospholipid metabolism in AD brains and suggest that reduced PLA2 activity may contribute to the cholinergic deficit and to the production of amyloidogenic peptides in the disease.

Dynamics and Participation of Type II Phospholipase A2 in Rat Zymosan-Induced Pleurisy1

Intrapleural injection of zymosan into rats induces acute inflammation characterized by plasma leakage and cellular influx. The level of type II phospholipase A2 (PLA2) increased in the pleural fluid as well as in exudating leukocytes after the injection of zymosan. Rather low PLA2 activity was found in cell lysates, though treatment of such lysates at low pH increased the PLA2 activity drastically. The appearance of "acid-extracted" PLA2 activity in leukocytes preceded that of the extracellular enzyme activity, suggesting that pleural leukocytes might be one of the origins of the extracellular enzyme. Treatment of exudating pleural leukocytes with purified rat type II PLA2 elicited the production of prostaglandin E2, but not platelet-activating factor appreciably. These findings indicate that type II PLA2 might play a role in the progression of inflammation through the production of eicosanoids in the present inflammation model.

Purification and Amino Acid Sequence of Basic Protein II, a Lysine-49-Phospholipase A2 with Low Activity, from Trimeresurus flavoviridis Venom1

A basic protein (pI 10.3), named basic protein II, was purified to homogeneity from the venom of Trimeresurus flavoviridis (Habu snake) after four chromatographic steps. The amino acid sequence of this protein was determined by sequencing the S-pyridylethylated derivative and its peptides produced by chemical (cyanogen bromide) and enzymatic (chymotrypsin, clostripain, and Staphylococcus aureus V8 protease) cleavages. The protein consisted of 122 amino acid residues and was found to be identical in sequence to basic protein I from the same source except that Asp-58 of basic protein I is replaced by asparagine. Like basic protein I, the structural feature of basic protein II is that Tyr-28 and Asp-49 common in phospholipases A2 from snake venoms and mammalian pancreas are replaced by asparagine and lysine, respectively. Thus, basic protein II belongs to the category of lysine-49-phospholipase A2. The action of basic protein II on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine released only oleic acid, indicating that it has phospholipase A2 activity. Its molar activity toward 1,2-dilauroyl-sn-glycero-3-phosphorylcholine, however, was only 1.7% of that of T. flavoviridis phospholipase A2 isolated previously. Affinity for Ca2+ and reactivity toward p-bromophenacyl bromide of basic protein II were 8 and 5.3 times, respectively, smaller than those of phospholipase A2 from the same source, substantiating the low phospholipase A2 activity of basic protein II.

Effect of phorbolmyristate acetate on fatty acid uptake and distribution in human promyelocytic leukemia (HL-60) cells in vitro

Human promyelocytic leukemia (HL-60) cells can be induced to differentiate to macrophages in vitro by phorbolmyristate acetate (PMA). HL-60 cells, unlike normal cells incorporated a major portion of linoleic acid (LA) and arachidonic acid (AA) in the ether lipid fraction. On exposure to PMA, similar to the normal cells tested, the fatty acids were incorporated mainly in the phospholipid fraction. Since, ether lipid pool is metabolically inert and considered as a storage pool where as the phospholipid fraction is a metabolically active pool this may explain, at least in part, the low metabolic rate of AA and the low phospholipase A2 activity in HL-60 cells.

Effects of a toxic phospholipase A2 (AgTx) from the venom of the Pit viper (Agkistrodon halys (Pallas)) on the crayfish stretch receptor neuron.

Neurotoxicity of an isolated fraction (V) of the venom from the snake Agkistrodon halys (Pallas) was studied on the crayfish stretch receptor using a two-electrode voltage-clamp technique. The toxin was previously shown to have less phospholipase A2 activity but to be more toxic in mice compared with bee venom phospholipase A2. At 5 micrograms ml-1 (0.35 microM) the AgTx had very little effects on the electrophysiological properties of the receptor neuron whereas at 50 micrograms ml-1 (3.5 microM) the leak conductance was increased about three times and the net outward (K) current was reduced to 70% of control. The net inward (Na) current was not affected except for a small (+ 10 mV) shift in the I-V relationship. The resting membrane potential and the membrane capacitance were not changed by AgTx. These effects of AgTx were similar to those seen after exposure to bee venom phospholipase A2 at concentrations 10-20 times lower. At concentrations of AgTx which presynaptically affect the frog neuromuscular transmission (5-10 micrograms ml-1) the effects on ion channels in the stretch receptor neuron are very small and probably reflect the low phospholipase A2 activity compared with that of bee venom phospholipase A2.

Etude des phospholipases A des membranes plasmiques de foie de rat

The plasma membranes phospholipase A2 studied in situ shows very little sensitivity for pH variations ; the optimal concentration for Ca++ is 5 mM ; the enzymatic kinetics are of the Michaelis type. An inactive state of the phospholipase A2 exists : when rats are injected with heparin, their plasma membranes contain a very low phospholipase A2 activity. These membranes recover a high A2 activity if they are incubated in the presence of a rat platelets lysate. Treatment of membranes by NaCl 1 M displaces phospholipase A1 and phospholipase A2 but for the latter only when being in active state. Treatment of animals, conditions of preparation and of incubation of membranes influence the respective amounts of phospholipases A1 and A2 present in these membranes and are discussed in this paper. The localisation of these enzymatic proteins inside the membrane according to the membranous model proposed by Singer et al. [18] is also discussed.