Adult ; Antibodies, Anticardiolipin ; blood ; Antibodies, Antiphospholipid ; blood ; Autoantibodies ; blood ; Female ; Humans ; Male ; Middle Aged ; Phosphatidic Acids ; immunology ; Phosphatidylcholines ; immunology ; Phosphatidylethanolamines ; immunology ; Phosphatidylinositols ; immunology ; Phosphatidylserines ; immunology ; Reference Values

A lipidomic study on extensive plasma lipids in bacterial peritonitis (cecal ligation and puncture, CLP)-induced sepsis in mice was done at 24 h post-CLP. The effects of administration of lysophosphatidylcholine (LPC) and lysophosphatidic acid (LPA), compounds known to have beneficial effects in CLP, on the sepsis-induced plasma lipid changes were also examined. Among the 147 plasma lipid species from 13 lipid subgroups (fatty acid [FA], LPA, LPC, lysophosphatidylethanolamine [LPE], phosphatidic acid [PA], phosphatidylcholine [PC], phosphatidylethanolamine [PE], phosphatidylinositol [PI], monoacylglyceride [MG], diacylglyceride [DG], triacylglyceride [TG], sphingomyelin [SM], and ceramide [Cer]) analyzed in this study, 40 and 70 species were increased, and decreased, respectively, in the CLP mice. Treatments with LPC and LPA affected 14 species from 7 subgroups, and 25 species from 9 subgroups, respectively. These results could contribute to finding the much needed reliable biomarkers of sepsis.
Animals ; Biomarkers ; Ligation ; Lysophosphatidylcholines ; Mice* ; Peritonitis ; Phosphatidic Acids ; Phosphatidylcholines ; Phosphatidylinositols ; Plasma* ; Punctures ; Sepsis

PURPOSE: Phospholipase D (PLD) catalyzes the hydrolytic cleavage of terminal phosphate diester bond of glycerophopholipids to produce phosphatidic acid (PA). PLD plays an important role in signal transduction and is known to be involved closely in cancer promotion, inflammation, and other cell responses. In order to evaluate radiation effect in tumor cells, various cells were screened for PLD activities and examined their radiation effects on PLD following gamma- ray irradiation. MATERIALS AND METHODS: PLD activities in 19 species of cell were measured by radioactive isotope method with 1,2 - di [1-14C] phosphatidylcholine in the presence of oleate. Among the cell lines examined, VERO 76, L 1210 and P 388 were selected and examined for their effects of metal ions and agonists on PLD activities before and after irradiation by Co-60 teletheraphy unit. RESULTS: The activities of oleate-PLD were observed in 11 species among 19 cell lines examined. VERO 76 and L 1210 cells showed that the PLD activity increased immediately after irradiation and reached to 150~200% of the control levels. The activation of PLD in response to gamma-ray was maximum at 20 Gy. In irradiated VERO 76, the stimulatory effect of Mg2+ was reduced and the activation of PLD by agonists in irradiated cells vary from those of the control cells. CONCLUSION: The activation effect of irradiation on PLD activity observed strongly implies that the PLD activity is closely related to the phenomena of cell necrosis. Therefore the cell lines examined here could provide a good source for the study of radiobiology that cover from cell death to cancer promotion.
Cell Death ; Cell Line* ; Inflammation ; Ions ; Necrosis ; Oleic Acid ; Phosphatidic Acids ; Phosphatidylcholines ; Phospholipase D ; Radiation Effects ; Radiobiology ; Signal Transduction

Ganoderma lingzhi is widely recognized as a medicinal basidiomycetes. Triterpene acids (TAs) are the key bioactive medicinal components of G. lingzhi. Our previous studies have shown that phospholipid acid (PA) produced by phospholipase D (PLD) plays a regulatory role in TA synthesis. In order to further elucidate the molecular mechanism how PA regulates TA synthesis in G. lingzhi, PA beads enrichment combined with LC-MS/MS technology was used to identify PA interacting proteins in G. lingzhi. A total of 19 PA interacting proteins were identified, including cytochrome P450 monooxygenase (GL22084), specific protein kinase MAPK (GL23765), catalase and cell surface hydrophobicity-associated protein. GST tagged GL22084 and GL23765 proteins were obtained through gene cloning, heterologous expression, and purification. The interactions between GL22084/GL23765 and PA were verified by GST pull down assay. The identification of PA interacting proteins provides a basis for further understanding the molecular mechanism how PLD-mediated PA signaling molecules regulates the TA synthesis in G. lingzhi. Moreover, the PA interacting proteins identified in this study can also provide clues for the research of PLD/PA signaling pathway in other species.
Chromatography, Liquid ; Ganoderma ; Phosphatidic Acids ; Tandem Mass Spectrometry

BACKGROUND: GH3 cells are a well characterized and widely used model used for the in vitro study of growth hormone (GH) secretion. Thyrotropin releasing hormone (TRH) binds to receptors belonging to the family of G protein-coupled receptors, and secrets both GH & prolactin. Phospholipase D (PLD) is an enzyme that hydrolyses phosphatidylcholine to yield phosphatidic acid and choline, and plays important roles in cellular proliferation and hormonal secretion. To elucidate the pathway of the action of TRH in GH3 cells, we investigated the activities of PLC and PLD in GH3 cells treated with TRH or phorbor 12-myristate 13-acetate (PMA). METHODS: GH3 cells were labeled with [3H] myristate, followed by incubation of with 0.3% ethanol, prior to before the addition of the agonists. The total lipids were extracted from the harvested cells following treatment with the agonists. The PLD activity was assessed by measuring [3H] phosphatidylethanol from the [3H] phospholipid using thin layer chromatography. RESULTS: TRH (1 muM) stimulated the PLC activity by 44-fold over that of the control values. TRH (1 microM), mastoparan (5 muM), and PMA (500 muM) for 30 minutes increased PLD activity by 1.9, 1.5 and 2.2 fold, respectively, in comparison to the controls. The PLD activities after 15, 30, 60, 120 and 240 min treatments of TRH (1 microM) were 142%, 170%, 172%, 160% and 115%, respectively. CONCLUSION: These results suggest that TRH stimulates not only the PLC activity, but also the PLD activity in GH3 cells.
Cell Proliferation ; Choline ; Chromatography, Thin Layer ; Ethanol ; Growth Hormone ; Humans ; Myristic Acid ; Phosphatidic Acids ; Phosphatidylcholines ; Phospholipase D* ; Phospholipases* ; Prolactin ; Thyrotropin-Releasing Hormone

BACKGROUND: Phospholipase C(PLC) plays a central role in cellular signal transduction and is important in cellular growth, differentiation and transformation. There are currently ten known mammalian isozymes of PLC reported to this date. Hydrolysis of phosphatidylinositol 4,5-bisphosphate(PIP2) by PLC produces two important second messengers, inositol 1,4,5-trisphosphate(IP3) and diacylglycerol. PLC-gamma1, previously, was known to be activated mainly through growth factor receptor tyrosine kinase. Other mechanisms of activating PLC-gamma1 have been reported such as activation through tau protein in the presence of arachidonic acid in bovine brain and activation by IP3, phosphatidic acid, etc. Very recently, another PLC-gamma1 activator protein such as tau has been found in bovine lung tissue, which now is considered to be AHNAK protein. But there has been no report concerning AHNAK and its associated disease to this date. In this study, we examined the expression of the PLC-gamma1 activator, AHNAK, in lung cancer specimens and their paired normal. METHODS: From surgically resected human lung cancer tissues taken from twenty-eight patients and their paired normal counterparts, we evaluated expression level of AHNAK protein using immunoblot analysis of total tissue extract. Immunohistochemical stain was performed with primary antibody against AHNAK protein. RESULTS: Twenty-two among twenty-eight lung cancer tissues showed over expression of AHNAK protein(eight of fourteen squamous cell lung cancers, all of fourteen adenocarcinomal). the resulting bands were multiple ranging from 70 to 200 kDa in molecular weight and each band was indistinct and formed a smear, reflecting mobility shift mainly due to proteolysis during extraction process. On immunohistochemistry, lung cancer tissues showed a very heavy, dense staining with anti-AHNAK protein antibody as compared to the surrounding normal lung tissue, coresponding well with the results of the western blot. CONCLUSION: The overexpression of PLC-gamma1 activator protein, AHNAK in lung cancer may provide evidence that the AHNAK protein and PLC-gamma1 act in concerted manner in carcinogenesis.
Arachidonic Acid ; Blotting, Western ; Brain ; Carcinogenesis ; Humans* ; Hydrolysis ; Immunohistochemistry ; Inositol ; Isoenzymes ; Lung Neoplasms* ; Lung* ; Molecular Weight ; Phosphatidic Acids ; Phosphatidylinositols ; Phospholipases* ; Protein-Tyrosine Kinases ; Proteolysis ; Second Messenger Systems ; Signal Transduction ; tau Proteins

Phosphatide acid (PA) is a kind of multifunctional bioactive phospholipid. It has been proved that PA produced by lysophosphatide acid acyltransferase (LPAATbeta) was involved in several signalling pathways in tumor cells, leading to the proliferation, apoptosis, migration, invasion, respiratory burst, expression and release of cytokine form tumor cells. The fact that expression of LPAATbeta was higher in tumor tissues than in their homologous normal tissues, and that antitumor effect of inhibitng LPAATbeta on solid tumor and hematological malignancy suggested that the targeting LPAATbeta would be a promising method of antitumor treatment. In this paper, the relevant basic and preclinical researches of LPAATbeta on antitumor treatment were summarized.
Acyltransferases ; antagonists & inhibitors ; genetics ; metabolism ; Humans ; Neoplasms ; drug therapy ; enzymology ; Phosphatidic Acids ; metabolism ; physiology

BACKGROUND: Phospholipase D (PLD) is a widely distributed enzyme that hydrolyzes phosphatidylcholine, a major phospholipids in the cell membrane, to form phosphatidic acid (PA) which acts by itself as a cellular messenger. PLD can also be transformed by PA phosphohydrolase into diacylglycerol (DAG), which is essential for the activation of protein kinase C (PKC). PLD has been shown to induce the proliferation of T cells and to activate by Der p 1 in peripheral blood mononuclear cells from atopic dermatitis. Single nucleotide polymorphism (SNP) has recently served as a key marker to discover the genetic mechanism of special chronic diseases. METHODS: One hundred eighteen children with atopic dermatitis were recruited, and graded as 23 mild (<25), 48 moderate (25-50) and 47 severe (>50) by measuring SCORAD index. Genomic DNA were purified from blood and made into PCR primers attaching GC-Clamp, and 26 exons of PLD were amplified by PCR-DGGE (denaturing gradient gel electrophoresis). RESULTS: Polymorphism was found in four subjects. Of them, three PLD1 cSNP (Exon23: G2658A, T2664A, G2684A) were detected in exon 23 of 26 exons of PLD1. Four cases among 118 subjects had cSNP of G2658A (3.4%), two T2664A cases (1.7%), one G2684A case (0.8%). There were no significant correlations between IgE and detected cSNP. CONCLUSION: Three PLD1 gene cSNPs (G2658A, T2664A, G2684A) were detected in the blood of children with atopic dermatitis. Among them, G2658A polymorphism seems to be correlated to the serum IgE level, but PLD1 cSNP does not appear to contribute to the pathogenic processing of atopic dermatitis.
Cell Membrane ; Child* ; Chronic Disease ; Dermatitis, Atopic* ; DNA ; Exons ; Humans ; Immunoglobulin E ; Phosphatidic Acids ; Phosphatidylcholines ; Phospholipase D ; Phospholipases* ; Phospholipids ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; Protein Kinase C ; T-Lymphocytes

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline. A variety of signal molecules such as hormones, neurotransmitters, extracellular matrix molecules, and growth factors are known to induce the activation of PLD in a wide range of cell types. Hence PLD is implicated in a broad spectrum of physio-logical processes and diseases, including mitogenesis, cell differentiation, metabolic regulation, secretion, neural and cardiac stimulation, inflammation, oncogenesis, and diabetes. The signal-dependent activation of PLD has been observed in a variety of brain and neural-derived cells. In this paper, human chromosomal locations and developmental neural expression patterns in rat of PLD1 and PLD2 were investigated with fluorescent in situ hybridization (FISH) and in situ hybridization histochemistry, respectively. The PLD1 was assigned to human chromosome 3q26 and expressed most strikingly in selected ventricular neural cells lining spinal cord and brain during neuronal differentiation and migration period. The PLD2 was assigned to human chromosome 17p13.1 and expressed in differentiating ventricular neural cells and multiple regions of the postnatal rat brain.
Animals ; Brain ; Carcinogenesis ; Cell Differentiation ; Choline ; Chromosomes, Human* ; Extracellular Matrix ; Humans ; Humans* ; Hydrolysis ; In Situ Hybridization ; In Situ Hybridization, Fluorescence ; Inflammation ; Intercellular Signaling Peptides and Proteins ; Neurons ; Neurotransmitter Agents ; Phosphatidic Acids ; Phosphatidylcholines ; Phospholipase D* ; Phospholipases* ; Rats* ; Spinal Cord

PURPOSE: Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidyl choline to phosphatidic acid (PA) and choline. Recently, PLD has been drawing much attentions and considered to be associated with cancer process since it is involved in cellular signal transduction. In this experiment, oleate-PLD activities were measured in various tissues of the living rats after whole body irradiation. MATERIAL AND METHODS: The reaction mixture for the PLD assay contained 0.1microCi 1,2-di[1-14C]palmitoyl phosphatidylcholine, 0.5mM phosphatidylcholine, 5mM sodium oleate, 0.2% taurodeoxycholate, 50mM HEPES buffer (pH 6.5), 10mM CaCl2, and 25mM KF. phosphatidic acid, the reaction product, was separated by TLC and its radioactivity was measured with a scintillation counter. The whole body irradiation was given to the female Wistar rats via Cobalt 60 Teletherapy with field size of 10cm x 10cm and an exposure of 2.7Gy per minute to the total doses of 10Gy and 25Gy. RESULTS: Among the tissues examined, PLD activity in lung was the highest one and was followed by kidney, skeletal muscle, brain, spleen, bone marrow, thymus, and liver. Upon irradiation, alteration of PLD activity was observed in thymus, spleen, lung, and bone marrow. Especially PLD activities of the spleen and thymus revealed the highest sensitivity toward gamma-ray with more than two times amplification in their activities. In contrast, the PLD activity of bone marrow appears to be reduced to nearly 30%. Irradiation effect was hardly detected in liver which showed the lowest PLD activity. CONCLUSION: The PLD activities affected most sensitively by the whole-body irradiation seem to be associated with organs involved in immunity and hematopoiesis. This observation strongly indicates that the PLD is closely related to the physiological function of these organs. Furthermore, radiation stress could offer an important means to explore the phenomena covering from cell proliferation to cell death on these organs.
Animals ; Attention ; Bone Marrow ; Brain ; Cell Death ; Cell Proliferation ; Choline ; Cobalt ; Female ; Hematopoiesis ; HEPES ; Humans ; Hydrolysis ; Kidney ; Liver ; Lung ; Muscle, Skeletal ; Oleic Acid ; Phosphatidic Acids ; Phosphatidylcholines ; Phospholipase D* ; Phospholipases* ; Radiation Effects ; Radioactivity ; Rats* ; Rats, Wistar ; Scintillation Counting ; Signal Transduction ; Sodium ; Spleen ; Taurodeoxycholic Acid ; Thymus Gland ; Whole-Body Irradiation