ABSTRACT
OBJECTIVES@#Systemic lupus erythematosus (SLE) is a kind of autoimmune inflammatory connective tissue disease which seriously endangers human health. Genetic factors play a key role in the pathogenesis of SLE. This study aims to investigate a novel phospholipase D2 (PLD2) mutation associated with familial SLE, and further explore the underlying mechanism of the mutation in SLE.@*METHODS@#The blood samples from a SLE patient, the patient's parents, and 147 normal controls were collected and DNA was extracted. Whole genome high-throughput sequencing was performed in the patient and her parents and the results were further analyzed by various bioinformatics methods. The wild type (wt), mutant type (mu), and negative control PLD2 plasmids were further constructed and transfected into 293 cells. The expression level of HRAS protein in 293 cells was detected by Western blotting.@*RESULTS@#In this SLE family, the female SLE patient and her mother, 1 in generation II and 1 in generation III had typical clinical manifestations of SLE, and all of them had lupus nephritis at early stage. The genetic characteristics are consistent with autosomal dominant inheritance. A novel PLD2 heterozygous mutation (c.2722C>T) was found in the patient and her mother, but not in her father and other normal controls. Compared with wtPLD2 plasmid and negative control PLD2 plasmid, the expression of HRAS in 293 cells transfected with muPLD2 plasmid was significantly up-regulated (both @*CONCLUSIONS@#PLD2 c.2722C>T mutation may be one of the pathogeny of SLE in this family.
Subject(s)
Female , Humans , Case-Control Studies , High-Throughput Nucleotide Sequencing , Lupus Erythematosus, Systemic/genetics , Lupus Nephritis , Mutation , Phospholipase DABSTRACT
Background Loxoscelism is a severe human envenomation caused by Loxosceles spider venom. To the best of our knowledge, no study has evaluated the presence of antibodies against Loxosceles venom in loxoscelism patients without treatment with antivenom immunotherapy. We perform a comparative analysis for the presence of antibodies capable of recognizing Loxosceles venom in a group of patients diagnosed with loxoscelism and in a group of people without loxoscelism. Methods The detection of L. laeta venom, Sicarius venom and recombinant phospholipases D from Loxosceles (PLDs) in sera from people with loxoscelism (Group 1) and from healthy people with no history of loxoscelism (Group 2) was evaluated using immuno-dot blot, indirect ELISA, and Western blot. Results We found naturally heterophilic antibodies (IgG-type) in people without contact with Loxosceles spiders or any clinical history of loxoscelism. Either serum pools or single sera from Group 1 and Group 2 analyzed by dot blot tested positive for L. laeta venom. Indirect ELISA for venom recognition showed titles of 1:320 for Group 1 sera and 1:160 for Group 2 sera. Total IgG quantification showed no difference in sera from both groups. Pooled sera and purified IgG from sera of both groups revealed venom proteins between 25 and 32 kDa and the recombinant phospholipase D isoform 1 (rLlPLD1), specifically. Moreover, heterophile antibodies cross-react with PLDs from other Loxosceles species and the venom of Sicarius spider. Conclusions People without contact with the spider venom produced heterophilic antibodies capable of generating a cross-reaction against the venom of L. laeta and Sicarius spiders. Their presence and possible interference should be considered in the development of immunoassays for Loxosceles venom detection.
Subject(s)
Antibodies, Heterophile/analysis , Phospholipase D/immunology , Spider Venoms/immunology , Spider Bites/complicationsABSTRACT
Loxoscelism is a severe human envenomation caused by Loxosceles spider venom. To the best of our knowledge, no study has evaluated the presence of antibodies against Loxosceles venom in loxoscelism patients without treatment with antivenom immunotherapy. We perform a comparative analysis for the presence of antibodies capable of recognizing Loxosceles venom in a group of patients diagnosed with loxoscelism and in a group of people without loxoscelism. Methods The detection of L. laeta venom, Sicarius venom and recombinant phospholipases D from Loxosceles (PLDs) in sera from people with loxoscelism (Group 1) and from healthy people with no history of loxoscelism (Group 2) was evaluated using immuno-dot blot, indirect ELISA, and Western blot. Results We found naturally heterophilic antibodies (IgG-type) in people without contact with Loxosceles spiders or any clinical history of loxoscelism. Either serum pools or single sera from Group 1 and Group 2 analyzed by dot blot tested positive for L. laeta venom. Indirect ELISA for venom recognition showed titles of 1:320 for Group 1 sera and 1:160 for Group 2 sera. Total IgG quantification showed no difference in sera from both groups. Pooled sera and purified IgG from sera of both groups revealed venom proteins between 25 and 32 kDa and the recombinant phospholipase D isoform 1 (rLlPLD1), specifically. Moreover, heterophile antibodies cross-react with PLDs from other Loxosceles species and the venom of Sicarius spider. Conclusions People without contact with the spider venom produced heterophilic antibodies capable of generating a cross-reaction against the venom of L. laeta and Sicarius spiders. Their presence and possible interference should be considered in the development of immunoassays for Loxosceles venom detection.(AU)
Subject(s)
Humans , Male , Female , Adolescent , Adult , Middle Aged , Phospholipase D/isolation & purification , Spider Venoms/toxicity , Antibodies, Heterophile/blood , Antivenins/therapeutic use , Enzyme-Linked Immunosorbent Assay/methods , Immunoblotting/methodsABSTRACT
<p><b>OBJECTIVE</b>To assess the association of polymorphisms of N-acyl-phosphatidylethanolamine-phospholipase D (DAPE-PLD) and fatty acid amide hydrolase (FAAH) genes, as well as their interaction, with schizophrenia.</p><p><b>METHODS</b>Polymorphisms of NAPE-PLD rs12540583 and FAAH rs324420, rs2295633, and rs6429600 were determined with PCR - restriction fragment length polymorphism assay and Sanger sequencing. The genotypes of 345 subjects of Han Chinese origin diagnosed with schizophrenia and a 403 controls were compared. The results were analyzed with SPSS 17.0, and the interaction of the two genes was analyzed using a multifactor dimensionality reduction (MDR) method.</p><p><b>RESULTS</b>The frequency of NAPE-PLD rs12540583 polymorphism was significantly different between the two groups under both dominant and additive models (χ2=17.18 vs. χ2=18.94, P<0.0125). The frequencies of AC genotype and C allele of the patient group at rs12540583 were higher than those of the controls, and the interaction of NAPE-PLD and FAAH was associated with schizophrenia. A four-loci model (rs12540583, rs324420, rs2295633 and rs6429600) can best model the interaction between NAPE-PLD and FAAH.</p><p><b>CONCLUSION</b>The AC genotype and C allele of NAPE-PLD rs12540583 locus are risk factors for schizophrenia, and the interaction between NAPE-PLD rs12540583 and FAAH rs324420, rs2295633 and rs6429600 is associated with schizophrenia.</p>
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Amidohydrolases , Genetics , Asian People , Genetics , China , Ethnology , Genotype , Phospholipase D , Genetics , Polymorphism, Genetic , Schizophrenia , GeneticsABSTRACT
Background: Caseous lymphadenitis, caused by Corynebacterium pseudotuberculosis, is one of the most important diseases of sheep and goats, causing considerable losses for herd owners. Phospholipase D [PLD] is a potent exotoxin produced by C. pseudotuberculosis and it has been considered as the major virulence factor for this bacterium, possibly contributing to the spread of the bacteria from the initial site of infection to secondary sites within the host. Heat shock proteins [HSPs] are important candidates for the development of vaccines because they are usually able to promote both humoral and cellular immune re-sponses in mammals
Objectives: The aim of this study was the cloning and expression of the PLD and HSP genes of C. pseudotuberculosis
Methods: PLD and HSP[60] genes were cloned into pMAL-c2X vector and recombinant plasmids construct was transformed to DH[5] strain of E. coll. Expression of the proteins was shown by SDS-PAGE and accuracy of the cloned genes was confirmed by nucleo-tide sequence analysis
Results: The transformed E. coll strain DH[5] expressed PLD and HSP60 proteins effectively. The expressed fusion protein was found almost entirely in the soluble form
Conclusions: In the following studies the immunogenicity and protectivity of these recombinant proteins against C. pseudotuberculosis infections can be assessed
Subject(s)
Recombinant Proteins , Phospholipase D , Chaperonin 60ABSTRACT
Hypoxia-inducible factor-1alpha (HIF-1alpha) is a key transcriptional mediator that coordinates the expression of various genes involved in tumorigenesis in response to changes in oxygen tension. The stability of HIF-1alpha protein is determined by oxygen-dependent prolyl hydroxylation, which is required for binding of the von Hippel-Lindau protein (VHL), the recognition component of an E3 ubiquitin ligase that targets HIF-1alpha for ubiquitination and degradation. Here, we demonstrate that PLD2 protein itself interacts with HIF-1alpha, prolyl hydroxylase (PHD) and VHL to promote degradation of HIF-1alpha via the proteasomal pathway independent of lipase activity. PLD2 increases PHD2-mediated hydroxylation of HIF-1alpha by increasing the interaction of HIF-1alpha with PHD2. Moreover, PLD2 promotes VHL-dependent HIF-1alpha degradation by accelerating the association between VHL and HIF-1alpha. The interaction of the pleckstrin homology domain of PLD2 with HIF-1alpha also promoted degradation of HIF-1alpha and decreased expression of its target genes. These results indicate that PLD2 negatively regulates the stability of HIF-1alpha through the dynamic assembly of HIF-1alpha, PHD2 and VHL.
Subject(s)
Humans , Cell Line , HEK293 Cells , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Phospholipase D/metabolism , Prolyl Hydroxylases/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Interaction Maps , Proteolysis , Ubiquitin-Protein Ligases/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/metabolismABSTRACT
Endocytosis is differentially regulated by hypoxia-inducible factor-1alpha (HIF-1alpha) and phospholipase D (PLD). However, the relationship between HIF-1alpha and PLD in endocytosis is unknown. HIF-1alpha is degraded through the prolyl hydroxylase (PHD)/von Hippel-Lindau (VHL) ubiquitination pathway in an oxygen-dependent manner. Here, we show that PLD1 recovers the decrease in epidermal growth factor receptor (EGFR) endocytosis induced by HIF-1alpha independent of lipase activity via the Rab5-mediated endosome fusion pathway. EGF-induced interaction of PLD1 with HIF-1alpha, PHD and VHL may contribute to EGFR endocytosis. The pleckstrin homology domain (PH) of PLD1 itself promotes degradation of HIF-1alpha, then accelerates EGFR endocytosis via upregulation of rabaptin-5 and suppresses tumor progression. These findings reveal a novel role of the PLD1-PH domain as a positive regulator of endocytosis and provide a link between PLD1 and HIF-1alpha in the EGFR endocytosis pathway.
Subject(s)
Animals , Female , Humans , Blood Proteins/chemistry , Endocytosis , HEK293 Cells , HT29 Cells , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mice, Nude , Neoplasms/genetics , Phospholipase D/chemistry , Phosphoproteins/chemistry , Protein Structure, Tertiary , ErbB Receptors/metabolism , Signal Transduction , Up-Regulation , Vesicular Transport Proteins/genetics , rab5 GTP-Binding Proteins/metabolismABSTRACT
MicroRNAs (miRNAs) are a type of small non-coding RNAs that are often play important roles in carcinogenesis, but the carcinogenic mechanism of miRNAs is still unclear. This study will investigate the function and the mechanism of miR-638 in carcinoma (GC). The expression of miR-638 in GC and the DNA copy number of miR-638 were detected by real-time PCR. The effect of miR-638 on cell proliferation was measured by counting kit-8 assay. Different assays, including bioinformatics algorithms (TargetScan and miRanda), luciferase report assay and Western blotting, were used to identify the target gene of miR-638 in GC. The expression of miR-638 target gene in clinical CRC tissues was also validated by immunohistochemical assay. From this research, we found that miR-638 was downregulated in GC tissues compared with corresponding noncancerous tissues (NCTs), and the DNA copy number of miR-638 was lower in GC than NCTs, which may induce the corresponding downregulation of miR-638 in GC. Ectopic expression of miR-638 inhibited GC cell growth in vitro. Subsequently, we identified that PLD1 is the target gene of miR-638 in GC, and silencing PLD1 expression phenocopied the inhibitory effect of miR-638 on GC cell proliferation. Furthermore, we observed that PLD1 was overexpressed in GC tissues, and high expression of PLD1 in GC predicted poor overall survival. In summary, we revealed that miR-638 functions as a tumor suppressor in GC through inhibiting PLD1.
Subject(s)
Humans , 3' Untranslated Regions , Genetics , Apoptosis , Genetics , Base Sequence , Cell Line, Tumor , Cell Proliferation , Genetics , Down-Regulation , Genetics , MicroRNAs , Genetics , Phospholipase D , Genetics , Prognosis , Stomach Neoplasms , Diagnosis , Genetics , PathologyABSTRACT
OBJECTIVE@#To clarify the effect of glycosylphosphatidylinositol-specific phospholipase D (GPIPLD) on hepatoma cells HepG2 and the possible molecular mechanism.@*METHODS@#MTT, fluorescent staining and Western blot were applied to analyze the effect and molecular mechanism of GPI-PLD on hepatoma cells by transfected high expression GPI-PLD model. We inoculated HepG2 in nude mice models to further clarify the effect of GPI-PLD on hepatoma cells in vivo.@*RESULTS@#Compared with the control groups, PI3K-Akt signaling pathway activity and proliferation of hepatoma cells were significantly inhibited in the GPI-PLD group. Nude mice models showed that the tumor growth and tumor weight [(1.87 ± 0.09) g] of the GPI-PLD group were significantly less than those of the blank control group [(2.20 ± 0.17) g] and the negative control group [(2.15 ± 0.09) g]. AST, ALT and AFP serum concentration in the GPI-PLD group were significantly lower than those of the control groups (P<0.05).@*CONCLUSION@#GPI-PLD can inhibit the proliferation of hepatoma cells and growth in vivo, and promote the apoptosis of hepatoma cells by reducing the activity of PI3K-Akt signaling pathway.
Subject(s)
Animals , Humans , Mice , Apoptosis , Carcinoma, Hepatocellular , Metabolism , Pathology , Cell Line , Down-Regulation , Hep G2 Cells , Liver Neoplasms , Metabolism , Pathology , Mice, Nude , Phosphatidylinositol 3-Kinases , Phospholipase D , Metabolism , Proto-Oncogene Proteins c-akt , Metabolism , Signal Transduction , TransfectionABSTRACT
Autophagy is a conserved lysosomal self-digestion process used for the breakdown of long-lived proteins and damaged organelles, and it is associated with a number of pathological processes, including cancer. Phospholipase D (PLD) isozymes are dysregulated in various cancers. Recently, we reported that PLD1 is a new regulator of autophagy and is a potential target for cancer therapy. Here, we investigated whether PLD2 is involved in the regulation of autophagy. A PLD2-specific inhibitor and siRNA directed against PLD2 were used to treat HT29 and HCT116 colorectal cancer cells, and both inhibition and genetic knockdown of PLD2 in these cells significantly induced autophagy, as demonstrated by the visualization of light chain 3 (LC3) puncta and autophagic vacuoles as well as by determining the LC3-II protein level. Furthermore, PLD2 inhibition promoted autophagic flux via the canonical Atg5-, Atg7- and AMPK-Ulk1-mediated pathways. Taken together, these results suggest that PLD2 might have a role in autophagy and that its inhibition might provide a new therapeutic basis for targeting autophagy.
Subject(s)
Humans , Autophagy/drug effects , Cell Line, Tumor , Colorectal Neoplasms/enzymology , Genetic Therapy , HCT116 Cells , Phospholipase D/antagonists & inhibitors , Quinolines/pharmacology , RNA Interference , RNA, Small Interfering/genetics , Signal Transduction/drug effects , Spiro Compounds/pharmacologyABSTRACT
Growth factor-stimulated phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC), generating phosphatidic acid (PA) which may act as a second messenger during cell proliferation and survival. Therefore, PLD is believed to play an important role in tumorigenesis. In this study, a potential mechanism for PLD-mediated tumorigenesis was explored. Ectopic expression of PLD1 or PLD2 in human glioma U87 cells increased the expression of hypoxia-inducible factor-1alpha (HIF-1alpha) protein. PLD-induced HIF-1 activation led to the secretion of vascular endothelial growth factor (VEGF), a HIF-1 target gene involved in tumorigenesis. PLD induction of HIF-1alpha was significantly attenuated by 1-butanol which blocks PA production by PLD, and PA per se was able to elevate HIF-1alpha protein level. Inhibition of mTOR, a PA-responsive kinase, reduced the levels of HIF-1alpha and VEGF in PLD-overexpressed cells. Epidermal growth factor activated PLD and increased the levels of HIF-1alpha and VEGF in U87 cells. A specific PLD inhibitor abolished expression of HIF-1alpha and secretion of VEGF. PLD may utilize HIF-1-VEGF pathway for PLD-mediated tumor cell proliferation and survival.
Subject(s)
Humans , Cell Line, Tumor , Epidermal Growth Factor/metabolism , Gene Expression Regulation, Neoplastic , Glioma/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Phosphatidic Acids/metabolism , Phospholipase D/genetics , Signal Transduction , Transfection , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Interstitial cells of Cajal (ICCs) are the pacemaker cells in the gastrointestinal tract, and histamine is known to regulate neuronal activity, control vascular tone, alter endothelial permeability, and modulate gastric acid secretion. However, the action mechanisms of histamine in mouse small intestinal ICCs have not been previously investigated, and thus, in the present study, we investigated the effects of histamine on mouse small intestinal ICCs, and sought to identify the receptors involved. Enzymatic digestions were used to dissociate ICCs from small intestines, and the whole-cell patch-clamp configuration was used to record potentials (in current clamp mode) from cultured ICCs. Histamine was found to depolarize resting membrane potentials concentration dependently, and whereas 2-PEA (a selective H1 receptor agonist) induced membrane depolarizations, Dimaprit (a selective H2-agonist), R-alpha-methylhistamine (R-alpha-MeHa; a selective H3-agonist), and 4-methylhistamine (4-MH; a selective H4-agonist) did not. Pretreatment with Ca(2+)-free solution or thapsigargin (a Ca(2+)-ATPase inhibitor in endoplasmic reticulum) abolished the generation of pacemaker potentials and suppressed histamine-induced membrane depolarization. Furthermore, treatments with U-73122 (a phospholipase C inhibitor) or 5-fluoro-2-indolyl des-chlorohalopemide (FIPI; a phospholipase D inhibitor) blocked histamine-induced membrane depolarizations in ICCs. On the other hand, KT5720 (a protein kinase A inhibitor) did not block histamine-induced membrane depolarization. These results suggest that histamine modulates pacemaker potentials through H1 receptor-mediated pathways via external Ca2+ influx and Ca2+ release from internal stores in a PLC and PLD dependent manner.
Subject(s)
Animals , Mice , Carbazoles , Cyclic AMP-Dependent Protein Kinases , Dimaprit , Domperidone , Estrenes , Gastric Acid , Gastrointestinal Tract , Hand , Histamine , Indoles , Interstitial Cells of Cajal , Intestine, Small , Membrane Potentials , Membranes , Methylhistamines , Neurons , Permeability , Phospholipase D , Pyridoxal , Pyrroles , Pyrrolidinones , Thapsigargin , Type C PhospholipasesABSTRACT
Radiation and drug resistance remain the major challenges and causes of mortality in the treatment of locally advanced, recurrent and metastatic breast cancer. Dysregulation of phospholipase D (PLD) has been found in several human cancers and is associated with resistance to anticancer drugs. In the present study, we evaluated the effects of PLD inhibition on cell survival, cell death and DNA damage after exposure to ionizing radiation (IR). Combined IR treatment and PLD inhibition led to an increase in the radiation-induced apoptosis of MDA-MB-231 metastatic breast cancer cells. The selective inhibition of PLD1 and PLD2 led to a significant decrease in the IR-induced colony formation of breast cancer cells. Moreover, PLD inhibition suppressed the radiation-induced activation of extracellular signal-regulated kinase and enhanced the radiation-stimulated phosphorylation of the mitogen-activated protein kinases p38 and c-Jun N-terminal kinase. Furthermore, PLD inhibition, in combination with radiation, was very effective at inducing DNA damage, when compared with radiation alone. Taken together, these results suggest that PLD may be a useful target molecule for the enhancement of the radiotherapy effect.
Subject(s)
Female , Humans , Breast Neoplasms/drug therapy , Cell Death/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , DNA Damage , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Extracellular Signal-Regulated MAP Kinases/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Phospholipase D/antagonists & inhibitors , Radiation Tolerance/drug effects , Radiation, Ionizing , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Fourteen isolates of Corynebacteruim pseudotuberculosis of them 7 were isolated from sheep with Caseous Lymphadenitis "biotype 1" and 7 isolated from buffaloes with Oedematous Skin Disease "biotype 2". All isolates were identified by standard microbiological techniques and by polymerase chain reaction targeting, 16S rRNA and phospholipase D genes. Synergistic haemolytic titers of all isolates were assayed by plate technique. The presences of phospholipase D gene in supernatants of all isolates were performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblot technique by using hyperimmune serum raised in rabbit immunized with recombinant phospholipase D gene antigen. The concentration of phospholipase D gene was assayed by scanning the bound phospholipase D gene with specific antibodies that appeared at 31.5 kDa. Results presented that there is no correlation between titer of Synergistic haemolytic activity and the actual phospholipase D genes concentration in culture supernatants. Also results presented that Synergistic haemolytic activity and phospholipase D genes produced by biotype 2 (buffalo isolates) was generally higher than those by biotype 1(sheep isolates).
Subject(s)
Animals , Cattle , Rabbits , Corynebacterium Infections , Corynebacterium pseudotuberculosis/enzymology , Corynebacterium pseudotuberculosis/isolation & purification , Phospholipase D/genetics , Phospholipase D/isolation & purification , Gene Expression Regulation , In Vitro Techniques , Lymphadenitis , RNA , Buffaloes , Electrophoresis , Enzyme Activation , Methods , Rabbits , SheepABSTRACT
Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to generate the lipid second messenger, phosphatidic acid. PLD is localized in most cellular organelles, where it is likely to play different roles in signal transduction. PLD1 is primarily localized in vesicular structures such as endosomes, lysosomes and autophagosomes. However, the factors defining its localization are less clear. In this study, we found that four hydrophobic residues present in the N-terminal HKD catalytic motif of PLD1, which is involved in intramolecular association, are responsible for vesicular localization. Site-directed mutagenesis of the residues dramatically disrupted vesicular localization of PLD1. Interestingly, the hydrophobic residues of PLD1 are also involved in the interruption of its nuclear localization. Mutation of the residues increased the association of PLD1 with importin-beta, which is known to mediate nuclear importation, and induced the localization of PLD1 from vesicles into the nucleus. Taken together, these data suggest that the hydrophobic amino acids involved in the interdomain association of PLD1 are required for vesicular localization and disturbance of its nuclear localization.
Subject(s)
Humans , Amino Acid Motifs , Amino Acid Sequence , Amino Acids/chemistry , Cell Nucleus/enzymology , Endosomes/enzymology , HEK293 Cells , Hydrophobic and Hydrophilic Interactions , Lysosomes/enzymology , Phagosomes/enzymology , Phospholipase D/chemistry , Protein Interaction Domains and Motifs , Protein Transport , Transport Vesicles/enzymologyABSTRACT
The pathogenesis and pathophysiology of Acanthamoeba infections remain incompletely understood. Phos-pholipases are known to cleave phospholipids, suggesting their possible involvement in the host cell plasma membrane disruption leading to host cell penetration and lysis. The aims of the present study were to determine phospholipase activities in Acanthamoeba and to determine their roles in the pathogenesis of Acanthamoeba. Using an encephalitis isolate (T1 genotype), a keratitis isolate (T4 genotype), and an environmental isolate (T7 genotype), we demonstrated that Acanthamoeba exhibited phospholipase A2 (PLA2) and phospholipase D (PLD) activities in a spectrophotometry-based assay. Interestingly, the encephalitis isolates of Acanthamoeba exhibited higher phospholipase activities as compared with the keratitis isolates, but the environmental isolates exhibited the highest phospholipase activities. Moreover, Acanthamoeba isolates exhibited higher PLD activities compared with the PLA2. Acanthamoeba exhibited optimal phospholipase activities at 37degrees C and at neutral pH indicating their physiological relevance. The functional role of phospholipases was determined by in vitro assays using human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. We observed that a PLD-specific inhibitor, i.e., compound 48/80, partially inhibited Acanthamoeba encephalitis isolate cytotoxicity of the host cells, while PLA2-specific inhibitor, i.e., cytidine 5'-diphosphocholine, had no effect on parasite-mediated HBMEC cytotoxicity. Overall, the T7 exhibited higher phospholipase activities as compared to the T4. In contract, the T7 exhibited minimal binding to, or cytotoxicity of, HBMEC.
Subject(s)
Humans , Acanthamoeba/enzymology , Cell Adhesion , Cells, Cultured , Endothelial Cells/parasitology , Keratitis/parasitology , Phospholipase D/genetics , Phospholipases A2/genetics , Protozoan Proteins/genetics , Soil/parasitologyABSTRACT
This study was conducted to investigate the effects of extremely low frequency electromagnetic fields (EMF) on signal pathway in plasma membrane of cultured cells (RAW 264.7 cells and RBL 2H3 cells), by measuring the activity of phospholipase A2 (PLA2), phospholipase C (PLC) and phospholipase D (PLD). The cells were exposed to the EMF (60 Hz, 0.1 or 1 mT) for 4 or 16 h. The basal and 0.5 microM melittin-induced arachidonic acid release was not affected by EMF in both cells. In cell-free PLA2 assay, we failed to observe the change of cPLA2 and sPLA2 activity. Also both PLC and PLD activities did not show any change in the two cell lines exposed to EMF. This study suggests that the exposure condition of EMF (60 Hz, 0.1 or 1 mT) which is 2.4 fold higher than the limit of occupational exposure does not induce phospholipases-associated signal pathway in RAW 264.7 cells and RBL 2H3 cells.
Subject(s)
Arachidonic Acid , Cell Line , Cell Membrane , Cells, Cultured , Electromagnetic Fields , Magnets , Occupational Exposure , Phospholipase D , Phospholipases , Phospholipases A2 , Pyridoxal , Signal Transduction , Type C PhospholipasesABSTRACT
This study was purposed to investigate the expression and significance of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) in bone marrow mononuclear cells (BMMNC) isolated from patients with acute myeloid leukemia (AML), GPI-PLD activity in BMMNC isolated from 78 patients with AML and 15 normal persons was measured by using GPI-anchored placental alkaline phosphatase (PLAP) as a substrate and Triton X-114 phase partitioning. The GPI-PLD mRNA expression was measured by semi-quantitive reverse transcription-polymerase chain reaction (RT-PCR). The results showed that the mRNA expression level and activity of GPI-PLD in BMMNC from de novo AML patients were 1.86 +/- 0.32 and 46.96 +/- 7.15% respectively; the mRNA expression level and activity of GPI-PLD in BMMNC from completely remission and refractory or relapsed patients were 1.26 +/- 0.29, 33.36 +/- 5.13%and 1.79 +/- 0.19, 44.31 +/- 7.22%, while those in BMMNC from normal controls were 1.27 +/- 0.23, 35.38 +/- 5.15% respectively. The mRNA expression level and activity of GPI-PLD in de novo and refractory or relapsed patients were obviously higher than those in normal controls with significant difference (p < 0.01), while the comparison between remitted patients and normal controls showed no statistical difference (p > 0.05). It is concluded that the expression level of GPI-PLD mRNA coincides with GPI-PLD activity. The mRNA expression and activity of GPI-PLD in de novo and refractory or relapsed patients are obviously higher than those in normal controls. It is worthy of further exploring whether GPI-PLD plays a certain role in process of leukemia pathogenesis.
Subject(s)
Adolescent , Adult , Child , Female , Humans , Male , Middle Aged , Young Adult , Bone Marrow Cells , Cell Biology , Metabolism , Case-Control Studies , Leukemia, Myeloid, Acute , Metabolism , Pathology , Phospholipase D , Genetics , Metabolism , RNA, Messenger , GeneticsABSTRACT
Rebamipide a gastroprotective drug, is clinically used for the treatment of gastric ulcers and gastritis, but its actions on gastric cancer are not clearly understood. Phospholipase D (PLD) is overexpressed in various types of cancer tissues and has been implicated as a critical factor in inflammation and carcinogenesis. However, whether rebamipide is involved in the regulation of PLD in gastric cancer cells is not known. In this study, we showed that rebamipide significantly suppressed the expression of both PLD1 and PLD2 at a transcriptional level in AGS and MKN-1 gastric cancer cells. Downregulation of PLD expression by rebamipide inhibited its enzymatic activity. In addition, rebamipide inhibited the transactivation of nuclear factor kappa B (NFkappaB), which increased PLD1 expression. Rebamipide or PLD knockdown significantly suppressed the expression of genes involved in inflammation and proliferation and inhibited the proliferation of gastric cancer cells. In conclusion, rebamipide-induced downregulation of PLD may contribute to the inhibition of inflammation and proliferation in gastric cancer.
Subject(s)
Humans , Alanine/analogs & derivatives , Cell Line, Tumor , Cell Proliferation/drug effects , Down-Regulation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Inflammation/enzymology , Isoenzymes/genetics , NF-kappa B/metabolism , Phospholipase D/genetics , Promoter Regions, Genetic/genetics , Quinolones/pharmacology , Stomach Neoplasms/enzymology , Transcription, Genetic/drug effectsABSTRACT
As glucose is known to induce insulin secretion in pancreatic beta cells, this study investigated the role of a phospholipase D (PLD)-related signaling pathway in insulin secretion caused by high glucose in the pancreatic beta-cell line MIN6N8. It was found that the PLD activity and PLD1 expression were both increased by high glucose (33.3 mM) treatment. The dominant negative PLD1 inhibited glucose-induced Beta2 expression, and glucose-induced insulin secretion was blocked by treatment with 1-butanol or PLD1-siRNA. These results suggest that high glucose increased insulin secretion through a PLD1-related pathway. High glucose induced the binding of Arf6 to PLD1. Pretreatment with brefeldin A (BFA), an Arf inhibitor, decreased the PLD activity as well as the insulin secretion. Furthermore, BFA blocked the glucose-induced mTOR and p70S6K activation, while mTOR inhibition with rapamycin attenuated the glucose induced Beta2 expression and insulin secretion. Thus, when taken together, PLD1 would appear to be an important regulator of glucose-induced insulin secretion through an Arf6/PLD1/mTOR/p70S6K/Beta2 pathway in MIN6N8 cells.