Screening for target toxins of the antiophidic protein DM64 through a gel-based interactomics approach.

DM64 is a glycosylated protein with antivenom activity isolated from the serum of the opossum Didelphis aurita. It binds non-covalently to myotoxins I (Asp49) and II (Lys49) from Bothrops asper venom and inhibits their myotoxic effect. In this study, an affinity column with immobilized DM64 as bait was used to fish potential target toxins. All ten isolated myotoxins tested were able to effectively bind to the DM64 column. To better access the specificity of the inhibitor, crude venoms from Bothrops (8 species), Crotalus (2 species) and Naja naja atra were submitted to the affinity purification. Venom fractions bound and nonbound to the DM64 column were analyzed by two-dimensional gel electrophoresis and MALDI-TOF/TOF MS. Although venom fractions bound to the column were mainly composed of basic PLA2, a few spots corresponding to acidic PLA2 were also observed. Some unexpected protein spots were also identified: C-type lectins and CRISP may represent putative new targets for DM64, whereas the presence of serine peptidases in the venom bound fraction is likely a consequence of nonspecific binding to the column matrix. The present results contribute to better delineate the inhibitory potential of DM64, providing a framework for the development of more specific antivenom therapies. BIOLOGICAL SIGNIFICANCE: Local tissue damage induced by myotoxic PLA2 remains a serious consequence of snake envenomation, since it is only partially neutralized by traditional antivenom serotherapy. Myotoxin inhibition by highly specific molecules offers great promise in the treatment of snakebites, a health problem largely neglected by governments and pharmaceutical industries. Bioactive compounds such as DM64 can represent a valuable source of scaffolds for drug development in this area. The present study has systematically profiled the binding specificity of DM64 toward a variety of snake venom toxin classes and therefore can lead to a better understanding of the structure-function relationship of this important antivenom protein.

Development of a high-throughput assay for measuring phospholipase A activity using synthetic 1,2-α-eleostearoyl-sn-glycero-3-phosphocholine coated on microtiter plates.

To date, several sensitive methods, based on radiolabeled elements or sterically hindered fluorochrome groups, are usually employed to screen phospholipase A (PLA) activities. With the aim of developing a convenient, specific, sensitive, and continuous new ultraviolet (UV) spectrophotometric assay for PLA, we have synthesized a specific glycerophosphatidylcholine (PC) esterified at the sn-1 and sn-2 positions, with α-eleostearic acid (9Z, 11E, 13E-octadecatrienoic acid) purified from Aleurites fordii seed oil. The conjugated triene present in α-eleostearic acid constitutes an intrinsic chromophore and, consequently, confers the strong UV absorption properties of this free fatty acid as well as of the glycerophospholipids harboring it. This coated PC film cannot be desorbed by the various buffers used during PLA assays. Following the action of PLA at the oil-water interface, α-eleostearic acid is freed and desorbed from the film and then solubilized with ß-cyclodextrin. The UV absorbance of the α-eleostearic acid is considerably enhanced due to the transformation from an adsorbed to a water-soluble state. The PLA activity can be measured continuously by recording the variations with time of the UV absorption spectra. The rate of lipolysis was monitored by measuring the increase of absorption at 272 nm, which was found to be linear with time and proportional to the amount of added PLA. This continuous high-throughput PLA assay could be used to screen new PLA and/or PLA inhibitors present in various biological samples.

Enriquecimento ambiental como estratégia para promover a neurogênese na doença de Alzheimer: possível participação da fosfolipase A2: [revisão] / Environmental enrichment as strategy to promote neurogenesis in Alzheimer disease: possible participation of phospholipase A2: [review]

CONTEXTO: Com a descoberta de que a neurogênese constitutiva persiste no cérebro adulto, surgiu a hipótese na literatura de que a doença de Alzheimer (DA) poderia ser superada, ou pelo menos melhorada, visto que a geração de novos neurônios poderia ajudar a compensar a perda de neurônios na doença. OBJETIVOS: Neste trabalho, foi revisada a literatura sobre a neurogênese endógena no cérebro de sujeitos com DA e modelos animais de DA, os efeitos de atividade cognitiva sobre a neurogênese, e a relação entre a enzima fosfolipase A2 (PLA2) e a neurogênese. MÉTODOS: A base de dados MedLine foi pesquisada utilizando as palavras-chave doença de Alzheimer, atividade cognitiva, fosfolipase A2, neurogênese e neuritogênese. RESULTADOS: A revisão da literatura evidenciou neuroproliferação aumentada no cérebro com DA, no entanto, os novos neurônios falham em se diferenciar em neurônios maduros. Uma estratégia não farmacológica, ambiente enriquecido, aumenta a neurogênese (incluindo amadurecimento neuronal) em animais experimentais. Relação entre PLA2 e neurogênese tem sido demonstrada em modelos experimentais in vitro e in vivo. CONCLUSÃO: Os dados indicam que o enriquecimento ambiental (com estimulações cognitiva e física) poderia ser uma estratégia apropriada para promover a neurogênese endógena na DA e sugerem a participação da PLA2 na neurogênese promovida por estimulação cognitiva.

BACKGROUND: With the discovery that constitutive neurogenesis persists in the adult brain, has emerged the hypothesis in the literature that Alzheimer disease (AD) could be overcome, or at least ameliorated, since the generation of new neurons might help to compensate for the loss of neurons in the disease. OBJECTIVES: In this work the literature on endogenous neurogenesis in the brain of subjects with AD and animal models of AD, the effects of cognitive activity on neurogenesis, and the relationship between the enzyme phospholipase A2 (PLA2) and neurogenesis was reviewed. METHODS: MedLine database was searched using the keywords Alzheimer disease, cognitive activity, phospholipase A2, neurogenesis, and neuritogenesis. RESULTS: The literature review evidenced increased neuroproliferation in AD brain, however, the new neurons fail to differentiate into mature neurons. A non-pharmacological strategy, enriched environment, increases neurogenesis (including neuronal maturation) in experimental animals. Relationship between PLA2 and neurogenesis has been demonstrated in in vitro and in vivo experimental models. DISCUSSION: The data indicate that environmental enrichment (with cognitive and physical stimulations) might be a suitable strategy to promote endogenous neurogenesis in AD, and suggest the participation of PLA2 in the neurogenesis promoted by cognitive stimulation.

Detection of honeybee venom in envenomed tissues by direct MALDI MSI.

A new analytical approach using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) for the study of honeybee venom is shown. In vitro and in vivo models simulating the bee sting have been developed using live honeybees and, as the envenomation sites, pig ears and rat legs; MALDI MSI has been used to map, over time, the diffusion and distribution of three venom allergens (Api m 1, Api m 4, and Api m 6) and two venom toxins (apamine and mast cell degranulating peptide). In conjunction with other classical biochemical techniques and high resolution mass spectrometry (HRMS), structural data have been obtained that contribute to current understanding of honeybee venom composition. Initial data have also been obtained demonstrating the feasibility of mapping the organism's response to the sting. The opportunity to monitor venom diffusion and the organism's response at the same time might open new pathways for in vivo preclinical studies in designing and testing new venom immunotherapy (VIT).

Toxic activities of Brazilian centipede venoms.

Centipedes have a venom gland connected to a pair of forceps, which are used to arrest preys. Human victims bitten by centipedes usually manifest burning pain, paresthesia and edema, which may develop into superficial necrosis. The aim of this work was to characterize and compare toxic activities found in venoms of three species of Brazilian centipedes-Otostigmus pradoi, Cryptops iheringi and Scolopendra viridicornis. By SDS-PAGE (4-20%), important differences were noticed among venoms (between 7 and 205kDa). Few bands showed feeble caseinolytic, fibrinogenolytic and gelatinolytic activities by zymography, but strong hyaluronidase activity was observed in S. viridicornis and O. pradoi venoms. In addition, such activities could be inhibited by o-phenanthroline, indicating that these enzymes are metalloproteinases. All venoms induced nociception, edema and myotoxicity in mice, but only S. viridicornis induced mild hemorrhagic activity. No coagulant activity was detected in centipede venoms. Low phospholipase A(2) activity was observed exclusively in S. viridicornis and O. pradoi venoms, but these venoms had intense direct hemolytic activity on human erythrocytes. Cross-reactivity among venoms was observed using species-specific sera raised in rabbits. Differences were noticed among centipede venoms, but S. viridicornis is indeed the most toxic venom and thereby it could induce a more severe envenomation.

Complementary DNA sequencing and identification of mRNAs from the venomous gland of Agkistrodon piscivorus leucostoma.

To advance our knowledge on the snake venom composition and transcripts expressed in venom gland at the molecular level, we constructed a cDNA library from the venom gland of Agkistrodon piscivorus leucostoma for the generation of expressed sequence tags (ESTs) database. From the randomly sequenced 2112 independent clones, we have obtained ESTs for 1309 (62%) cDNAs, which showed significant deduced amino acid sequence similarity (scores >80) to previously characterized proteins in National Center for Biotechnology Information (NCBI) database. Ribosomal proteins make up 47 clones (2%) and the remaining 756 (36%) cDNAs represent either unknown identity or show BLASTX sequence identity scores of <80 with known GenBank accessions. The most highly expressed gene encoding phospholipase A(2) (PLA(2)) accounting for 35% of A. p. leucostoma venom gland cDNAs was identified and further confirmed by crude venom applied to sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis and protein sequencing. A total of 180 representative genes were obtained from the sequence assemblies and deposited to EST database. Clones showing sequence identity to disintegrins, thrombin-like enzymes, hemorrhagic toxins, fibrinogen clotting inhibitors and plasminogen activators were also identified in our EST database. These data can be used to develop a research program that will help us identify genes encoding proteins that are of medical importance or proteins involved in the mechanisms of the toxin venom.

Attenuated free cholesterol loading-induced apoptosis but preserved phospholipid composition of peritoneal macrophages from mice that do not express group VIA phospholipase A2.

Mouse macrophages undergo ER stress and apoptosis upon free cholesterol loading (FCL). We recently generated iPLA(2)beta-null mice, and here we demonstrate that iPLA(2)beta-null macrophages have reduced sensitivity to FCL-induced apoptosis, although they and wild-type (WT) cells exhibit similar increases in the transcriptional regulator CHOP. iPLA(2)beta-null macrophages are also less sensitive to apoptosis induced by the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin and the scavenger receptor A ligand fucoidan, and restoring iPLA(2)betaexpression with recombinant adenovirus increases apoptosis toward WT levels. WT and iPLA(2)beta-null macrophages incorporate [(3)H]arachidonic acid ([(3)H]AA]) into glycerophosphocholine lipids equally rapidly and exhibit identical zymosan-induced, cPLA(2)alpha-catalyzed [(3)H]AA release. In contrast, although WT macrophages exhibit robust [(3)H]AA release upon FCL, this is attenuated in iPLA(2)beta-null macrophages and increases toward WT levels upon restoring iPLA(2)beta expression. Recent reports indicate that iPLA(2)beta modulates mitochondrial cytochrome c release, and we find that thapsigargin and fucoidan induce mitochondrial phospholipid loss and cytochrome c release into WT macrophage cytosol and that these events are blunted in iPLA(2)beta-null cells. Immunoblotting studies indicate that iPLA(2)beta associates with mitochondria in macrophages subjected to ER stress. AA incorporation into glycerophosphocholine lipids is unimpaired in iPLA(2)beta-null macrophages upon electrospray ionization-tandem mass spectrometry analyses, and their complex lipid composition is similar to WT cells. These findings suggest that iPLA(2)beta participates in ER stress-induced macrophage apoptosis caused by FCL or thapsigargin but that deletion of iPLA(2)beta does not impair macrophage arachidonate incorporation or phospholipid composition.

Butyrate inhibits oral cancer cell proliferation and regulates expression of secretory phospholipase A2-X and COX-2.

Although surgical resection is the first choice for oral cancer, the development of new anti-cancer drugs is of great interest. The effect of the histone deacetylation inhibitor, sodium butyrate (NaBu) on oral cancer cell (OCC) HSC-3 and HSC-4 proliferation in vitro was investigated. The synthesis of rate-limiting enzymes such as sPLA2 (-IIA, -V, -X) and COX-2 was examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, as well as PGE2 by ELISA. NaBu acted in a concentration-dependent manner. Over 3 mM, it inhibited OCC proliferation, due to increased p21 expression and cell cycle arrest in the G2/M-phase. At low concentration (< or = 1 mM), NaBu showed no effects or enhanced cell proliferation. NaBu also regulated COX-2 and sPLA2-X expression, and augmented PGE2 synthesis in OCC. These results indicate that NaBu is a novel candidate agent for the treatment of oral cancer. The treatment efficacy must be investigated in additional experiments considering NaBu concentration and tumor cell heterogeneity.

A new photoprobe for studying biological activities of secreted phospholipases A2.

Ammodytoxin (Atx) is a snake venom phospholipase A2 (sPLA2s) with presynaptic toxicity, anticoagulant activity and the ability to influence cell cycle progression. These multiple physiological activities make this molecule a promising tool for studying processes influenced by the highly homologous mammalian sPLA2s-for example cell proliferation and apoptosis. Secreted PLA2s can act on cells as enzymes or as ligands for cellular receptors. To further characterize the sPLA2-binding molecules in cells we have developed a new method based on AtxC and a biotin-containing cross-linking reagent sulfo-SBED which possesses both an amine-reactive and a photo-reactive site, together with a biotin moiety that enables specific detection and affinity-based concentration. The biological activity of the AtxC derivatized by sulfo-SBED was demonstrated by biotin-tagging of calmodulin and R25, both known AtxC targets, but not of other proteins. In addition, using the new protocol we specifically labelled 14-3-3 proteins, protein disulfide isomerase and two unknown proteins of 45 and 46kDa in the mitochondrial-synaptosomal fraction of porcine cerebral cortex, none of which could be tagged by the previously used methods. The new methodology, which can be used for any sPLA2, constitutes a novel approach to discovering and purifying sPLA2-binding proteins, to studying the topology of their respective complexes and to following sPLA2s in different biological systems.

Role of phospholipase A2 in the induction of drip loss in porcine muscle.

The role of phospholipase A2 in the induction of drip loss from pig muscle has been investigated. In samples from porcine M. longissimus dorsi, total PLA2 activity as well as mRNA and protein levels of the group VIA iPLA2 (iPLA2-VIA) increased during the initial 4 h post-mortem period. Morphological studies of porcine muscle showed that at 4 h post-mortem, gaps had formed between muscle fibers and that the sarcolemma membrane borders appeared blurred. At the same time iPLA2-VIA protein levels were increased inside muscle fibers and at the sarcolemma. iPLA2-VIA mRNA abundance in samples from different breeds of pigs with variations in drip loss revealed no clear correlation between drip loss level and iPLA2-VIA expression. Together, these data indicate that during the post-mortem period, iPLA2-VIA expression and activity is increased at the muscle fiber membranes. PLA2 activity may affect membrane permeability and consequently the progression of drip formation in porcine muscle.

Effect of chronic CB1 cannabinoid receptor antagonism on livers of rats with biliary cirrhosis.

Recent studies have shown that the activated endocannabinoid system participates in the increase in IHR (intrahepatic resistance) in cirrhosis. The increased hepatic production of vasoconstrictive eicosanoids is involved in the effect of endocannabinoids on the hepatic microcirculation in cirrhosis; however, the mechanisms of these effects are still unknown. The aim of the present study was to investigate the effects of chronic CB(1) (cannabinoid 1) receptor blockade in the hepatic microcirculation of CBL (common bile-duct-ligated) cirrhotic rats. After 1 week of treatment with AM251, a specific CB(1) receptor antagonist, IHR, SMA (superior mesenteric artery) blood flow and hepatic production of eicosanoids [TXB(2) (thromboxane B(2)), 6-keto PGF(1alpha) (prostaglandin F(1alpha)) and Cys-LTs (cysteinyl leukotrienes)] were measured. Additionally, the protein levels of hepatic COX (cyclo-oxygenase) isoforms, 5-LOX (5-lipoxygenase), CB(1) receptor, TGF-beta(1) (transforming growth factor beta(1)), cPLA(2) [cytosolic PLA(2) (phospholipase A(2))], sPLA(2) (secreted PLA(2)) and collagen deposition were also measured. In AM251-treated cirrhotic rats, a decrease in portal venous pressure was associated with the decrease in IHR and SMA blood flow. Additionally, the protein levels of hepatic CB(1) receptor, TGF-beta(1), cPLA(2) and hepatic collagen deposition, and the hepatic levels of 5-LOX and COX-2 and the corresponding production of TXB(2) and Cys-LTs in perfusates, were significantly decreased after 1 week of AM251 treatment in cirrhotic rats. Furthermore, acute infusion of AM251 resulted in a decrease in SMA blood flow and an increase in SMA resistance in CBL rats. In conclusion, the chronic effects of AM251 treatment on the intrahepatic microcirculation were, at least partly, mediated by the inhibition of hepatic TGF-beta(1) activity, which was associated with decreased hepatic collagen deposition and the activated PLA(2)/eicosanoid cascade in cirrhotic livers.

LPS exacerbates endothelin-1 induced activation of cytosolic phospholipase A2 and thromboxane A2 production from Kupffer cells of the prefibrotic rat liver.

BACKGROUND/AIMS: Thromboxane A2 (TXA2) has been suggested to play a significant role in the development of portal hypertension in fibrosis, and Kupffer cell (KC) derived TXA2 has been shown to mediate the hyperresponsiveness of the portal circulation to the vasoconstrictive actions of endothelin-1 (ET-1) during endotoxemia. The aim of this study was to determine whether the double stresses of prefibrotic changes and endotoxemia additively activate KC to increase release of TXA2 in response to ET-1, resulting in elevated portal resistance. METHODS: One week Bile duct ligation (BDL) rats and sham-operated controls were subjected to isolated liver perfusions following LPS or saline for 6h. In a separate experiment, KC were isolated from BDL or sham rats and incubated with LPS or saline for 6h before the ET-1 treatment. RESULTS: The double stresses of early fibrosis and LPS resulted in a greater sustained increase in portal pressure in response to ET-1 in BDL rats, and this increase correlated well with the much enhanced release of TXA2 in the perfusate. Media from the cultured KC showed significantly greater TXA2 release in response to ET-1 in BDL group than those in sham group, and LPS exacerbated this effect. Protein levels of cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2, and thromboxane synthase were also significantly elevated in KC from BDL rats. ET-1 produced a marked increase in cPLA2 activation as measured by the phosphorylation of cPLA2 in KC of both BDL and sham groups. LPS greatly exacerbated the activation of cPLA2. CONCLUSIONS: The data suggest that the double stresses additively activate KC with an upregulation of the key enzymes in the TXA2 biosynthesis and release increased amount of TXA2 via the augmented activation of cPLA2 in response to ET-1, which leads to the increased portal resistance and ultimately hepatic microcirculatory dysfunction.

Seeing is believing: real-time cellular activity assay for phospholipase A2.

Quantitative real-time in situ activity assays are necessary for determining the physiological function and regulation of enzymes. A paper in this issue reports the synthesis of a series of new fluorogenic phospholipids that allow fast real-time measurements of cellular activity and head group selectivity of an important family of enzyme, phospholipases.

Differential expression of cyclooxygenase 1 and cyclooxygenase 2 in the bovine oviduct.

The aim of the present study was to investigate the enzymes for the local prostaglandin (PG) biosynthesis present in the bovine oviduct during the estrous cycle to influence early reproductive events. Bovine oviducts were classified into four phases: pre-ovulatory, post-ovulatory, early-to-mid luteal, and late luteal phase, subdivided further into ipsi- or contralateral site and separated into ampulla or isthmus. Oviductal cells were gained by flushing the oviductal regions. Quantitative real-time reverse transcriptase-PCR was performed for the secretory and cytosolic phospholipases A(2) (sPLA(2)IB, cPLA(2)alpha, and cPLA(2)beta) and cyclooxygenases (COX-1 and COX-2) as the first step enzymes of PG synthesis. COX-1 and cPLA(2)beta showed significant highest mRNA expression around and before ovulation compared with the luteal phase respectively. sPLA(2)IB and cPLA(2)alpha mRNA expression was unregulated during the estrous cycle. Regional differences in mRNA content were found for sPLA(2)IB with higher mRNA expression in the ampulla than in the isthmus. Western blot analysis revealed the highest COX-1 protein content in the early-to-mid luteal phase. Immunohistochemistry demonstrated that COX-1 was localized in epithelial and smooth muscle cells, whereas COX-2 was only localized in epithelial cells. COX-2 showed a differential distribution within the epithelial cell layer suggesting a regulation on a cellular level, although the COX-2 mRNA and protein amounts did not vary throughout the estrous cycle. A COX activity assay of oviductal cells revealed that COX activity originated predominantly from COX-1 than from COX-2. Treatment of primary oviductal cells with 10 pg/ml 17beta-estradiol or 10 ng/ml progesterone resulted in a higher expression of COX-2 and cPLA(2)alpha, but not of the other enzymes. The expression pattern of these enzymes suggests that an estrous-cycle dependent and region-specific PG synthesis in the bovine oviduct may be required for a successful reproduction.

PCB 50 stimulates release of arachidonic acid and prostaglandins from late gestation rat amnion fibroblast cells.

Amniotic phospholipase A2 activity contributes to elevated levels of arachidonic acid and prostaglandins observed during labor. Polychlorinated biphenyls (PCBs) activate PLA2 and have been associated with shortened gestation length. To determine if PCBs stimulate amniotic PLA2, cell cultures of rat amnion fibroblasts (RAF) were established from gestation day (gd) 20 rats and labeled with 0.5 micro Ci [3H]-arachidonic acid prior to a 0.5- or 4-h exposure to 0.1% DMSO (solvent control), PCB 50 (1-50 micro M) or TNFalpha (positive control). PCB 50 and TNFalpha induced significant release of [3H]-arachidonic acid from amnion fibroblast cells in time-dependent manners (p<0.001), an effect associated with a significant increase in iPLA2 expression (p<0.05). PCB 50 also stimulated prostaglandin production from RAF cells independent of changes in immunoreactive COX-2. These data suggest that amnion may serve as a target for PCB-induced release of arachidonic acid and uterotonic prostaglandins, with a potential for adverse pregnancy outcomes.

Expression profiling of auxin-treated Arabidopsis roots: toward a molecular analysis of lateral root emergence.

Treating Arabidopsis roots with exogenous auxin results in dramatic changes in cellular processes including de novo induction of lateral roots which later emerge through the overlying cells. Microarray experiments reveal approximately 80 genes that are substantially up-regulated in the root over the first 12 h following auxin treatment. We hypothesize that the observed increase in expression of pectate lyase family genes leads to degradation of the pectin-rich middle lamellae, allowing cells in the parent root to separate cleanly. Differences in the degree of pectin methylation in lateral and parent roots may explain why lateral roots are not degraded themselves.

A continuous fluorescence assay for phospholipase A2 with nontagged lipid.

Human nonpancreatic secreted phospholipase A2 (hnps PLA2) is considered to be an important drug target for antiinflammation therapy. We have established a new fluorescence assay by using 1-anilinonaphthalene-8-sulfonate (ANS) as an interfacial probe for hydrophobic environment detection. The fitted apparent k(cat)/K(m) of hnps PLA2 is 0.0181 +/- 0.0005 RFU/microMs. Tests on known synthesized inhibitor gave IC50 values similar to those from isotope-labeled assay. Because ANS is a commonly used probe for hydrophobic environment detection that needs no modification in the current assay, this strategy may be widely applicable for interfacial catalytic reactions.

Expression of phospholipase A2 group IVA (PLA2G4A) is upregulated by human chorionic gonadotropin in bovine granulosa cells of ovulatory follicles.

Prostaglandins are required for the ovulatory process, and their biosynthesis depends on the initial release of arachidonic acid from membrane phospholipids. We hypothesized that phospholipase A2 group IVA (PLA2G4A) expression is upregulated in granulosa cells (GC) at ovulation. We have characterized bovine PLA2G4A cDNA, and investigated its spatiotemporal regulation at the mRNA and protein levels in hCG-induced ovulatory follicles and in vitro, using forskolin-stimulated GC. Regulation of PLA2G4A mRNA expression was studied in GC obtained from bovine follicles collected at different developmental stages: small follicles (2-4 mm), dominant follicles at Day 5 (D5) of the estrous cycle, ovulatory follicles 24 h following injection of hCG, and corpus luteum at D5. PLA2G4A mRNA increased by 14-fold in GC of hCG-stimulated versus dominant follicles (P < 0.0001). Follicular walls obtained from ovulatory follicles recovered at 0, 6, 12, 18, and 24 h post-hCG injection showed an initial 16-fold increase in PLA2G4A transcript at 12 h that reached a 45-fold increase at 24 h, as compared to 0 h (P < 0.0001). Immunoblots of GC extracts showed an initial induction of the PLA2G4A protein at 18 h post-hCG, reaching a maximum at 24 h. Immunohistochemistry observations showed that PLA2G4A signal was mainly observed in mural GC compared to antral GC in hCG-stimulated follicles. Stimulation of cultured bovine GC with 10 microM of forskolin caused an increase in PLA2G4A mRNA and protein. Ovulation is associated with an LH/hCG-dependent induction of PLA2G4A in GC via the adenylyl cyclase/cAMP pathway.

Early diet-induced non-alcoholic steatohepatitis in APOE2 knock-in mice and its prevention by fibrates.

BACKGROUND/AIMS: The molecular mechanisms leading to Non-Alcoholic Steatohepatitis (NASH) are not fully understood. In mice, NASH can be inhibited by fenofibrate, a synthetic agonist for the nuclear receptor peroxisome proliferator activated receptor alpha, which regulates hepatic triglyceride metabolism. This study aimed to elucidate the relation between steatosis and inflammation in NASH in a human-like hyperlipidemic mouse model. METHODS: Liver phenotype and gene expression were assessed in APOE2 knock-in mice that were fed a western-type high fat diet with or without co-administration of fenofibrate. RESULTS: In response to a western diet, APOE2 knock-in mice developed NASH characterized by steatosis and inflammation. Strikingly, macrophage accumulation in the liver preceded the steatosis during progression of the disease. This phenotype was in line with gene expression patterns, which showed regulation of two major groups of genes, i.e. inflammatory and lipid genes. Fenofibrate treatment decreased hepatic macrophage accumulation and abolished steatosis. Moreover, a marked reduction in the expression of inflammatory genes occurred immediately after fenofibrate treatment. CONCLUSIONS: These data indicate that inflammation might play an instrumental role during the development of NASH in this mouse model. Inhibition of NASH by fenofibrate may be due, at least in part, to its inhibitory effect on pro-inflammatory genes.

Crystal structure analysis of phosphatidylcholine-GM2-activator product complexes: evidence for hydrolase activity.

GM2-activator protein (GM2AP) is a lysosomal lipid transfer protein with important biological roles in ganglioside catabolism, phospholipid metabolism, and T-cell activation. Previous studies of crystal structures of GM2AP complexed with the physiological ligand GM2 and platelet activating factor (PAF) have shown binding at two specific locations within the spacious apolar pocket and an ordering effect of endogenous resident lipids. To investigate the structural basis of phospholipid binding further, GM2AP was cocrystallized with phosphatidylcholine (PC), known to interact with GM2AP. Analysis of three crystal forms revealed binding of single chain lipids and fatty acids only and surprisingly not intact PC. The regions of best defined electron density are consistent with the presence of lyso-PC and oleic acid, which constitute deacylation products of PC. Their acyl tails are in stacking contact with shorter, less well-defined stretches of electron density that may represent resident fatty acids. The GM2AP associated hydrolytic activity that generates lyso-PC was further confirmed by mass spectrometry and enzymatic assays. In addition, we report the structures of (i) mutant Y137S, assessing the role of Tyr137 in lipid transfer via the hydrophobic cleft, and (ii) apo-mouse GM2AP, revealing a hydrophobic pocket with a constricted opening. Our structural results provide new insights into the biological functions of GM2AP. The combined effect of hydrolytic and lipid transfer properties has profound implications in cellular signaling.