Isolation and gene expression analysis of Phospholipase C in response to abiotic stresses from Vigna radiata (L.) Wilczek.

Phosphatidylinositol (PtdIns) is a major phospholipid in eukaryotic cells. Many studies have revealed that the phosphoinositide (PI) signaling pathway plays an important role in plant growth and development. Phospholipase C (PLC) is reported to have a crucial role in the PI pathway. This work focuses on the isolation and investigation of PLC in response to abiotic stress factors in green gram. The PLC cDNA, designated VrPLC, encoding a protein of 591 amino acids was cloned and expressed in E. coli.The predicted isoelectric point (pI) and molecular weight were 5.96 and 67.3 kDa, respectively. The tertiary structure of the PLC was also predicted and found to be mainly composed of random coils. In addition, VrPLC expression analysis was performed under environmental stress and the results showed that the expression of VrPLC was rapidly induced in an abscisic acid independent manner in response to drought and salt stress. PLC expression was found to be up-regulated by SA and down-regulated by wound in leaf tissues; however, there was no significant difference in the expression of PLC in plants subjected to high temperature and H2O2. Our results suggest that a close link/relationship between PLC expression and stress responses in green gram.

Increased activity of goat liver plasma membrane alkaline phosphatase upon release by phosphatidylinositol-specific phospholipase C.

Mammalian alkaline phosphatase (ALP) is attached to the plasma membrane by a unique glycosylphosphatidylinositol (GPI) anchor. The influence of such a complex anchoring device on the enzyme function is not fully understood. Here, we report the effect of cleavage of the GPI anchor on the activity of goat liver plasma membrane ALP (GLPM-ALP). Phosphatidylinositol-specific phospholipase C (PI-PLC) purified from Bacillus cereus was used for the cleavage of the GPI anchor (delipidation) and hence for release of ALP from the membrane. Detergents — octyl-β-D-glucopyranoside (OG) and triton X100 (TX100) were also used for solubilization of ALP from the membrane. Resistance to solubilization by TX100 suggested the association of GPI-ALP with lipid rafts. Solubilization of GLPM-ALP with OG had no effect on the enzyme activity; however, delipidation with PI-PLC resulted in enhanced ALP activity. Kinetic analysis showed catalytic activation of PI-PLC-treated GLPM-ALP with an increase in Vmax (35%) without a significant change in Km. Moreover, this change in Vmax was observed to be independent of pH and buffer. The results suggested the implication of GPI anchor in modulating the catalytic property of GLPM-ALP, thus indicating the role of this special anchoring structure in the enzyme regulation.

Role of PLC-PIP2 and cAMP-PKA signal pathways in radiation-induced immune-suppressing effect / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>The purpose of the present study was to observe the changes in CD4+CD25+Nrp1+Treg cells after irradiation with different doses and explore the possible molecular mechanisms involved.</p><p><b>METHODS</b>ICR mice and mouse lymphoma cell line (EL-4 cells) was used. The expressions of CD4, CD25, Nrp1, calcineurin and PKC-α were detected by flow cytometry. The expressions of TGF-β1, IL-10, PKA and cAMP were estimated with ELISA.</p><p><b>RESULTS</b>At 12 h after irradiation, the expression of Nrp1 increased significantly in 4.0 Gy group, compared with sham-irradiation group (P<0.05) in the spleen and thymus, respectively, when ICR mice received whole-body irradiation (WBI). Meanwhile the synthesis of Interleukin 10 (IL-10) and transforming growth factor-β1 (TGF-β1) increased significantly after high dose irradiation (HDR) (> or = 1.0 Gy). In addition, the expression of cAMP and PKA protein increased, while PKC-α, calcineurin decreased at 12h in thymus cells after 4.0 Gy X-irradiation. While TGF-β1 was clearly inhibited when the PLC-PIP2 signal pathway was stimulated or the cAMP-PKA signal pathway was blocked after 4.0 Gy X-irradiation, this did not limit the up-regulation of CD4+CD25+Nrp1+Treg cells after ionizing radiation.</p><p><b>CONCLUSION</b>These results indicated that HDR might induce CD4+CD25+Nrp1+Treg cells production and stimulate TGF-β1 secretion by regulating signal molecules in mice.</p>

Expression of phospholipase C epsilon-1 in gastric cancer and its association with prognosis / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To study the expression of phospholipase C epsilon-1(PLCE1) and its clinical significance in gastric cancer.</p><p><b>METHODS</b>Surgical specimens were collected from 125 patients who underwent radical gastrectomy between 2005 and 2007 in the Zhejiang Provincial Peoples' Hospital. Expression level of PLCE1 protein was measured by immunohistochemistry in these 125 surgical specimens, which included primary gastric cancer and matched adjacent normal gastric mucosa tissues, and then 41 pairs of above specimens were selected randomly to examine the expression level of PLCE1 mRNA by quantitative reverse transcription PCR(qRT-PCR).</p><p><b>RESULTS</b>Immunohistochemistry and qRT-PCR showed that both protein and mRNA level of PLCE1 were up-regulated in gastric cancer compared with paired normal gastric mucosa. Univariate analysis demonstrated that the expression of PLCE1 was significantly associated with differentiation degree, invasion depth, lymph node metastasis, distant metastasis, and TNM stage (all P<0.01). The 5-year survival rate of positive PLCE1 group was significantly lower as compared to negative group (31.2% vs. 54.0%, P<0.01). However, the expression of PLCE1 was not an independent prognostic factor for gastric cancer (P>0.05).</p><p><b>CONCLUSIONS</b>PLCE1 is up-regulated in gastric cancer, which is associated with the malignant biological behaviors of gastric cancer. High expression of PLCE1 suggests poor prognosis.</p>

The promoting research of phospholipase C epsilon-1 on nasal Th2 cell polarization / 临床耳鼻咽喉头颈外科杂志

Phospholipase C epsilon-1 (PLCE1) is a phospholipase C isoenzyme encoded by PLCE1 gene, and has more complicated molecular structure and function than other subtypes. Phospholipase C epsilon-1 is accepted the dual regulation by the upstream G proteins and GTP enzymes of Ras family. The downstream signal of PLCE1 is not only cause the Ca2+ flow and protein kinase C(PKC) activation, but also can be used as the GTP enzyme guanylic acid conversion factor of Ras superfamily, so as to regulate the expression of certain genes, adjusting cell growth and differentiation processes. PLCE1 plays a very important role in the signal transduction in the regulation of cell growth, differentiation, proliferation and apoptosis. Previous studies showed that phospholipase C epsilon-1 played an important role in the development of malignant tumors (especially the digestive tumors), heart disease, nephrotic syndrome and other diseases, but there are some questions about the mechanisms of PLCE1 involved in allergic rhinitis, this article will make an overview about PLCE1 promotes allergic rhinitis CD4+ T cells differentiate to Th2 cells by PKC-NF-κB pathway and Ras-MAPK pathway.

Genetic polymorphism of the phospholipase C epsilon 1 gene and risk of gastric cancer / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The pathogenesis of gastric cancer (GC) involves environmental and genetic factors. Recently, two genome-wide association studies found that phospholipase C epsilon 1 (PLCE1) polymorphisms might be related to GC risk, and several studies further validated this finding. However, these studies yielded inconsistent results.</p><p><b>METHODS</b>A comprehensive database search was performed to identify eligible studies. Odds ratios with 95% confidence intervals were calculated to assess the strength of the association between PLCE1 rs2274223, rs753724, and rs11187842 and risk of GC. Subgroup analyses, publication bias, and sensitivity analyses were also conducted.</p><p><b>RESULTS</b>Eleven studies (12 cohorts) were included in the meta-analysis. Based on 13 676 cases and 23 569 controls, a significant association between PLCE1 rs2274223 and GC risk was detected under various genotypic models. In the subgroup analyses, the association was significant for cardia GC, but weak for non-cardia GC. The association under the heterozygote model was detected for PLCE1 rs753724 and rs11187842 based on three studies involving 2768 cases and 3890 controls.</p><p><b>CONCLUSIONS</b>Our findings demonstrate that the presence of the G allele at rs2274223 of the PLCE1 gene may contribute to susceptibility to GC, especially cardia GC. PLCE1 rs753724 and rs11187842 are associated with GC risk under the heterozygote model. Further well-designed large studies are warranted to validate these findings.</p>

Relationship between rs2274223 and rs3765524 polymorphisms of PLCE1 and risk of esophageal squamous cell carcinoma in a Kazakh Chinese population / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the association between the rs2274223 and rs3765524 polymorphism of phospholipase C epsilon 1 (PLCE1) gene and the susceptibility to develop esophageal squamous cell carcinoma (ESCC) in a pure Kazakh Chinese population.</p><p><b>METHODS</b>Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) was utilized to genotype the potentially functional single nucleotide polymorphism rs2274223 A>G and rs3765524 C>T of PLCE1 in an ongoing hospital-based and case-control study of 200 ESCC cases with 300 cancer-free age ( ± 5 years) and sex matched controls. Statistical analyses were performed with Statistical Products and Services Solutions software (version 13.0). Adjusted odds ratios (OR) and 95% confidence evaluation intervals (95%CI) measured by multivariate logistic regression analysis were adopted to study the correlation of the gene polymorphism with the susceptibility to ESCC.</p><p><b>RESULTS</b>The genotype frequencies observed for rs2274223 was consistent with Hardy-Weinberg equilibrium in controls. Univariate analysis revealed significant differences between cases and controls with respect to genotype distribution for rs2274223 (P = 0.006). The variants of rs2274223 were found to confer significantly increased risk of ESCC (GG vs AA: OR = 3.17, 95%CI = 1.45-6.93; AG/GG vs AA: OR = 1.55, 95%CI = 1.08-2.22) in the Kazakh Chinese population. Moreover, AG/GG genotype of rs2274223 was found to be significantly associated with poorly-differentiated ESCC (OR = 2.48, 95%CI = 1.10-5.60). When the ESCC patients were divided into two subgroups, stage I/II and stage III/IV according to the AJCC TNM classification, the GT/GG genotype of rs2274223 was significantly associated with stage III/IV ESCC (OR = 1.85, 95%CI = 1.05-3.25). No significant association was found between rs3765524 and Kazakh ESCC.</p><p><b>CONCLUSIONS</b>These results indicate that rs2274223 site polymorphism of the PLCE1 gene is strongly associated with risk of ESCC in a Kazakh Chinese population, especially the poorly-differentiated and stage III/IV ESCC.</p>

Syringaresinol causes vasorelaxation by elevating nitric oxide production through the phosphorylation and dimerization of endothelial nitric oxide synthase

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an important role in vascular functions, including vasorelaxation. We here investigated the pharmacological effect of the natural product syringaresinol on vascular relaxation and eNOS-mediated NO production as well as its underlying biochemical mechanism in endothelial cells. Treatment of aortic rings from wild type, but not eNOS-/- mice, with syringaresinol induced endothelium-dependent relaxation, which was abolished by addition of the NOS inhibitor NG-monomethyl-L-arginine. Treatment of human endothelial cells and mouse aortic rings with syringaresinol increased NO production, which was correlated with eNOS phosphorylation via the activation of Akt and AMP kinase (AMPK) as well as elevation of intracellular Ca2+ levels. A phospholipase C (PLC) inhibitor blocked the increases in intracellular Ca2+ levels, AMPK-dependent eNOS phosphorylation, and NO production, but not Akt activation, in syringaresinol-treated endothelial cells. Syringaresinol-induced AMPK activation was inhibited by co-treatment with PLC inhibitor, Ca2+ chelator, calmodulin antagonist, and CaMKKbeta siRNA. This compound also increased eNOS dimerization, which was inhibited by a PLC inhibitor and a Ca2+-chelator. The chemicals that inhibit eNOS phosphorylation and dimerization attenuated vasorelaxation and cGMP production. These results suggest that syringaresinol induces vasorelaxation by enhancing NO production in endothelial cells via two distinct mechanisms, phosphatidylinositol 3-kinase/Akt- and PLC/Ca2+/CaMKKbeta-dependent eNOS phosphorylation and Ca2+-dependent eNOS dimerization.

NYD-SP27, a novel intrinsic decapacitation factor in sperm / 亚洲男科学杂志(英文版)

Prior to fertilization sperm has to undergo an activation process known as capaciation, leading to the acrosome reaction. Till now, little is known about the mechanism for preventing premature capacitation in sperm although decapacitation factors from various sources have been thought to be involved. In this study, we report that NYD-SP27, an isoform of phospholipase C Zeta 1 (PLCZ1), is localized to the sperm acrosome in mouse and human spermatozoa by immunofluorescence using a specific antibody. Western blot and double staining analyses show NYD-SP27 becomes detached from sperm, as they undergo capacitation and acrosome reaction. The absence of HCO3-, a key factor in activating capacitation, from the capacitation-inducing medium prevents the loss of NYD-SP27 from sperm. The anti-NYD-SP27 antibody also prevents the loss of NYD-SP27 from sperm, reduced the number of capacitated sperm, inhibited the acrosome reaction induced by ATP and progesterone, and inhibited agonist-induced PLC-coupled Ca2+ mobilization in sperm, which can be mimicked by the PLC inhibitor, U73122. These data strongly suggest that NYD-SP27 is a physiological inhibitor of PLC that acts as an intrinsic decapacitation factor in sperm to prevent premature capacitation and acrosome reaction.