Phosphatidylinositol-specific Phospholipase C Research Articles

Effect of membrane-bound complement regulatory proteins on tumor cell sensitivity to complement-dependent cytolysis triggered by heterologous expression of the α-gal xenoantigen.

Engineering malignant cells to express a heterologous α-gal antigen can induce heterograft hyperacute rejection, resulting in complement-dependent cytolysis (CDC) of tumor cells, which has been considered as a novel strategy for antitumor therapy. A549 cells engineered to express Galα1-3Galβ1-4GlcNAc-R (α-gal) epitope exhibited strong resistance to CDC treated by normal human serum (NHS) in a previous study. We hypothesized that the expression of membrane-bound complement regulatory proteins (mCRPs) decay accelerating factor (CD55) and protectin (CD59) influenced the efficacy of the α-gal/NHS-mediated antitumor effect to tumor cells in vitro. The present study confirmed that A549 cells expressed high levels of CD55 and CD59, whereas Lovo cells expressed relatively low levels of these proteins. A549 and Lovo cells transfected with plasmids containing or lacking the α-gal epitope were evaluated for their susceptibility to CDC by NHS and detected using a trypan blue exclusion assay. α-gal-expressing Lovo (Lovo-GT) cells were almost completely killed by α-gal-mediated CDC following incubation with 50% NHS, whereas no cytolysis was observed in α-gal expressing A549 (A549-GT) cells. Abrogating CD55 and CD59 function from A549-GT cells by various concentrations of phosphatidylinositol-specific phospholipase C (PI-PLC) or blocking antibodies increased the susceptibility of cells to CDC, and the survival rate decreased significantly comparing to the controls (P<0.05). The findings of the present study indicated that using the α-gal/NHS system to eliminate tumor cells via inducing the complement cascade reaction might represent a feasible approach for the treatment of cancer. However, high levels of mCRP expression may limit the efficacy of this approach. Therefore, an improved efficacy of cancer cell killing may be achieved by combining strategies of heterologous α-gal expression and mCRP downregulation.

A novel assay to measure scrambling of natural phospholipids in reconstituted proteoliposomes

Scramblases transport phospholipids between the leaflets of a membrane bilayer in an ATP‐independent and bidirectional way to facilitate membrane growth and maintain lipid homeostasis. Members of two protein families, Class A G protein‐coupled receptors and the TMEM16 family of ion channels, have been identified to act as scramblases. However, the mechanisms by which they translocate phospholipids are still poorly understood. Synthetic glycerophospholipid analogs carrying fluorescent nitrobenzoxadiazole (NBD) groups, such as NBD‐phosphatidylcholine (NBD‐PC), have proven useful to identify and characterize phospholipid scramblases in membrane preparations and reconstituted proteoliposomes.We now have established a novel biochemical assay to measure the activity of scramblase proteins using radiolabeled natural phosphatidylinositol ([3H]PI) as probe. As sources of scramblase activity we used crude extracts from yeast containing ER membrane proteins, and also purified bovine opsin. We reconstituted symmetric large unilamellar liposomes containing trace amounts of [3H]PI. Addition of PI‐specific phospholipase C (PI‐PLC) resulted in hydrolysis of 46–52% of [3H]PI, i.e. the portion present in the outer leaflet of the bilayer. The time to reach maximal [3H]PI hydrolysis depended on the amount of PI‐PLC used. In contrast, PI‐PLC treatment of [3H]PI‐containing liposomes reconstituted with yeast extract containing scramblase activity or purified opsin resulted in protein‐dependent hydrolysis of 60–80% of [3H]PI, indicating that the [3H]PI located in the inner leaflet of the proteoliposomes became accessible to cleavage by PI‐PLC during the course of the experiment.To allow direct comparison of opsin's ability to scramble [3H]PI and NBD‐PC, we then prepared opsin proteoliposomes containing both [3H]PI and NBD‐PC and measured scrambling using PI‐PLC and dithionite, respectively, as probes. The results showed that opsin readily scrambled both NBD‐PC and [3H]PI, albeit with different characteristics. The results of these experiments, as well as experiments that directly compare opsin‐mediated scrambling of NBD‐PI and [3H]PI will be presented.apoproteinSupport or Funding InformationThis research is supported by the Velux StiftungThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

F202. ABNORMAL REMODELING PROCESSING IN NEURAL GPI-APS SECRETORY PATHWAY IN SCHIZOPHRENIA

BackgroundAbnormalities in post translational modifications (PTMs) such as glycosylation have become targets of schizophrenia (SCZ) research and are implicated in the neuropathophysiology of this illness. Glycosylphosphatidylinositol (GPI) attachment to proteins and glycoproteins events; proteins essential to cellular function, including neurotransmitter rreceptors, adhesion molecules, and enzymes, are all modified by GPI. Biosynthesis of GPI -APs occurs in the endoplasmic reticulum (ER). Once GPI-APs are synthesized, they are transported from the ER to the cell surface through the Golgi apparatus. Inositol deacylation of the GPI-APs by is common PTMs. The GPI-anchored proteins (GPI-APs) play an essential role in many biological PGAP1 is required for efficient export from the ER and acts a molecular mechanism for quality control of GPI-APs. The p24 complex binds specifically to GPI-APs and plays a role in their selective trafficking by sensing the status of the GPI anchor and to promote efficient ER exit of remodeled GPI-APs. In this study, we identified abnormalities of proteins associated with the ER exit of GPI-APs in SCZ. To address mechanisms of GPI-APs ER exit, we measured expression of proteins of the GPI-APs ER exit and targeting pathway. We also measured expression of GPI-APs which have been previously implicated in SCZ, including GPC1, NCAM, MDGA2 and EPHA1.MethodsWe assessed the total expression and subcellular localization of proteins involved in ER export processing of GPI-APs from the DLPFC of 15 matched pairs of SCZ and comparison subjects. Specifically, we measured levels of PGAP1 and Tmp21 (p24). Additionally, we performed a Triton X-114 phase separation to distinguish between membrane-associated and cytosolic forms of protein substrates. We confirmed the sensitivity of each target GPI-AP to phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme that specifically cleaves GPI from GPI-APs.ResultsWe found a significant decrease in p24 in total tissue homogenates and PGAP1 in an ER enriched fraction from subjects with SCZ. We also identified diminished sensitivity of the GPI-APs, GPC1 and NCAM, to PI-PLC treatment in SCZ.DiscussionDecreased PGAP1 in an ER enriched fraction in consistent with reduced inositol deacylation and potential dysfunction as the gatekeeper of GPI-AP ER exit in SCZ. This also suggests that the GPI-anchor is not correctly modified. Decreased p24 levels suggest downregulation of transport between the Golgi and the ER in SCZ. Additionally, we observed unchanged total level of GPI-APs in Triton X-114 phase separation, but a significant decrease in the amount of NCAM and GPC1 that was sensitive to PI-PLC in SCZ. This finding may be consistent with abnormal GPI modification of these two candidate proteins. Together, these findings suggest dysregulation of the GPI-APs remodeling system in SCZ, which may impact the structure of the GPI-anchor for SCZ-relevant proteins like NCAM and GPC1.

The Phosphoinositide Signal Transduction Pathway in the Pathogenesis of Alzheimer's Disease.

During aging and in age-associated disorders, such as Alzheimer's Disease (AD), learning abilities decline. Probably, disturbances in signal transduction in brain cells underlie the cognitive decline. The phosphorylation/dephosphorylation imbalance occurring in degenerating neurons was recently related to abnormal activity of one or more signal transduction pathways. AD is known to be associated with altered neuronal Ca2+ homeostasis, as Ca2+ accumulates in affected neurons leading to functional impairment. It is becoming more and more evident the involvement of signal transduction pathways acting upon Ca2+ metabolism and phosphorylation regulation of proteins. A growing interest raised around the role of signal transduction systems in a number of human diseases including neurodegenerative diseases, with special regard to the systems related to the phosphoinositide (PI) pathway and AD. The PI signal transduction pathway plays a crucial role, being involved in a variety of cell functions, such as hormone secretion, neurotransmitter signal transduction, cell growth, membrane trafficking, ion channel activity, cytoskeleton regulation, cell cycle control, apoptosis, cell and tissue polarity, and contributes to regulate the Ca2+ levels in the nervous tissue. A number of observations indicated that PI-specific phospholipase C (PLC) enzymes might be involved in the alteration of neurotransmission. To understand the role and the timing of action of the signalling pathways recruited during the brain morphology changes during the AD progression might help to elucidate the aetiopathogenesis of the disease, paving the way to prognosis refinement and/or novel molecular therapeutic strategies.

Ceratocystis cacaofunesta genome analysis reveals a large expansion of extracellular phosphatidylinositol-specific phospholipase-C genes (PI-PLC)

BackgroundThe Ceratocystis genus harbors a large number of phytopathogenic fungi that cause xylem parenchyma degradation and vascular destruction on a broad range of economically important plants. Ceratocystis cacaofunesta is a necrotrophic fungus responsible for lethal wilt disease in cacao. The aim of this work is to analyze the genome of C. cacaofunesta through a comparative approach with genomes of other Sordariomycetes in order to better understand the molecular basis of pathogenicity in the Ceratocystis genus.ResultsWe present an analysis of the C. cacaofunesta genome focusing on secreted proteins that might constitute pathogenicity factors. Comparative genome analyses among five Ceratocystidaceae species and 23 other Sordariomycetes fungi showed a strong reduction in gene content of the Ceratocystis genus. However, some gene families displayed a remarkable expansion, in particular, the Phosphatidylinositol specific phospholipases-C (PI-PLC) family. Also, evolutionary rate calculations suggest that the evolution process of this family was guided by positive selection. Interestingly, among the 82 PI-PLCs genes identified in the C. cacaofunesta genome, 70 genes encoding extracellular PI-PLCs are grouped in eight small scaffolds surrounded by transposon fragments and scars that could be involved in the rapid evolution of the PI-PLC family. Experimental secretome using LC–MS/MS validated 24% (86 proteins) of the total predicted secretome (342 proteins), including four PI-PLCs and other important pathogenicity factors.ConclusionAnalysis of the Ceratocystis cacaofunesta genome provides evidence that PI-PLCs may play a role in pathogenicity. Subsequent functional studies will be aimed at evaluating this hypothesis. The observed genetic arsenals, together with the analysis of the PI-PLC family shown in this work, reveal significant differences in the Ceratocystis genome compared to the classical vascular fungi, Verticillium and Fusarium. Altogether, our analyses provide new insights into the evolution and the molecular basis of plant pathogenicity.

CD55 and CD59 expression protects HER2-overexpressing breast cancer cells from trastuzumab-induced complement-dependent cytotoxicity

A large proportion (40–60%) of patients with human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer do not benefit from trastuzumab treatment, potentially due to the lack of complement-dependent cytotoxicity (CDC) activation. In the present study, the effect of complement decay-accelerating factor (CD55) and CD59 glycoprotein precursor (CD59) expression on trastuzumab-induced CDC in HER2-positive breast cancer cell lines was investigated. The CD55 and CD59-overexpressing and HER2-positive cell lines SK-BR-3 and BT474 were selected for subsequent experiments. Blocking CD55 and CD59 function using targeting monoclonal antibodies significantly enhanced the cell lysis of SK-BR-3 and BT474 cells following treatment with trastuzumab. In addition, following treatment with 0.1 U/ml phosphatidylinositol-specific phospholipase C (PI-PLC) for 1 h, CD55 and CD59 surface expression was significantly decreased, and the cell lysis rate was further enhanced. Treatment of SK-BR-3 cells with short hairpin RNA (shRNA) targeting CD55 and CD59 downregulated CD55 and CD59 expression at the mRNA and protein levels, and resulted in significantly enhanced trastuzumab-induced CDC-dependent lysis. The data from the present study suggested that CD55 and CD59 serve roles in blocking trastuzumab-induced CDC, therefore strategies targeting CD55 and CD59 may overcome breast cancer cell resistance to trastuzumab. The results from the present study may provide a basis for developing suitable, personalized treatment strategies to improve the clinical efficacy of trastuzumab for patients with HER2-positive breast cancer.

PLC-Mediated Signaling Pathway in Pollen Tubes Regulates the Gametophytic Self-incompatibility of Pyrus Species.

Among the Rosaceae species, the gametophytic self-incompatibility (GSI) is controlled by a single multi-allelic S locus, which is composed of the pistil-S and pollen-S genes. The pistil-S gene encodes a polymorphic ribonuclease (S-RNase), which is essential for identifying self-pollen. However, the S-RNase system has not been fully characterized. In this study, the self-S-RNase inhibited the Ca2+-permeable channel activity at pollen tube apices and the selectively decreased phospholipase C (PLC) activity in the plasma membrane of Pyrus pyrifolia pollen tubes. Self-S-RNase decreased the Ca2+ influx through a PLC-mediated signaling pathway. Phosphatidylinositol-specific PLC has a 26-amino acid insertion in pollen tubes of the ‘Jinzhuili’ cultivar, which is a spontaneous self-compatible mutant of the ‘Yali’ cultivar. ‘Yali’ plants exhibit a typical S-RNase-based GSI. Upon self-pollination, PLC gene expression is significantly higher in ‘Jinzhuili’ pollen tubes than that in ‘Yali’ pollen tubes. Moreover, the PLC in pollen tubes can only interact with one of the two types of S-RNase from the style. In the Pyrus x bretschneideri Rehd, the PLC directly interacted with the S7-RNase in the pollen tube, but not with the S34-RNase. Collectively, our results reveal that the effects of S-RNase on PLC activity are required for S-specific pollen rejection, and that PLC-IP3 participates in the self-incompatibility reaction of Pyrus species.

Genome-wide identification and characterization of phospholipase C gene family in cotton (Gossypium spp.).

Phospholipase C (PLC) are important regulatory enzymes involved in several lipid and Ca2+-dependent signaling pathways. Previous studies have elucidated the versatile roles of PLC genes in growth, development and stress responses of many plants, however, the systematic analyses of PLC genes in the important fiber-producing plant, cotton, are still deficient. In this study, through genome-wide survey, we identified twelve phosphatidylinositol-specific PLC (PI-PLC) and nine non-specific PLC (NPC) genes in the allotetraploid upland cotton Gossypium hirsutum and nine PI-PLC and six NPC genes in two diploid cotton G. arboretum and G.raimondii, respectively. The PI-PLC and NPC genes of G. hirsutum showed close phylogenetic relationship with their homologous genes in the diploid cottons and Arabidopsis. Segmental and tandem duplication contributed greatly to the formation of the gene family. Expression profiling indicated that few of the PLC genes are constitutely expressed, whereas most of the PLC genes are preferentially expressed in specific tissues and abiotic stress conditions. Promoter analyses further implied that the expression of these PLC genes might be regulated by MYB transcription factors and different phytohormones. These results not only suggest an important role of phospholipase C members in cotton plant development and abiotic stress response but also provide good candidate targets for future molecular breeding of superior cotton cultivars.

Development of a highly efficient oil degumming process using a novel phosphatidylinositol-specific phospholipase C enzyme.

Enzymatic degumming using phospholipase C (PLC) enzymes may be used in environmentally friendly processes with improved oil recovery yields. In this work, phosphatidylinositol-specific phospholipase C (PIPLC) candidates obtained from an in silico analysis were evaluated for oil degumming. A PIPLC from Lysinibacillus sphaericus was shown to efficiently remove phosphatidylinositol from crude oil, and when combined with a second phosphatidylcholine and phosphatidylethanolamine-specific phospholipase C, the three major phospholipids were completely hydrolyzed, providing an extra yield of oil greater than 2.1%, compared to standard methods. A remarkably efficient fed-batch Escherichia coli fermentation process producing ∼14g/L of the recombinant PIPLC enzyme was developed, which may facilitate the adoption of this cost-effective oil-refining process.

Prevalence, Virulence Potential, and Antibiotic Susceptibility Profile of Listeria monocytogenes Isolated From Bovine Raw Milk Samples Obtained From Rajasthan, India.

Listeriosis is a serious foodborne disease of a global concern, and can effectively be controlled by a continuous surveillance of the virulent and multidrug-resistant strains of Listeria monocytogenes. This study was planned to investigate prevalence of L. monocytogenes in bovine raw milk samples. A total of 457 raw milk samples collected from 15 major cities in Rajasthan, India, were analyzed for the presence of L. monocytogenes by using standard microbiological and molecular methods. Five of the 457 samples screen tested positive for L. monocytogenes. Multiplex serotyping showed that 3/5 strains belonged to serotype 4b followed by one strain each to 1/2a and to 1/2c. Further virulence potential assessment indicated that all strains possessed inlA and inlC internalins, and, in addition, two strains also possessed the gene for inlB. All strains were positive for Listeriolysin O (LLO) and showed phosphatidylinositol-specific phospholipase C (PI-PLC) activity on an in vitro agar medium with variations in production levels among the strains. A good correlation between the in vitro pathogenicity test and the chick embryo test was observed, as the strains showing higher LLO and PI-PLC activity were found to be lethal to fertilized chick embryos. All strains were resistant to the majority of antibiotics and were designated as multidrug-resistant strains. However, these strains were susceptible to 9 of the 22 tested antibiotics. The maximum zone of inhibition (mm) and acceptable minimum inhibitory concentration were observed with azithromycin, and thus it could be the first choice of a treatment. Overall, the presence of multidrug-resistant L. monocytogenes strains in the raw milk of Rajasthan region is an indicator of public health hazard and highlighting the need of consumer awareness in place and implementation of stricter food safety regulations at all levels of milk production.

Isoflurane neurotoxicity involves activation of hypoxia inducible factor-1α via intracellular calcium in neonatal rodents

ObjectivePreviously, we found that the inhaled anesthetic isoflurane up-regulated the transcriptional factor hypoxia inducible factor (HIF)-1α protein levels during induction of neurodegeneration in the brain of neonatal rats. Here, we investigated the role of HIF-1α and the underlying signaling pathway in the neurodegenration induced by isoflurane in rodent developing brain. MethodsPrimary hippocampal neurons were exposed to isoflurane (0.4mM) for 12h. Neuron injury was analyzed by 3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyl-tetra-zolium bromide (MTT) test and quantification of lactate dehydrogenaserelease. HIF-1α gene expression and transcriptional activity, cleaved caspase-3 and phosphoinositide phospholipase C (PLC)-γ gene expression were quantified. Cytosolic calcium concentration was measured by calcium imagining. The role of HIF-1α on juvenile learning and memory impairment induced by isoflurane was studied by fear conditioning and extinction test and Morris water maze test. ResultsIsoflurane induced HIF-1α gene expression and transcriptional activity in vitro. Furthermore, pharmacological inhibition of HIF-1α or knockdown of HIF-1α by shRNA counteracted the neurotoxicity of isoflurane. Ca2+ signaling pathways involving PLC-γ activation are required for isoflurane-evoked HIF-1α accumulation. Finally, partial deficiency of HIF-1α attenuated isoflurane-induced neurodegeneration in developing brain, and alleviated juvenile learning and memory impairment induced by isoflurane. ConclusionHIF-1α activation via cytosolic Ca2+ signaling pathway play a role in the mechanism of isoflurane-induced neurodegeneration in neonatal rodents, suggesting HIF-1α as a potential therapeutic target for the prevention of the deleterious effects of prolonged exposures to anesthetics.

In silico identification and characterization of a hypothetical protein of Mycobacterium tuberculosis EAI5 as a potential virulent factor.

Tuberculosis, a life threatening disease caused by different strains of Mycobacterium tuberculosis is creating an alarming condition due to the emergence of increasing multi drug resistance (MDR) trait. In this study, in silico approach was used for the identification of a conserved novel virulent factor in Mycobacterium tuberculosis EAI5 (Accession no.CP006578) which can also act as potential therapeutic target. Systematic comparative search of genes that are common to strain EAI5 and other human pathogenic strains of M. tuberculosis enlisted 408 genes. These were absent in the non-pathogenic Mycobacterium smegmatis MC2155 and in the human genome. Among those genes, only the protein coding hypothetical genes (97 out of 408) and their corresponding products were selected for further exploration. Of these, 11 proteins were found to have notable conserved domains, of which one hypothetical protein (NCBI Acc No. AGQ35418.1) was selected for further in silico exploration which was found to have two functional domains, one having phosphatidylinositol specific phospholipase C (PI-PLC) activity while the other short domain with weak lectin binding activity. As PI-PLC contributes virulence property in some pathogenic bacteria with a broad range of activities, different bioinformatic tools were used to explore its physicochemical and other important properties which indicated its secretary nature. This PI-PLC was previously not reported as drug/vaccine target to the best of our knowledge. Its predicted 3D structure can be explored for development of inhibitor for novel therapeutic strategies against MDR-TB.

Comparison of Phosphoinositide-Specific Phospholipase C Expression Panels of Human Osteoblasts Versus MG-63 and Saos Osteoblast-Like Cells

Background: A large number of phospholipase C (PLC) enzymes, both mRNA transcripts and proteins, have been detected in osteoblasts, corroborating the importance of calcium regulation in bone tissue. MG-63 and SaOS-2 human osteosarcoma cell lines are actually considered osteoblast-like cells, and are therefore widely used as experimental models for osteoblasts. Objectives: Our aim was to verify whether MG-63 or SaOS-2 cells might also represent appropriate experimental osteoblast models for signal transduction studies, with special regard to the phosphoinositide (PI) pathway. We analyzed the expression and the subcellular distribution of enzymes related to calcium signal transduction (the PI-specific PLC family), which are known to possess high cell/tissue specificity. Materials and Methods: The expression of PLC genes was analyzed by performing RT-PCR experiments. The presence of PLC enzymes and their subcellular distribution within the cells was analyzed with immunofluorescence experiments. Conclusions: MG-63 and SaOS-2 osteosarcoma cell lines might not represent appropriate experimental models for studies that aim to analyze signal transduction in osteoblasts.

Phosphatidic Acid-Mediated Signaling Regulates Microneme Secretion in Toxoplasma.

The obligate intracellular lifestyle of apicomplexan parasites necessitates an invasive phase underpinned by timely and spatially controlled secretion of apical organelles termed micronemes. In Toxoplasma gondii, extracellular potassium levels and other stimuli trigger a signaling cascade culminating in phosphoinositide-phospholipase C (PLC) activation, which generates the second messengers diacylglycerol (DAG) and IP3 and ultimately results in microneme secretion. Here we show that a delicate balance between DAG and its downstream product, phosphatidic acid (PA), is essential for controlling microneme release. Governing this balance is the apicomplexan-specific DAG-kinase-1, which interconverts PA and DAG, and whose depletion impairs egress and causes parasite death. Additionally, we identify an acylated pleckstrin-homology (PH) domain-containing protein (APH) on the microneme surface that senses PA during microneme secretion and is necessary for microneme exocytosis. As APH is conserved in Apicomplexa, these findings highlight a potentially widely used mechanism in which key lipid mediators regulate microneme exocytosis.

U-73122 reduces the cell growth in cultured MG-63 ostesarcoma cell line involving Phosphoinositide-specific Phospholipases C.

The definition of the number and nature of the signal transduction pathways involved in the pathogenesis and the identification of the molecules promoting metastasis spread might improve the knowledge of the natural history of osteosarcoma, also allowing refine the prognosis and opening the way to novel therapeutic strategies. Phosphatydil inositol (4,5) bisphosphate (PIP2), belonging to the Phosphoinositide (PI) signal transduction pathway, was related to the regulation of ezrin, an ezrin–radixin–moesin protein involved in metastatic osteosarcoma spread. The levels of PIP2 are regulated by means of the PI-specific Phospholipase C (PLC) enzymes. Recent literature data suggested that in osteosarcoma the panel of expression of PLC isoforms varies in a complex and unclear manner and is related to ezrin, probably networking with Ras GTPases, such as RhoA and Rac1. We analyzed the expression and the subcellular localization of PLC enzymes in cultured human osteosarcoma MG-63 cells, commonly used as an experimental model for human osteoblasts, using U-73122 PLC inhibitor, U-73343 inactive analogue, and by silencing ezrin. The treatment with U-73122 significantly reduces the number of MG-63 viable cells and contemporarily modifies the expression and the subcellular localization of selected PLC isoforms. U-73122 reduces the cell growth in cultured MG-63 ostesarcoma cell line involving PI-specific Phospholipases C.

The Role of Phosphoinositide Phospholipase C in Expression Regulation of DREB2

The Role of Phosphoinositide Phospholipase C in Expression Regulation of DREB2

The sperm phospholipase C-ζ and Ca2+ signalling at fertilization in mammals.

A series of intracellular oscillations in the free cytosolic Ca(2+) concentration is responsible for activating mammalian eggs at fertilization, thus initiating embryo development. It has been proposed that the sperm causes these Ca(2+) oscillations after membrane fusion by delivering a soluble protein into the egg cytoplasm. We previously identified sperm-specific phospholipase C (PLC)-ζ as a protein that can trigger the same pattern of Ca(2+) oscillations in eggs seen at fertilization. PLCζ appears to be the elusive sperm factor mediating egg activation in mammals. It has potential therapeutic use in infertility treatments to improve the rate of egg activation and early embryo development after intra-cytoplasmic sperm injection. A stable form of recombinant human PLCζ could be a prototype for use in such invitro fertilization (IVF) treatments. We do not yet understand exactly how PLCζ causes inositol 1,4,5-trisphosphate (InsP3) production in eggs. Sperm PLCζ is distinct among mammalian PI-specific PLCs in that it is far more potent in triggering Ca(2+) oscillations in eggs than other PLCs, but it lacks a PH domain that would otherwise be considered essential for binding to the phosphatidylinositol 4,5-bisphosphate (PIP2) substrate. PLCζ is also unusual in that it does not appear to interact with or hydrolyse plasma membrane PIP2. We consider how other regions of PLCζ may mediate its binding to PIP2 in eggs and how interaction of PLCζ with egg-specific factors could enable the hydrolysis of internal sources of PIP2.

Biochemical characterization of the tomato phosphatidylinositol-specific phospholipase C (PI-PLC) family and its role in plant immunity

Plants possess effective mechanisms to quickly respond to biotic and abiotic stresses. The rapid activation of phosphatidylinositol-specific phospholipase C (PLC) enzymes occurs early after the stimulation of plant immune-receptors. Genomes of different plant species encode multiple PLC homologs belonging to one class, PLCζ. Here we determined whether all tomato homologs encode active enzymes and whether they can generate signals that are distinct from one another. We searched the recently completed tomato (Solanum lycopersicum) genome sequence and identified a total of seven PLCs. Recombinant proteins were produced for all tomato PLCs, except for SlPLC7. The purified proteins showed typical PLC activity, as different PLC substrates were hydrolysed to produce diacylglycerol. We studied SlPLC2, SlPLC4 and SlPLC5 enzymes in more detail and observed distinct requirements for Ca2+ ions and pH, for both their optimum activity and substrate preference. This indicates that each enzyme could be differentially and specifically regulated in vivo, leading to the generation of PLC homolog-specific signals in response to different stimuli. PLC overexpression and specific inhibition of PLC activity revealed that PLC is required for both specific effector- and more general “pattern”-triggered immunity. For the latter, we found that both the flagellin-triggered response and the internalization of the corresponding receptor, Flagellin Sensing 2 (FLS2) of Arabidopsis thaliana, are suppressed by inhibition of PLC activity. Altogether, our data support an important role for PLC enzymes in plant defence signalling downstream of immune receptors.This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

Inhibitory effect of fermented Arctium lappa fruit extract on the IgE-mediated allergic response in RBL‑2H3 cells.

Arctium lappa fruit has been used in traditional medicine, and it is known to exert beneficial effects, such as antioxidant, anti-inflammatory and anticancer effects. However, the effects of the Arctium lappa fruit on the allergic response remain unknown. In this study, we evaluated the anti-allergic effects of Arctium lappa fruit extract (AFE) and its fermented form (F-AFE) using immunoglobulin E (IgE)-activated RBL‑2H3 cells. To investigate the anti-allergic effects of AFE or F-AFE, we examined the release of β-hexosaminidase, a key biomarker of degranulation during an allergic reaction, and the production of pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) in the cells treated with or without the above-mentioned extracts. AFE weakly inhibited the release of β-hexosaminidase, whereas F-AFE significantly suppressed the release of β-hexosaminidase in a dose-dependent manner. Consistently, F-AFE suppressed the production of TNF-α and PGE2 in a dose-dependent manner. F-AFE exerted an inhibitory effect on the production of β-hexosaminidase, TNF-α and PGE2 with an IC50 value of 30.73, 46.96 and 36.27 µg/ml, respectively. Furthermore, F-AFE inhibited the phosphorylation of Lyn, Fyn and Syk, which are involved in the FcεRI signaling pathway, that of phosphoinositide phospholipase C (PLC)γ1/2 and protein kinase C (PKC)δ, which are associated with the degranulation process, as well as that of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase 1/2 (JNK), p38 and Akt, which are associated with cytokine expression. In the late phase, F-AFE partially suppressed the phosphorylation of cytosolic phospholipase A2 (cPLA2), but not the expression of cyclooxygenase (COX)-2. To compare and identify the major components of the two extracts, we used high-performance liquid chromatography. The levels of arctigenin, one of the major compounds, were elevated 6-fold in F-AFE compared with AFE, whereas the levels of arctiin, an arctigenin glycoside, were decreased in F-AFE by approximately 57.40%. These results suggest that arctigenin plays an important role in the anti-allergic effects of F-AFE. Taken together, F-AFE containing anti-allergic phytochemicals, including arctigenin, inhibited the activation of the FcεRI receptor induced by the antigen‑IgE complex. Such effects may provide further information for the development of a phytomedicine for allergic diseases.

Intracellular postsynaptic cannabinoid receptors link thyrotropin-releasing hormone receptors to TRPC-like channels in thalamic paraventricular nucleus neurons

In rat thalamic paraventricular nucleus of thalamus (PVT) neurons, activation of thyrotropin-releasing hormone (TRH) receptors enhances excitability via concurrent decrease in G protein-coupled inwardly-rectifying potassium (GIRK)-like and activation of transient receptor potential cation (TRPC)4/5-like cationic conductances. An exploration of intracellular signaling pathways revealed the TRH-induced current to be insensitive to phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitors, but reduced by D609, an inhibitor of phosphatidylcholine-specific PLC (PC-PLC). A corresponding change in the I–V relationship implied suppression of the cationic component of the TRH-induced current. Diacylglycerol (DAG) is a product of the hydrolysis of PC. Studies focused on the isolated cationic component of the TRH-induced response revealed a reduction by RHC80267, an inhibitor of DAG lipase, the enzyme involved in the hydrolysis of DAG to the endocannabinoid 2-arachidonoylglycerol (2-AG). Further investigation revealed enhancement of the cationic component in the presence of either JZL184 or WWL70, inhibitors of enzymes involved in the hydrolysis of 2-AG. A decrease in the TRH-induced response was noted in the presence of rimonabant or SR144528, membrane permeable CB1 and CB2 receptor antagonists, respectively. A decrease in the TRH-induced current by intracellular, but not by bath application of the membrane impermeable peptide hemopressin, selective for CB1 receptors, suggests a postsynaptic intracellular localization of these receptors. The TRH-induced current was increased in the presence of arachidonyl-2′-chloroethylamide (ACEA) or JWH133, CB1 and CB2 receptor agonists, respectively. The PI3-kinase inhibitor LY294002, known to inhibit TRPC translocation, decreased the response to TRH. In addition, a TRH-induced enhancement of the low-threshold spike was prevented by both rimonabant, and SR144528. TRH had no influence on excitatory or inhibitory miniature postsynaptic currents, suggesting presynaptic CB receptors are not involved in this situation. Collectively, the data imply that activation of TRH receptors in these midline thalamic neurons engages novel signaling pathways that include postsynaptic intracellular CB1 and CB2 receptors in the activation of TRPC4/5-like channels.