Identification of phosphatidic acid interacting proteins in Ganoderma lingzhi / 生物工程学报

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.

Lipidomic analysis of plasma lipids composition changes in septic mice

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.

Phospholipase D activates HIF-1-VEGF pathway via phosphatidic acid

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.

STAT3 is involved in phosphatidic acid-induced Bcl-2 expression in HeLa cells

Phosphatidic acid (PA), the product of a PLD-mediated reaction, is a lipid second messenger that participates in various intracellular signaling events and is known to regulate a growing list of signaling proteins. We found that Bcl-2 was upregulated by PA treatment in HeLa cells. However, how PA upregulates Bcl-2 expression has not yet been studied. In this study, we tried to discover the mechanisms of Bcl-2 up-regulation by PA treatment in HeLa cells. Treatment with PA resulted in significantly increased expression of Bcl-2 in HeLa cells. Moreover, PA-induced Bcl-2 expression was blocked by mepacrine, an inhibitor of PLA2, but not by propranolol, an inhibitor of PA phospholyhydrolase (PAP). Treatment of 1,2-dipalmitoryl-sn-glycero-3-phosphate (DPPA) also increased Bcl-2 expression. These results indicate that Bcl-2 expression is mediated by lysophosphatidic acid (LPA), not by arachidonic acid (AA). Thereafter, we used MEK1/2 inhibitor, PD98059 to investigate the relationship between ERK1/2 MAPK and PA-induced Bcl-2 expression. PA-induced Bcl-2 expression was decreased when ERK1/2 was inhibited by PD98059. The transcription factor such as STAT3 which is controlled by ERK1/2 MAPK was increased along with Bcl-2 expression when the cells were treated with PA. Furthermore, STAT3 siRNA treatments inhibited PA-induced Bcl-2 expression, suggesting that STAT3 (Ser727) is involved in PA-induced Bcl-2 expression. Taken together, these findings indicate that PA acts as an important mediator for increasing Bcl-2 expression through STAT3 (Ser727) activation via the ERK1/2 MAPK pathway.

Evidence for histidine residues on plasma membrane phosphatidate phosphohydrolase from rat liver

Phosphatidate phosphohydrolase [PAP] catalyzes the dephosphorylation of phosphatidic acid to yield P[i], and diacylglycerol. Two different forms of PAP in rat hepatocyte have been reported. PAP[1] is located in cytosolic and microsomal fractions and participates in the synthesis of triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine, whereas the other form of phosphatidate phosphohydrolase [PAP[2]] is primarily involved in lipid signaling pathways. In rat liver, PAP2 has two isoforms; one PAP[2a] and another PAP2b- In this study, essential histidine residues were investigated in native form of rat purified PAP[2b] with diethylpyrocarbonate. PAP[2b] purified from rat liver plasma membrane by solubilizing with n-octyle glucoside and several chromatography steps. Gel electrophoresis [SDS-PAGE] performed on purified enzyme in order to evaluate its purity and to measure the molecular weight of the enzyme subunit. The enzyme inactivated with diethylpyrocarbonate [DEPC] and the number of moles of histidine residues modified per mol of enzyme determined. The specific activity of purified enzyme was 7350mU/mg protein and it showed only a single band on SDS-PAGE with a MW of about 33.8 kDa. The PAP[2b] inactivated by DEPC. The maximum 6 moles of histidine residues modified per mole of PAP[2b], when about 90% of enzyme activity is lost with DEPC. The data showed that the incubation of PAP[2b] by DEPC can inhibit enzyme activity. Our findings also, revealed the presence of essential histidines in the structure of PAP[2b] which involve in its activity. This enzyme is likely to have a similar hydrolysis catalytic mechanism as its super family through a phosphohistidine intermediate

Relationship between lysophosphatide acid acyltransferase beta and tumor - review / 中国实验血液学杂志

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.

Detection of SNP of Phospholipase D1 in Children with Atopic Dermatitis / 소아알레르기및호흡기학회지

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 (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.

The role of mapk and pkc-delta in phosphatidic acid-mediated intercellular adhesion molecule-1 expression

BACKGROUND: Phosphatidic acid (PA), an important second messenger, is involved in inflammation. Notably, cell-cell interactions via adhesion molecules play a central role in inflammation. This thesis show that PA induces expression of intercellular adhesion molecule-1 (ICAM-1) on macrophages and describe the signaling pathways. MATERIALS AND METHODS: Macrophages were cultured in the presence of 10% FBS and assayed cell to cell adhesion using HUVEC. For the gene and protein analysis, RT-PCR, Western blot and flow cytometry were performed. In addition, overexpressed cell lines for dominant negative PKC-delta mutant established and tested their effect on the promoter activity and expression of ICAM-1 protein by PA. RESULTS: PA-activated macrophages significantly increased adhering to human umbilical vein endothelial cell and this adhesion was mediated by ICAM-1. Pretreatment with rottlerin (PKC-delta inhibitor) or expression of a dominant negative PKC-delta mutant, but not Go6976 (classical PKC-alpha inhibitor) and myristoylated PKC-zeta inhibitor, attenuated PA-induced ICAM-1 expression. The p38 mitogen-activated protein kinase (MAPK) inhibitor blocked PA-induced ICAM-1 expression in contrast, ERK upstream inhibitor didn't block ICAM-1. CONCLUSION: These data suggest that PA-induced ICAM-1 expression and cell-cell adhesion in macrophages requires PKC-delta activation and that PKC-delta activation is triggers to sequential activation of p38 MAPK.

Column chromatographic fractionation of soybean phospholipid and chemical analysis of isolated phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid and cerebroside

Phospholipid mixtures were isolated from crude soybean oil and laboratory prepared soy lecithin were fractionated on two silicic acid columns with subsequent thin layer chromatography identification of isolated fractions. The percentages of the isolated four eluted fractions from each of the two columns were calculated. Phosphatidylcholine, phosphatidylethanolamine phosphatidylinositol, phosphatidic acid and cerebroside were isolated. Fatty acid composition and total phosphorus of the isolated phospholipids were determined

Trp-Lys-Tyr-Met-Val-Met stimulates phagocytosis via phospho-lipase D-dependent signaling in mouse dendritic cells

Dendritic cells (DCs) play a key role in activating the immune response against invading pathogens as well as dying cells or tumors. Although the immune response can be initiated by the phagocytic activity by DCs, the molecular mechanism involved in this process has not been fully investigated. Trp-Lys-Tyr-Met-Val-Met-NH2 (WKYMVM) stimulates the activation of phospholipase D (PLD) via Ca2+ increase and protein kinase C activation in mouse DC cell line, DC2.4. WKYMVM stimulates the phagocytic activity, which is inhibited in the presence of N-butanol but not t-butanol in DC2.4 cells. Furthermore, the addition of phosphatidic acid, an enzymatic product of PLD activity, enhanced the phagocytic activity in DC2.4 cells. Since at least two of formyl peptide receptor (FPR) family (FPR1 and FPR2) are expressed in DC2.4 as well as in mouse bone marrow-derived dendritic cells, this study suggests that the activation of FPR family by WKYMVM stimulates the PLD activity resulting in phagocytic activity in DC2.4 cells.

Avaliação da indução de VEGF por ácido lipoteicóico em macrófagos e células pulpares / Lipoteichoic acid up-regulates VEGF expression in macrophages and pulp cells

O fator de crescimento vascular endotelial (VEGF) é um potente indutor de angiogênese, permeabilidade vascular e edema. O aumento da expressão de VEGF na polpa dentária pode resultar em pressão intra-pulpar aumentada e contribuir para dor e dano pulpar irreversível. O ácido lipoteicóico (LTA) é uma molécula anfifílica de bactérias gram-positivas que tem sido associada a patogênese da pulpite. Objetivo: Pesquisar se LTA regula a expressão de VEGF em células pulpares ou macrófagos; e avalliar se LT A causa morte celular em células pulpares ou macrófagos. Métodos: Células tipo odontoblasto (MOPC-23), células pulpares indiferenciadas (00-21), fibroblastos gengivais ou macrófagos foram estimulados com 0-80 µg/ml S. sanguis or S. mutans LTA, e a expressão de VEGF foi avaliada por ELlSA e RT-PCR. Foi também realizado o teste de exclusão "Trypan blue", o Teste de Citometria de Fluxo, e a análise do Ciclo Celular, e avaliado se as células morreram por consequência de necrose ou apoptose. As análises estatísticas realizadas foram ANOVA e o "Student t-test" com p' < ou = '0.05. Resultados: LTA induziu um aumento de 9 vezes na expressão proteica de VEGF em macrófagos, um aumento de 2.4 em MOPC-23, de 1.6 em 00-21 quando comparado aos controles. Em contraste, o LTA não induziu a expressão de VEGF em fibroblastos gengivais. A expressão VEGF mRNA permaneceu constante após exposição ao LTA, o que sugere que a regulação de VEGF nessas células é primariamente pós-transcricional. O LTA não causou morte celular significante nas concentrações utilizadas neste trabalho. MDPC-23 foi a única população celular que mostrou um aumento na proporção de células apoptóticas após exposição ao L T A, comparadas com o controle. Conclusão: LTA de streptococci induz elevação de VEGF em macrófagos e células pulpares, e aumento de apoptose em células tipo odontoblasto. Este trabalho constitui a primeira demonstração de que o LTA é suficiente para induzir a expressão de um fator pró-angiogênico.

Vascular endothelial growth factor (VEGF) is a potent inducer of angiogenesis, vascular permeability, and edema. Upregulation of VEGF expression in the dental pulp may result in increased intra-pulpal pressure, and contribute to pain and irreversible tissue damage. Lipoteichoic acid (LTA) is an amphiphilic molecule from Gram-positive bacteria that has been associated with the pathogenesis of pulpitis. Objective: To investigate if LTA regulates expression of VEGF in pulp cells or macrophages; and to evaluate if lTA causes cell death in pulp cells or macrophages. Methods: We stimulated mouse odontoblast-like cells (MDPC-23), undifferentiated pulp cells (OD-21), gingival fibroblasts or macrophages with 0-80 µg/ml S. sanguis or S. mutans LTA, and evaluated VEGF expression by ELlSA and RT -PCR. We also performed Trypan Blue Exclusion Assay, Flow Cytometry Assay, Cell Cycle analyses and evaluated if the cells have died as a consequence of necrosis or apoptosis. Statistical analyses were performed by one-way ANOVA and student t-tests at p' < OR ='0.05. Results: LTA induced up to 9-fold increase in VEGF protein expression in macrophages, 2.4-fold increase in MDPC-23, and 1.6-fold increase in OD-21 as compared to controls. In contrast, L T A did not induce VEGF expression in gingival fibroblasts. VEGF mRNA expression remained constant upon exposure to LTA, which suggests that VEGF regulation in these cells is primarily post-transcriptional. LTA did not cause significant cell death, at the concentrations used here. MDPC-23 was the only cell population that showed an increase in the proportion of apoptotic cells upon exposure to L TA, as compared to controls. Conclusion: Streptococcal LTA induces VEGF upregulation in macrophages and pulp cells, and increase in apoptosis of odontoblast-like cells. This work constitutes the first demonstration that LTA is sufficient to induce expression of a pro-angiogenic factor.

Effect of TRH on Phospholipase D Activity in GH3 Cell / 대한내분비학회지

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.

Localization of phospholipase D genes on the human chromosome and various tissues of rat / 대한해부학회지

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.

Increased Expression of Phospholipase C-gamma1 Activator Protein, AHNAK in Human Lung Cancer Tissues / 결핵

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.

Phospholipase D, MMP-2 and TIMP-2 Expression in the Human Colorectal Cancer

BACKGROUND: Tumor invason and metastasis are the major causes of morbidity and death for cancer patients. Metastatic spread depends critically upon the invasiveness of the tumor cells, i.e., their ability to breach basement membrane by profusely secreting specific proteolytic enzymes such as MMP-2. TIMP-2 has a high affinity for progelatinase A and will form a 1:1 complex with either the latent or activated forms of the enzyme and has inhibitory activity against MMP-2. Laminin induced activation of Phospholipase D (PLD) and consequent generation of phosphatidic acid are involved in a signal propagation pathway leading to induction of MMP-2 in metastatic HT 1080 fibrosarcoma cells. We also studied a expression of PLD, MMP-2 and TIMP-2 in colorectal adenocarcinoma. METHODS: Colorectal adenocarcinomas from 13 patients in our hospital were studied for immunohistochemical expression of PLD, MMP-2, and TIMP-2 to assess their diagnostic and prognostic importance as well as relation between PLD and MMP-2. RESULTS: 1) Expression of PLD-2 was detected in 77% of the cases in colorectal adenocarcinomas. 2) MMP-2 expression was significantly associated with the presence of lymph-node metastasis, with moderated to strong expression present in 100% of the cases compared with 28.6% of the non-metastatic cases (P-value=0.017). 3) For colorectal adenocarcinomas, a strong correlation between PLD and MMP-2 expression was detected (P-value=0.008). CONCLUSION: PLD-2 can be used as a potential marker for malignant disease in colorectal adenocarcinomas. MMP-2 expression was significantly associated with the presence of lymph-nodemetastasis. A strong correlation between PLD and MMP-2 expression was also detected in colorectal carcinoma.

Reforço radicular interno para colocaçäo de núcleo e coroa / The internal radicular reforce for using of the post and crown

O presente trabalho vem demonstrar um meio alternativo de aproveitamento de dentes com conduto radicular amplo, antes considerados perdidos, através da reconstruçäo da parte interna da raiz com a utilizaçäo de adesivo dentinário, resina composta e pino plástico transmissor de luz (LTP-Luminex)

Alteration of Oleate-Phospholipase D Activities in Some Cell Lines after Irradiation / 대한암학회지

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.

Alteration of Phospholipase D Activity in the Rat Tissues by Irradiation / 대한치료방사선과학회지

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.

Influence of mancozeb on mitogenically responsive lipids in rat cerebrum and liver.

Mancozeb, a commonly used fungicide, has been shown to induce tumours in mouse skin and maneb, unit constituent of mancozeb, is reported to induce tumours in rats. The mechanism by which mancozeb induced tumorigenicity is not known. Since the levels of inositol phospholipids and phosphatidic acid have roles in the regulation of cell proliferation, the effects of mancozeb on the levels of these lipids were studied in rats. Daily oral administration of commercial grade mancozeb at a concentration of 50 mg/kg body wt for 30 days (5 days a week) caused no significant change in the levels of inositol phospholipids and phosphatidic acid (PA) in both cerebrum and liver, while at high concentration (250 mg/kg body wt) under the same treatment schedule mancozeb increased the levels of these lipids. In cerebrum, the levels of phosphatidylinositol (PI) and PA were increased by 36 and 43% respectively without affecting the levels of polyphosphoinositides, whereas in liver the levels of not only PI (50%) and PA (49%) but also those of polyphosphoinositides were increased. These results show that mancozeb influences the levels of PA and inositol phospholipids, involved in phospholipase C-pathway of signalling.