High-level soluble expression of phospholipase D from Streptomyces chromofuscus in Escherichia coli by combinatorial optimization

BACKGROUND: Phospholipase D (PLD) is used as the biocatalyst for phosphatidylserine (PS) production. In general, PLD was expressed in insoluble form in Escherichia coli. High-level soluble expression of PLD with high activity in E. coli is very important for industrial production of PLD. RESULTS: Streptomyces chromofuscus PLD coding gene was codon-optimized, cloned without signal peptide, and expressed in E. coli. The optimal recombinant E. coli pET-28a+PLD/BL21(DE3) was constructed with pET-28a without His-tag. The highest PLD activity reached 104.28 ± 2.67 U/mL in a 250-mL shake flask after systematical optimization. The highest PLD activity elevated to 122.94 ± 1.49 U/mL by feeding lactose and inducing at 20 C after scaling up to a 5.0-L fermenter. Substituting the mixed carbon source with 1.0 % (w/v) of cheap dextrin and adding a feeding medium could still attain a PLD activity of 105. 81 ± 2.72 U/mL in a 5.0-L fermenter. Fish peptone from the waste of fish processing and dextrin from the starch are both very cheap, which were found to benefit the soluble PLD expression. CONCLUSIONS: After combinatorial optimization, the high-level soluble expression of PLD was fulfilled in E. coli. The high PLD activity along with cheap medium obtained at the fermenter level can completely meet the requirements of industrial production of PLD.

Synergistic haemolytic activity and its correlation to phospholipase D productivity by Corynebacteruim pseudotuberculosis Egyptian isolates from sheep and buffaloes

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).

Pharmacokinetics of pegylated liposomal doxorubicin in Chinese tumor patients / 中国药学杂志

OBJECTIVE: To investigate the pharmacokinetics of pegylated liposomal doxorubicin (PLD) in Chinese tumor patients. METHODS: Ten patients with malignant tumors were given PLD (20-35 mg · m-2) combined chemotherapy. Serial plasma samples were obtained after completion of the infusion. The doxorubicin concentrations in plasma were measured using high-performance liquid chromatography (HPLC). The pharmacokinetic parameters were estimated by DAS software. The correlations between those parameters and gender were analyzed. RESULTS: The doxorubicin plasma concentration in the 10 patients ranged from 1.68 to 10.10 mg · L-1 during 0 - 168 h after administration. The AUC0-∞, CL, t1/2 and Vd were (918.6 ± 447.4) mg · L · h-1, (0.037 ± 0.02) L · h · m-2, (64.9 ± 28.4) h and (2.95 ± 0.88) L · m-2, respectively. The differences of the pharmacokinetic parameters, ie AUC0-∞, CL, t1/2 and Vd, between genders were not statistically significant. CONCLUSION: The pharmacokinetics of PLD fits one-compartment model, with long retention time, low clearance and small volume of distribution. Copyright 2012 by the Chinese Pharmaceutical Association.

Phospholipase D and Pathogenic Microorganisms Invasion / 微生物学通报

Phospholipase D(PLD) is ubiquitous in bacteria,fungi,and mammal.In pathogenic microorganisms,PLD can be pathogenic determinant and play a role in spore generation.In mammalian cells,PLD functions in several signal transduction pathways,such as membrane transportation,mitosis regulation,and actin cytoskeleton regulation.In the process of pathogens invasion host cells,both of the pathogen and host cells’ PLD will be activated and a series of cascade reaction will be generated.During this process,pathogen’s PLD can regulate the polymerization and reorganization of its own actin filaments and induce the polymerization or reorganization of the host cell actin filaments near the foci,thus to promote the phagocytosis of the pathogen by host cell.Investigating the role of PLD activation in the infection will be significance for further understanding the molecular mechanism of pathogen-host cell interaction.

Recombinant Human PLD2(rhPLD2)May Significantly Inhibit Expression of GPI-PLD of Guinea Pigs of Chronic Asthma in vivo / 中国生物化学与分子生物学报

The effect of recombinant human phospholipase D2(rhPLD2)in vivo was investigated on the secretion of serum glycosyl phosphatidylinositol-specific phospholipase D(GPI-PLD)in guinea pigs of chronic asthma．Ater treating the guinea pigs attacked by chronic asthma with rhPLD2,the GPI-PLD activity detection was canrried out by phase separation of human placental alkaline phosphatase in Triton X-114．Compared with the healthy guinea pigs(NS group),the serum GPI-PLD in the guinea pigs of chronic asthma are much higher than that of control groups,P≤0．01．Our results showed that rhPLD2 could significantly reduce the secretion of GPI-PLD when the guinea pigs were attacked by chronic asthma．

Phospholipase D is involved in oxidative stress-induced migration of vascular smooth muscle cells via tyrosine phosphorylation and protein kinase C

Oxidative stress has been implicated in mediation of vascular disorders. In the presence of vanadate, H2O2 induced tyrosine phosphorylation of PLD1, protein kinase C-a (PKC-a), and other unidentified proteins in rat vascular smooth muscle cells (VSMCs). Interestingly, PLD1 was found to be constitutively associated with PKC-a in VSMCs. Stimulation of the cells by H2O2 and vanadate showed a concentration-dependent tyrosine phosphorylation of the proteins in PLD1 immunoprecipitates and activation of PLD. Pretreatment of the cells with the protein tyrosine kinase inhibitor, genistein resulted in a dose-dependent inhibition of H2O2-induced PLD activation. PKC inhibitor and down-regulation of PKC abolished H2O2-stimulated PLD activation. The cells stimulated by oxidative stress (H2O2) caused increased cell migration. This effect was prevented by the pretreatment of cells with tyrosine kinase inhibitors, PKC inhibitors, and 1-butanol, but not 3-butanol. Taken together, these results suggest that PLD might be involved in oxidative stress-induced migration of VSMCs, possibly via tyrosine phosphorylation and PKC activation.

Phospholipase D activity is elevated in hepatitis C virus core protein-transformed NIH3T3 mouse fibroblast cells

Hepatitis C Virus (HCV) is associated with a severe liver disease and increased frequency in the development of hepatocellular carcinoma. Overexpression of HCV core protein is known to transform fibroblast cells. Phospholipase D (PLD) activity is commonly elevated in response to mitogenic signals, and has also been overexpressed and hyperactivated in some human cancer cells. The aim of this study was to understand how PLD was regulated in the HCV core protein-transformed NIH3T3 mouse fibroblast cells. We observed that PLD activity was elevated in the NIH3T3 cells overexpressing HCV core protein over the vector alone-transfected control cells, however, expression levels of PLD protein and protein kinase C (PKC) in the HCV core protein-transformed cells was similar to the control cells. Phorbol 12-myristate 13-acetate (PMA), which is known to activate PKC, stimulated PLD activity significantly more in the core protein-transformed cells, in comparison with that of the control cells. PLD activity assay using PKC isozyme-specific inhibitor and PKC translocation experiment showed that PKC-delta was mainly involved in the PMA- induced PLD activation in the core-transformed cells. Moreover, in cells overexpressing HCV core protein, PMA also stimulated p38 kinase more potently than that of the control cells, and an inhibitor of p38 kinase abolished PMA-induced PLD activation in cells overexpressing HCV core protein. Taken together, these results suggest that PLD might be implicated in core protein-induced transformation.

Comparative Analysis of Phospholipase D2 Localization in the Pancreatic Islet of Rat and Guinea Pig

To examine the localization pattern of phospholipase D2 (PLD2) in the pancreatic islet (the islet of Langerhans) depending on species, we conducted a morphological experiment in the rat and guinea pig. Since individual islets display a typical topography with a central core of B cell mass and a peripheral boundary of A, D, and PP cells, double immunofluorescent staining with a panel of antibodies was performed to identify PLD2-immunoreactive cells in the islets PLD2 immunoreactivity was mainly present in A and PP cells of the rat pancreatic islets. And yet, in the guinea pig, PLD2 immunoreactivity was exclusively localized in A cells, and not in PP cells. These findings suggest a possibility that PLD2 is mainly located in A cells of rodent pancreatic islets, and that the existence of PLD2 in PP cells is not universal in all species. Based on these results, it is suggested that PLD2 may play a significant role in the function of A and/or PP cells via a PLD-mediated signaling pathway.

Immunohistochemical Localization of Phospholipase D1 in Developing Rat Forebrain / 대한해부학회지

Phospholipase D (PLD), one of the intracellular signal transduction enzymes, may play an important role in developing brain. However, the developmental regulation of PLD protein has not been determined. In the present study, we investigated the temporal and spatial expression of PLD isozyme, PLD1 in the developing rat forebrain using an affinity-purified peptide antibody against PLD1. Our data showed that immunoreactivity for PLD1 was first seen in the germinal zone of the lateral ventricle, differentiating neurons and their processes at embryonic day 18 (E18). At E20, clusters of immunoreactive cells were observed in the medial germinal zone of the lateral ventricle, restricted zones of the frontal and parietal cortex, the nuclei of the medial septum and the diagonal band. During the first postnatal week, there was an increase in the number and staining intensity of the immunoreactive neurons in the cerebral cortex, which peaked at postnatal day 7 (P7). During the second postnatal week, there was an abrupt decrease in the number of immunoreactive cortical pyramidal neurons. By P14, only a few of the pyramidal neurons in cerebral cortex layer V were immunoreactive. These results revealed that expression of PLD1 protein at various stages of development of the septum and cerebral cortex is differentially regulated. This suggests that PLD1 may regulate the developmental processes of some neuronal populations.

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.

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.

Differential Effect of Genistein and Calphostin C on Phospholipase C Activation and Cell Proliferation in T98G Human Glioblastoma and Hs 683 Human Glioma Cells

Amajor role in sustaining tumors like gliomas has been attributed to growth factors. Many questions remain unanswered about how such external signals are transduced into a transformed phenotype. Growth factors such as PDGF and epidermal growth factor(EGF) activate PLC, and this activation requires the intrinsic tyrosine kinase activity of the growth factor receptor. There are only a few reports on PKC activity in astrocytoma cells, especially in human glioma cells. We focused on signal transduction of phospholipase C(PLC) and phospholipase D(PLD) in human glioma cells. In this study, using genistein and calphostin C, the regulation of PLC, PLD, and PKC was investigated. The results are as follows; 1) Genistein is a selective inhibitor of PDGF-induced PLC- and PLD activation in T98G glioblastoma cells but not in Hs 683 glioma cells. 2) Calphostin-C stimulates PLC and PLD, possibly through a PKC-independent pathway in both T98G and Hs 683 cells. 3) Both genistein and calphostin-C inhibit glioma cell proliferation, indicating that the pathway for activation of PLC and PLD is not relevant to the pathway of cell proliferation in glioma cells.

Design of plasma sterilization equipment and process control using H2O2 in low temperature / 医疗卫生装备

Objective To develop the plasma sterilization using H2O2 in low temperature. Methods According to the principle of plasma sterilization, the working process of the plasma sterilization using H2O2 in low temperature and its flow chart were designed. The requirement for system design was summarized, on the basis of which the structure frame was built up. The station diagram of movement of the gates controlled by PLD was put out by analyzing the process of their moving. The MCU acted as the main controller of the whole system. The action flow chart of the MCU controlling the process of the system was also given out. Results The process control in plasma sterilization equipment using H2O2 in low temperature achieved the stable running and low cost. Conclusion It is proved that this plan is feasible in implementation.

EFFECTS OF SELENIUM ON GENE EXPRESSION OF PHOSPHOLIPASE D GENE IN LIVER IN DIABETIC RATS / 营养学报

Objective: To study the regulation of selenium on gene expression of phospholipase D (PLD) gene in relation to liver metabolism of diabetic (DM) rats. Method: Differential display cDNA fragments were isolated from former research project in diabetic rats which were supplemented orally with 50 g/kg bw d selenium for 60d, and were cloned, sequenced, and the homology was analyzed. RT-PCR was made using the primers designed according to the sequences of cDNA fragments. Results: In differential display cDNA fragment, Se-4 and PLD were homologous with sequence identities of 100%.The expression levels of PLD mRNA in DM group and DM+Se group was higher obviously than that of NC group(P