The present study was undertaken to observe the quality and quantity of lipids in the adult worms of Chinese liverfluke, Clonorchis sinensis. Lipid extraction was done by the methods of Folch et a1. (l957) and Kenny (1952), and then the extracted lipid fractions of the worm were separated by thin layer chromatography. Those fractions were also subjected to perform the quantitative analyses of glycerides, cholesterols and phospholipids. The results obtained were summarized as follows: Total amount of glyceride was 37.56 mg per gram of worm tissue and the amount of monoglyceride was 8.34 mg per gm; diglyceride, 15.46 mg per gm; and triglyceride, 12.86 mg per gm. Total amount of cholesterol was 3.30 mg per gm of worm tissue, and the esterified cholesterol (1.72 mg/gm) was a little more than that of free cholesterol (1.26 mg/gm). The following 8 phospholipids were detected in the worm tissue of C. sinensis, i.e., lysophosphatidylcholine, phosphatidylcholine, phophatidylinositol, sphingomyelin, phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine and an unknown phospholipid.
parasitology-helminth-trematoda ; Clonorchis sinensis ; biochemistry ; glyceride ; cholesterol ; phospholipid ; lipid ; lysophosphatidylcholine ; phosphatidylcholine ; phophatidylinositol ; sphingomyelin ; phosphatidylglycerol ; phosphatidylserine ; phosphatidylethanolamine

OBJECTIVETo explore the mechanism of cell proliferation inhibition by transfecting phosphatidylethanolamine N-methyltransferase 2 gene (PEMT2).

METHODSThe expression and translocation of different isoforms of protein kinase C (PKC) in cells were observed with immunocytochemistry and Western blot techniques. The content of diacylglycerol (DAG) was analyzed with high performance thin layer chromatography (HPTLC) technique.

RESULTSTransfection of PEMT2 can inhibit the expression of cPKC alpha, but obviously promotes the expression and translocation from cytosol to plasma membrane of cPKC beta2. At the same time, the content of DAG was decreased in the transfected cells. Expression and translocation of other PKC isoforms were not changed by PEMT2 transfection.

CONCLUSIONEffects of overexpression of PEMT2 on the expression and translocation of different PKC isoforms might be related to the mechanism of cell proliferation inhibition and apoptosis induced by transfecting PEMT2.

Animals ; Liver Neoplasms, Experimental ; enzymology ; pathology ; Phosphatidylethanolamine N-Methyltransferase ; biosynthesis ; genetics ; Protein Isoforms ; Protein Kinase C ; biosynthesis ; genetics ; Rats ; Transfection

OBJECTIVE: To investigate whether endogenous nociceptin/orphanin FQ (N/OFQ) can inhibit arrhythmia and expression of β₁-adrenergic receptor (β₁-AR) on the surface of myocardial cell membrane in acute myocardial ischemia rats by Raf kinase inhibitory protein (RKIP). METHODS: (1) Experiment one: according to random number table method, 30 adult male Sprague-Dawley (SD) rats with only 6 weeks of age were divided into Sham group (open the chest but do not ligate the coronary artery), myocardial ischemia model group (coronary ligation of left anterior descending branch), and endogenous N/OFQ antagonists UFP-101 pretreatment group (UFP-101 group, preoperative 10 minutes after tail vein injection of 1 mL/kg UFP-101), with 10 rats in each group. Arrhythmia was recorded within 15 minutes after operation. The expression of phosphorylated RKIP (p-RKIP) was detected by Western Blot. (2) Experiment two: according to the random number table method, 30 4-week-old male SD rats were divided into UFP-101 control group, RKIP over expression group and RKIP antagonism group, with 10 rats in each group. The UFP-101 control group was intraperiton eally injected with corn oil every day, while the other two groups were injected with up adjuster of RKIP (Didymin). The rats in the three groups were all ligated after 4 weeks of feeding, and UFP-101 was injected through the tail vein 10 minutes before the operation. The RKIP antagonist group received intraperitoneal injection of the RKIP-specific antagonist locostatin 2 hours before surgery. Arrhythmia results were recorded within 15 minutes after operation. Western Blot was used to detect the expression of p-RKIP in myocardial tissue and expression of β₁-AR on the surface of myocardial cell membrane 15 minutes after surgery. RESULTS: (1) Experiment one: compared with Sham group, ventricular ectopic beat (VEB), ventricular tachycardia (VT) and ventricular fibrillation (VF) increased significantly in the model group and UFP-101 group, and arrhythmia score increased significantly. In addition, compared with the Sham group, p-RKIP expression was increased in the model group and decreased in the UFP-101 group. Compared with the model group, preconditioning with UFP-101 significantly reduced the occurrence of arrhythmia [arrhythmia score: 1.5 (0.3, 5.0) vs. 4.0 (2.0, 5.0), P < 0.05], and the expression of p-RKIP in myocardial tissue significantly decreased (p-RKIP/total RKIP: 0.20±0.11 vs. 0.43±0.11, P < 0.05). This indicated that antagonistic N/OFQ could reduce the phosphorylation of RKIP and the occurrence of arrhythmia. (2) Experiment two: compared with the UFP-101 control group, overexpression of RKIP significantly increased the occurrence of arrhythmia events, and the expression of β₁-AR on the surface of the myocardial cell membrane significantly increased. And antagonism RKIP overexpression could make the occurrence of arrhythmia eased [arrhythmia score: 3.0 (2.0, 3.0) vs. 4.0 (2.0, 5.0), P < 0.05], and significantly reduce the expression of myocardial cell membrane surface β₁-AR (β₁-AR/Na⁺-K⁺-ATPase: 0.88±0.09 vs. 1.02±0.08, P < 0.05), while there was no significant difference in total RKIP expression (total RKIP/GAPDH: 5.40±0.21 vs. 5.36±0.19, P > 0.05). This indicated that endogenous N/OFQ affected the expression of plasma β₁-AR on the surface of myocardial cell membrane and ischemic arrhythmia in rats through RKIP. CONCLUSIONS Endogenous N/OFQ can affect the expression of plasma β₁-AR on the membrane surface of ischemic myocardium and arrhythmia in rats via increased expression of RKIP phosphorylation.
Animals ; Arrhythmias, Cardiac ; Male ; Opioid Peptides/metabolism* ; Phosphatidylethanolamine Binding Protein ; Rats ; Rats, Sprague-Dawley ; Receptors, Opioid ; Nociceptin

This assay was designed to construct the prokaryotic expression vector, investigate the expression of PEBP-like in Escherichia coli and purify its product. The PEBP gene was inserted into the vector pET30a (+). The recombinant vector was transferred into E. coli BL21 (DE3)and induced the expression of protein by low concentration of IPTG and low temperature overnight. After purification, the supernatants were analyzed by SDS-PAGE and the results were identified by Western blotting. After IPTG induction, a new anticipating fusion protein of 28 kD appeared as an expected size, and its product was 26.8% in total protein, the fusion protein was positive by Western blotting. The prokaryotic expression system of PEBP-like is successfully constructed. It lays the foundation for the further application study on the antifreeze characters of the PEBP.
Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Plant ; Phosphatidylethanolamine Binding Protein ; genetics ; metabolism ; Plant Proteins ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Saussurea ; genetics

Phosphatidylethanolamine-binding protein (PEBP) is a cytoplasm soluble protein with a high conserved structure. It has been approved recently that PEBP is a multifunctional molecule regulating several important cellular signal pathways, including ERK cascade, NF-κB pathway, and signaling of G protein-coupled receptors. Furthermore, the role of PEBP in tumor metastasis also got a comprehensive attention in the field of clinical cancer research. Together, as a signal regulator at multiple paths in cell, PEBP is becoming a new focus in several research fields. This review is aimed to introduce the newest biological progress on PEBP.
Humans ; MAP Kinase Signaling System ; NF-kappa B ; physiology ; Neoplasms ; Phosphatidylethanolamine Binding Protein ; physiology ; Receptors, G-Protein-Coupled ; physiology ; Signal Transduction

To screen factors related to spermatogonial stem cell (SSC) proliferation, and to investigate the mechanism of infertility caused by cryptorchidism, ten-day-old Kunming (KM) mice were used and experimental cryptorchidism was conducted. On the 35th day after cryptorchid operation, the left testes were fixed in Bouin's fluid and used for histological analysis. The testes of 45-day-old mice were subjected to the same histological analysis, and it was found that they contained germ cells at every stage of development, from SSCs to sperm, indicating that the animals were fully sexually mature at this age. While in experimental cryptorchid mice, the spermatogenesis was arrested at the stage of spermatocytes, and only spermatogonia and primary spermatocytes were present in cryptorchid testes. The proportion of spermatogonia to other types of germ cells was much higher than that in sexually mature mice. On the other hand, the right testes were used for proteomic analysis. The total protein in testes was extracted on the 35th day after cryptorchid operation. The differentially expressed proteins in cryptorchid mice and sexually mature mice were screened and compared by the proteomic techniques. Through the separation of two-dimensional gel electrophoresis (2-DE), 20 differential protein spots were found, and 9 of them were digested and identified by the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrum. In cryptorchid mice, 6 out of 9 proteins were down-regulated, and 3 were up-regulated. Among these proteins, 4 proteins were identified, and they were Stathmin, phosphatidylethanolamine-binding protein1 (PEBP1), HES-related basic helix-loop-helix protein (HERP), and one unnamed protein (we temporarily named it Px). More Stathmin, PEBP1 and Px were expressed in sexually mature mice than in experimental cryptorchid mice. But HERP1 was the other way round. In the present study, we have screened 4 proteins related to cryptorchidism. It is helpful to study the mechanism of SSC proliferation and infertility caused by cryptorchidism.
Animals ; Cryptorchidism ; metabolism ; Male ; Membrane Proteins ; analysis ; Mice ; Phosphatidylethanolamine Binding Protein ; analysis ; Proteomics ; methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Stathmin ; analysis ; Testis ; chemistry

OBJECTIVE: To detect the expression of Raf kinase inhibitor protein (RKIP) and epithelial cadherin (E-cadherin) in human prostate cancer tissues, and their correlation. METHODS: We discussed the relationship between RKIP and E-cadherin and the clinical stage and pathological classification of prostate cancer by immunofluorescence histochemistry staining in the test of expression of RKIP in 26 prostate cancer tissues and 14 BPH tissues, and analyzed the correlation between them. RESULTS: The expression of RKIP and E-cadherin in prostate cancer tissues was obviously lower than that in the benign prostatic hypertrophy tissues. The expression of RKIP and E-cadherin in the dys-good differentiation group (Gleason 8-10) was significantly lower than that in the good differentiation group(GleasonCONCLUSION RKIP and E-cadherin are metastasis suppressor factors, whose decreased expression can increase the invasive capability of prostate cancer and suppress its differentiation. RKIP can suppress the metastasis of prostate cancer by increasing the E-cadherin expression in prostate cancer.
Adenocarcinoma ; metabolism ; pathology ; Aged ; Aged, 80 and over ; Cadherins ; genetics ; metabolism ; Fluorescent Antibody Technique ; Humans ; Male ; Middle Aged ; Neoplasm Metastasis ; Phosphatidylethanolamine Binding Protein ; genetics ; metabolism ; Prostatic Neoplasms ; metabolism ; pathology

OBJECTIVE: To investigate the effect of miR-4443 expression on migration and invasion of breast cancer. METHODS: We examined the expression of miR-4443 in breast carcinoma in situ and paired adjacent tissues from 3 breast cancer patients with high-throughput sequencing and verified the results using TCGA database. We also detected miR-4443 expressions using real-time quantitative PCR (RT-qPCR) in low invasive and highly invasive breast cancer cells (MCF-7 and MDA-MB-231 cells, respectively). The changes in apoptosis, migration and invasion of MCF-7 and MDA-MB-231 cells after transfection with miR-4443 mimics, mimics-NC, miR-4443 inhibitor or inhibitor-NC were analyzed using flow cytometry, wound healing assay and Transwell invasion assay. The target gene of miR-4443 was predicted by bioinformatics software and validated by a dual luciferase reporter gene system. RT-qPCR and Western blotting were performed to detect the expression of recombinant human phosphatidyl ethanolamine binding protein 1 (PEBP1) in the transfected cells. RESULTS: The expression of miR-4443 was significantly higher in the breast cancer tissues than in the adjacent tissues ( CONCLUSIONS MiR-4443 promotes the migration and invasion of breast cancer cells by inhibiting the expression of PEBP1, suggesting the possibility of suppressing miR-4443 expression as a potential therapeutic strategy for breast cancer.
Breast Neoplasms/genetics* ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Humans ; MCF-7 Cells ; MicroRNAs/genetics* ; Neoplasm Invasiveness/genetics* ; Phosphatidylethanolamine Binding Protein

OBJECTIVETo study the development of changes for Raf kinase inhibitor protein (RKIP) and its mRNA in rats hippocampus after electromagnetic radiation.

METHODSRats were exposed to X-band high power microwave (X-HPM), S-band high power microwave (S-HPM) and electromagnetic pulse (EMP) radiation source respectively. The animal model of electromagnetic radiation was established. Western blot was used to detect the expression of RKIP, and RT-PCR was applied to detect the expression of RKIP mRNA. The interaction of RKIP and Raf-1 was measured with co-immunoprecipitation method, and the expression of cerebral choline acetyltransferase (CHAT) was measured by immunohistochemistry.

RESULTSThe expression of RKIP significantly down-regulated at 6 h after radiation, and recovered at 1 d in group EMP, but the down-regulation continued during 1 approximately 7 d after radiation in the two microwave groups. The expression of RKIP mRNA changed wavily during 6 h approximately 7 d after radiation, which showed down-regulation at 6 h, and up-regulation at 3 d. The interaction of RKIP and Raf-1 decreased during 6 h approximately 7 d after radiation, most significantly at 7 d, and the two microwave groups were more significant. The expression of CHAT decreased continuously during 6 h approximately 7 d after radiation, and generally recovered on 14 d.

CONCLUSIONThe down-regulation of RKIP and its related proteins of hippocampus is induced by electromagnetic radiation.

Animals ; Electromagnetic Radiation ; Hippocampus ; metabolism ; radiation effects ; MAP Kinase Kinase Kinases ; metabolism ; Male ; Phosphatidylethanolamine Binding Protein ; genetics ; metabolism ; Proto-Oncogene Proteins c-raf ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar

The Raf kinase inhibitory protein (RKIP) has been demonstrated to modulate different intracellular signaling pathways in cancers. Studies have shown that RKIP is frequently downregulated in cancers; therefore, attempts have been made to upregulate the expression of RKIP using natural and synthetic agents for the treatment of human malignancies. Moreover, various regulators such as specific proteins and microRNAs (miRNAs) that are involved in the regulation of RKIP expression have also been identified. RKIP mechanistically modulates the apoptotic regulators of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling. Because of its critical role in human cancers, RKIP has drawn much research attention, and our understanding is expanding rapidly. Here, we summarize some of the biological complexities of RKIP regulation. However, we restrict our discussion to selected tumors by focusing on TRAIL, miRNAs and natural agents. Emerging evidence suggests a role for natural agents in RKIP regulation in cancer cells; therefore, naturally occurring agents may serve as cancer-targeting agents for cancer treatment. Although the literature suggests some advancement in our knowledge of RKIP biology, it is incomplete with regard to its preclinical and clinical efficacy; thus, further research is warranted. Furthermore, the mechanism by which chemotherapeutic drugs and novel compounds modulate RKIP and how nanotechnologically delivered RKIP can be therapeutically exploited remain to be determined.
Apoptosis ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; MicroRNAs/genetics ; Neoplasms/genetics/*metabolism ; Phosphatidylethanolamine Binding Protein/genetics/*metabolism ; Protein Interaction Maps ; *Signal Transduction ; TNF-Related Apoptosis-Inducing Ligand/genetics/metabolism