Toxoplasma gondii secretes a calcium-independent phospholipase A(2)

Phospholipases A(2) (PLA(2)) play an important role in Toxoplasma gondii host cell penetration. They are also key enzymes in the host cell response to the parasite invasion. PLA(2) hydrolyse cellular phospholipids, releasing multiple inflammatory lipidic mediators. We have investigated the biochemical characterisation of T. gondii PLA(2) activity in a mouse-cultured tachyzoite homogenate and in the peritoneal exudate from infected mice, using the hydrolysis of a fluorescent phosphatidylglycerol labelled at the sn-2 position. Spectrofluorimetry and thin-layer chromatography showed a PLA(2) activity (about 0.5-2 nmol/min per mg), calcium-independent, secreted into infected mice peritoneal exudate, with a broad pH activity ranging between 6.5 and 9.5 and resistant to a great number of potential PLA(2) inhibitors except dithio-nitrobenzoic acid (1 mM). An associated phospholipase A(1) activity was also displayed. These results suggest that Toxoplasma gondii displays specific phospholipases different from host enzymes and probably involved at critical steps of infectious cycle.     

New potent and selective polyfluoroalkyl ketone inhibitors of GVIA calcium-independent phospholipase A2

Group VIA calcium-independent phospholipase A₂ (GVIA iPLA₂) has recently emerged as an important pharmaceutical target. Selective and potent GVIA iPLA₂ inhibitors can be used to study its role in various neurological disorders. In the current work, we explore the significance of the introduction of a substituent in previously reported potent GVIA iPLA₂ inhibitors. 1,1,1,2,2-Pentafluoro-7-(4-methoxyphenyl)heptan-3-one (GK187) is the most potent and selective GVIA iPLA₂ inhibitor ever reported with a X_I(50) value of 0.0001, and with no significant inhibition against GIVA cPLA₂ or GV sPLA₂. We also compare the inhibition of two difluoromethyl ketones on GVIA iPLA₂, GIVA cPLA₂, and GV sPLA₂.     

Chemokine CCL2 modulation of neuronal excitability and synaptic transmission in rat hippocampal slices

In addition to its well-characterized effects in immune system, chemokine CC motif ligand 2 (CCL2, formerly known as monocyte chemoattractant protein-1) is believed to play an important role in brain physiological and pathological processes. It has been shown that CCL2 and its cognate receptor chemokine CC motif receptor 2 are constitutively expressed in several brain regions including the hippocampus, and the expression is up-regulated under pathological conditions. Whereas most investigations have so far focused on its involvement in CNS pathology, few studies have examined the effects of CCL2 on neuronal and synaptic physiology. In this study, we tested the effects of CCL2 on neuronal excitability and excitatory synaptic transmission in the CA1 region of rat hippocampal slices using whole-cell patch clamp techniques. Bath application of CCL2 depolarized membrane potential and increased spike firing in CA1 neuronal cells. Bath application of CCL2 also produced an increase of excitatory post-synaptic currents recorded in Schaffer-collateral fibers to CA1 synapses. Quantal analysis revealed that CCL2 increased the frequency of spontaneous excitatory post-synaptic current occurrence and mean quantal content. Taken together, our data indicate that CCL2 enhances neuronal excitability and synaptic transmission via pre-synaptic mechanisms. These results support the emerging concept that chemokines function as neuromodulators in the CNS.     

Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A2. Mechanism-based discrimination between calcium-dependent and -independent phospholipases A2

The majority of phospholipase A2 activity in myocardium is calcium-independent and selective for hydrolysis of plasmalogen substrate (Wolf, R. A., and Gross, R. W. (1985) J. Biol. Chem. 260, 7295-7303; Hazen, S. L., Stuppy, R. J., and Gross, R. W. (1990) J. Biol. Chem. 265, 10622-10630). Accordingly, identification of an inhibitor which selectively targets calcium-independent phospholipases A2 would facilitate elucidation of the biologic significance of this class of intracellular phospholipases. We now report that the haloenol lactone, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (Compound 1), is a potent, irreversible, mechanism-based inhibitor of myocardial calcium-independent phospholipase A2 which is greater than 1000-fold specific for inhibition of myocardial calcium-independent phospholipase A2 in comparisons with multiple calcium-dependent phospholipases A2. Mechanism-based inhibition of myocardial cytosolic calcium-independent phospholipase A2 by Compound 1 was established by demonstrating: 1) time-dependent irreversible inactivation; 2) covalent binding of [3H]Compound 1 to the purified phospholipase A2; 3) ablation of covalent binding of [3H]Compound 1 after chemical inactivation of phospholipase A2 enzymic activity; 4) identical inhibition of myocardial phospholipase A2 by Compound 1 in the absence or presence of nucleophilic scavengers; 5) Compound 1 is a substrate for myocardial calcium-independent phospholipase A2 resulting in the generation of the electrophilic alpha-bromomethyl ketone; 6) phospholipase A2 inhibition requires the in situ generation of the reactive electrophile (i.e. neither the alpha-bromomethyl ketone nor the diproteoenol lactone analog are inhibitory); and 7) concomitant attenuation of the inhibitory potency and the extent of covalent adduct formation in the presence of saturating substrate. Collectively, these results demonstrate that the haloenol lactone, Compound 1, is a substrate for, covalently binds to, and irreversibly inhibits canine myocardial cytosolic calcium-independent phospholipase A2.     

Naegleria fowleri amoebae express a membrane-associated calcium-independent phospholipase A(2)

Naegleria fowleri, a free-living amoeba, is the causative agent of primary amoebic meningoencephalitis. Previous reports have demonstrated that N. fowleri expresses one or more forms of phospholipase A(2) (PLA(2)) and that a secreted form of this enzyme is involved in pathogenesis. However, the molecular nature of these phospholipases remains largely unknown. This study was initiated to determine whether N. fowleri expresses analogs of the well-characterized PLA(2)s that are expressed by mammalian macrophages. Amoeba cell homogenates contain a PLA(2) activity that hydrolyzes the substrate that is preferred by the 85 kDa calcium-dependent cytosolic PLA(2), cPLA(2). However, unlike the cPLA(2) enzyme in macrophages, this activity is largely calcium-independent, is constitutively associated with membranes and shows only a modest preference for phospholipids that contain arachidonate. The amoeba PLA(2) activity is sensitive to inhibitors that block the activities of cPLA(2)-alpha and the 80 kDa calcium-independent PLA(2), iPLA(2), that are expressed by mammalian cells. One of these compounds, methylarachidonyl fluorophosphonate, partially inhibits the constitutive release of [(3)H]arachidonic acid from pre-labeled amoebae. Together, these data suggest that N. fowleri expresses a constitutively active calcium-independent PLA(2) that may play a role in the basal phospholipid metabolism of these cells.     

Catalytic residues of group VIB calcium-independent phospholipase A2 (iPLA2gamma)

Although human group VIB calcium-independent phospholipase A(2) (iPLA(2)gamma) contains the lipase-consensus sequence Gly-Xaa-Ser-Xaa-Gly in the C-terminal half, its overall sequence exhibits a week similarity to those of other PLA(2)s, and thus no information on the catalytic site has been available. Here we show that the C-terminal region of human iPLA(2)gamma is responsible for the enzymatic activity. Comparison of this catalytic domain with those of the mouse homologue, human cytosolic PLA(2) (cPLA(2)), and the plant PLA(2) patatin reveals that an amino acid sequence of a short segment around Asp-627 of iPLA(2)gamma is conserved among these PLA(2)s, in addition to the Ser-483-containing lipase motif; the corresponding serine and aspartate in cPLA(2) and patatin are known to form a catalytic dyad. Since substitution of alanine for either Ser-483 or Asp-627 results in a loss of the PLA(2) activity, we propose that Ser-483 and Asp-627 of human iPLA(2)gamma constitute an active site similar to the Ser-Asp dyad in cPLA(2) and patatin.     

一氧化氮对谷氨酸诱发的大鼠海马脑片CA1区神经元活动的抑制

用细胞外记录单位放电技术,在大鼠海马脑片上观察了Ｌ－精氨酸（Ｌ－ａｒｇ）、Ｎ－硝基Ｌ－精氨酸（Ｌ－ＮＮＡ）及ＳＩＮ－１对谷氨酸（ｇｌｕｔａｍａｔｅ,Ｇｌｕ）诱导的ＣＡ１区神经元放电的影响。旨在了解Ｌ－精氨酸：ＮＯ通路在谷氨酸诱发的海马放电中的作用及其可能的机制。结果如下：（１）用ＧｌＵ（０．５ｍｍｏｌ／Ｌ）灌流海马脑片１ｍｉｎ,１２个放电单位放电频率明显增加,表现为癫痫样放电;（２）海马脑片２ｍｉ     

A nuclear microscopic study of elemental changes in the rat hippocampus after kainate-induced neuronal injury

The effect of intracerebroventricular kainate injection on the elemental composition of the hippocampus was studied in adult Wistar rats, at 1 day and 1, 2, 3, and 4 weeks postinjection, using a nuclear microscope. An increase in calcium concentration was observed on the injected side from 1 day postinjection. The increase peaked at 3 weeks postinjection, reaching a concentration of 18 times normal. Large numbers of glial cells but no neurons were observed in the lesioned CA fields at this time, suggesting that an increased calcium level was present in glial cells. This was confirmed by high-resolution elemental maps of the lesioned areas, which showed very high intracellular calcium concentrations in almost all glial cells. It is possible that the high intracellular calcium level could activate calcium-dependent enzymes, including calpain II and cytosolic phospholipase A2, shown to be expressed in reactive glial cells after kainate injections. In addition to calcium, an increase in iron content was also observed at the periphery of the glial scar at 4 weeks postinjection. Because free iron could catalyze the formation of free radicals, the late increase in iron content may be related to oxygen radical formation during neurodegeneration.     

Snake inhibitors of phospholipase A(2) enzymes

Fragment 53--103 of bovine alpha-lactalbumin, prepared by limited peptic digestion of the protein at low pH, is a 51-residue polypeptide chain crosslinked by two disulfide bonds encompassing helix C (residues 86--98) of the native protein. Refolding of the fully reduced fragment (four--SH groups) is expected to lead to three fully oxidized isomers, the native (61--77, 73--91) and the two misfolded species named ribbon (61--91, 73--77) and beads (61--73, 77--91) isomers. The fragment with correct disulfide bonds was formed in approx. 30% yield when refolding was conducted in aqueous solution at neutral pH in the presence of the redox system constituted by reduced and oxidized glutathione. On the other hand, when the reaction was conducted in 30% (v/v) trifluoroethanol (TFE), the oxidative refolding to the native isomer was almost quantitative. To provide an explanation of the beneficial effect of TFE in promoting the correct oxidative folding, the conformational features of the various fragment species were analyzed by far-UV circular dichroism measurements. The fully reduced fragment is largely unfolded in water, but it becomes helical in aqueous TFE. Correctly refolded fragment is produced most when the helical contents of the reduced and oxidized fragment in aqueous TFE are roughly equal. It is proposed that 30% TFE promotes a native-like format of the fragment and thus an efficient and correct pairing of disulfides. Higher concentrations of TFE, instead, promote some non-native helical secondary structure in the fragment species, thus hampering correct folding.     

The expression and function of a group VIA calcium-independent phospholipase A2 (iPLA2beta) in beta-cells

Many cells express a Group VIA phospholipase A2, designated iPLA2beta, that does not require calcium for activation, is stimulated by ATP, and is sensitive to inhibition by a bromoenol lactone suicide substrate (BEL). Studies in various cell systems have led to the suggestion that iPLA2beta has a role in phospholipid remodeling, signal transduction, cell proliferation, and apoptosis. We have found that pancreatic islets, beta-cells, and glucose-responsive insulinoma cells express an iPLA2beta that participates in glucose-stimulated insulin secretion but is not involved in membrane phospholipid remodeling. Additionally, recent studies reveal that iPLA2beta is involved in pathways that contribute to beta-cell proliferation and apoptosis, and that various phospholipid-derived mediators are involved in these processes. Detailed characterization of the enzyme suggests that the beta-cells express multiple isoforms of iPLA2beta, and we hypothesize that these participate in different cellular functions.     

GABA对大鼠海马脑片缺氧损伤的保护作用

目的：研究GABA对大鼠海马脑片急性缺氧损伤的保护机制。方法：采用成年大鼠离体海马脑片,用胞外记录的电生理技术,观察GABA对急性缺氧后海马脑片诱发电位的影响。结果：（1）GABA可明显延迟PV的消失,但对PS却无影响;（2）给予GABAA受体拮抗剂荷包牡丹碱（bicuculine)以及Cl^-通道阻抗剂NPPB可阻断GABA的保护作用。结论：GAB可提高海马脑片耐缺氧能力,其机制可能与GABA通过GABAA受体提高Cl^-内流有关。     

Inhibitory effects of GABA on synaptic excitation in isolated rat hippocampal slices

In research on -aminobutyric acid (GABA) used at different concentrations on the amplitude of EPSP within populations (PEPSP), as recorded from dentrites in isolated hippocampal slices, GABA induced a dose-dependent reversible reduction in PEPSP amplitude with no noticeable signs of desensitization. Highest sensitivity to GABA was shown by PEPSP in hippocampal zone CA1 (threshold concentration: 3×10^–5–2×10^–4 M; (concentration at which the effect equal to 1/2 of maximum occurs) IC₅₀: 5×10^–4–1×10^–3 M). The effects of GABA on PEPSP were not blocked by bicuculline, picrotoxin, or penicillin. Action of GABA on dendritic antidromic population spike (DAPS — postynaptic effects) were slightly diminished by these blockers. Baclofen inhibited PEPSP more powerfully than GABA (threshold concentration: 1×10^–6 M: IC₅₀: 3×10^–6 M), although it only produced a minor reduction in DAPS amplitude even at high concentrations. It is concluded that the inhibitory effect of GABA on PEPSP in hippocampal zone CA1 may be put down mainly to its presynaptic action mediated by GABA_B receptors on axonal terminals of Schaffer collaterals.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 5, pp. 627–633, September–October, 1990.     

Identification of a calcium-independent phospholipase A2 in rat lung cytosol and differentiation from acetylhydrolase for 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF-acether)

The 100 000 X g supernatant of total rat lung homogenate was found to contain at least three phospholipase A2-type activities. Gel filtration separated a low molecular weight and Ca2+-requiring phospholipase A2 from Ca2+-independent acylhydrolase peak with an apparent higher molecular weight. Upon DEAE-cellulose chromatography this fraction was separated into a Ca2+-independent acylhydrolase and a Ca2+-independent platelet-activating factor-acetylhydrolase with no apparent overlap in acyl chain length specificity. The long-chain acylhydrolase was shown to exhibit specificity for the ester bond at the sn-2-position. Ca2+-independent phospholipase A2 activity was inhibited by p-bromophenacylbromide and was resistant to diisopropylfluorophosphate. In contrast, the Ca2+-independent acetylhydrolase activity was inhibited by diisopropylfluorophosphate but was unaffected by p-bromophenacylbromide.     

七氟醚对大鼠海马脑片缺血损伤的保护作用

目的:探讨七氟醚对脑缺血损伤的保护作用及其机制。方法:用电生理细胞外记录的方法和组织学检查的技术,观察对照组、2%七氟醚组和4%七氟醚组对缺氧无糖(OGD)及谷氨酸(Glu)损伤所致的大鼠海马脑片CA1区顺向群峰电位(OPS)的影响及各组脑片超微结构的变化。结果:对照组和2%七氟醚组在OGD和Glu损伤后海马脑片OPS很难恢复;4%七氟醚组明显改善OPS的恢复程度和恢复率,减轻海马CA1区神经元细胞损伤。电镜观察可见,对照组OGD和Glu损伤后海马CA1区锥体细胞明显水肿,核膜不完整,核染色加深,核内染色质凝聚成块,胞浆中内质网高度扩张,线粒体水肿;2%七氟醚组与对照组相似;4%七氟醚组细胞水肿不显,核膜完整,核内染色质轻度凝聚,内质网轻度扩张,线粒体无明显水肿。结论:4%七氟醚对大鼠海马脑片OGD损伤有保护作用,可能与减轻兴奋性Glu毒性有关。     

Benzene-di-N-octylcarbamates as conformationally constrained phospholipase A(2) inhibitors

Conformationally constrained 1,2-, 1,3-, and 1,4-benzene-di-N-octylcarbamates are potent reversible competitive inhibitors of Naja mocambique mocambique phospholipase A(2) with the K(i) values of 11, 4, and 15 microM, respectively. With the angle of 120(o) between two C(benzene)-O bonds, 1,3-benzene-di-N-octylcarbamate mimics the preferable eclipsed C(sn-2)-O/C(sn-3)-O conformer of phospholipid in the enzyme-phospholipid complex. Further, a three-step phospholipase A(2) inhibition mechanism by the inhibitor is proposed.     

Neuroprotective effects of Ptychopetalum olacoides Bentham (Olacaceae) on oxygen and glucose deprivation induced damage in rat hippocampal slices

Alcoholic infusions of Ptychopetalum olacoides Bentham (PO, Olacaceae) are used in traditional medicine by patients presenting age associated symptoms and those recovering from stroke. The aim of this study is to evaluate the neuroprotective properties of PO ethanol extract (POEE) using hippocampal slices from Wistar rats exposed to oxygen and glucose deprivation (OGD, followed by reoxygenation). Mitochondrial activity, an index of cell viability, was assessed by the MTT assay; in addition, the free radicals content was estimated by the use of dichlorofluorescein diacetate as probe. The OGD ischemic condition significantly impaired cellular viability, and increased free radicals generation. In non-OGD slices, incubation with POEE (0.6 microg/ml) increased (approximately 40%) mitochondrial activity, without affecting free radicals levels. In comparison to OGD controls, slices incubated with POEE (0.6 microg/ml) during and after OGD exposure had significantly increased cellular viability. In addition, at this same concentration, POEE prevented the increase of free radicals content induced by OGD. In view of the fact that respiratory chain inhibition and increased generation of free radicals are major consequences of the ischemic injury, this study suggests that Ptychopetalum olacoides contains useful neuroprotective compounds and, therefore, deserves further scrutiny.     

Differential regulation of calcium-dependent and calcium-independent cyclic nucleotide phosphodiesterases from heart by palmitoylcarnitine and palmitoyl coenzyme A

Regulation of Ca2+-dependent (peak I) and Ca2+-independent (peak II) phosphodiesterases from the heart by various fatty acyl esters and phospholipids were studied. DL-Palmitoylcarnitine stimulated the basal activity (in the absence of Ca2+) of peak I enzyme, while non-competitively inhibiting peak II enzyme with respect to cyclic AMP. It had no effect on other species of Ca2+-independent phosphodiesterases, including cyclic AMP- and cyclic GMP-specific enzymes from the lung, and cyclic CMP enzyme from the liver Palmitoyl-CoA and phosphatidylserine also stimulated the basal activity of peak I enzyme, but they were without effect on peak II enzyme. In comparison, DL-palmitoylcarnitine inhibited Ca2+-dependent activity of cardiac myosin light chain kinase, whereas phosphatidylserine was without effect. It is conceivable that differential regulation of phosphodiesterases by these lipids could profoundly alter the levels or effects, or both, of cyclic nucleotides and Ca2+ in the myocardium.     

A novel role of group VIB calcium-independent phospholipase A2 (iPLA2gamma) in the inducible expression of group IIA secretory PLA2 in rat fibroblastic cells

Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) is a prototypic sPLA(2) enzyme that may play roles in modification of eicosanoid biosynthesis as well as antibacterial defense. In several cell types, inducible expression of sPLA(2) by pro-inflammatory stimuli is attenuated by group IVA cytosolic PLA(2) (cPLA(2)alpha) inhibitors such as arachidonyl trifluoromethyl ketone, leading to the proposal that prior activation of cPLA(2)alpha is required for de novo induction of sPLA(2). However, because of the broad specificity of several cPLA(2)alpha inhibitors used so far, a more comprehensive approach is needed to evaluate the relevance of this ambiguous pathway. Here, we provide evidence that the induction of sPLA(2)-IIA by pro-inflammatory stimuli requires group VIB calcium-independent PLA(2) (iPLA(2)gamma), rather than cPLA(2)alpha, in rat fibroblastic 3Y1 cells. Results with small interfering RNA unexpectedly showed that the cytokine induction of sPLA(2)-IIA in cPLA(2)alpha knockdown cells, in which cPLA(2)alpha protein was undetectable, was similar to that in replicate control cells. By contrast, knockdown of iPLA(2)gamma, another arachidonyl trifluoromethyl ketone-sensitive intracellular PLA(2), markedly reduced the cytokine-induced expression of sPLA(2)-IIA. Supporting this finding, the R-enantiomer of bromoenol lactone, an iPLA(2)gamma inhibitor, suppressed the cytokine-induced sPLA(2)-IIA expression, whereas (S)-bromoenol lactone, an iPLA(2)beta inhibitor, failed to do so. Moreover, lipopolysaccharide-stimulated sPLA(2)-IIA expression was also abolished by knockdown of iPLA(2)gamma. These findings open new insight into a novel regulatory role of iPLA(2)gamma in stimulus-coupled sPLA(2)-IIA expression.