Lipocalin-type prostaglandin D synthase (L-PGDS), an N-glycosylated dual functional monomeric protein, acts as a PGD2-producing enzyme and also as a lipophilic ligand-binding protein. L-PGDS is localized in the central nervous system, male genital organs of various mammals and in the human and monkey heart, and secreted into the cerebrospinal fluid, seminal plasma and blood plasma. The L-PGDS concentrations in these body fluids are useful for the diagnosis of several neurological disorders, dysfunction of sperm formation, cardiovascular and renal diseases.
Amino Acid Sequence ; Animals ; Chromosome Mapping ; Humans ; Intramolecular Oxidoreductases ; analysis ; chemistry ; genetics ; Lipocalins ; Molecular Sequence Data

To compare the difference in tumor immunity and autoimmunity elicited by adenovirus (Ad) encoding human or murine tyrosinase-related protein 2 (AdhTRP2 or AdmTRP2), and to find the most effective way to induce immunity by AdhTRP2 or AdmTRP2, C57BL/6 mice were immunized with AdhTRP2 or AdmTRP2 intramuscularly at different doses of 10(5), 10(6), 10(7) and 10(8) separately (10 mice for each dose). Two weeks after the immunization, in vivo CTL assay and intracellular staining (ICS) of IFN-gamma were carried out to analyze the dose-effect relationship. Tumor growth and vitiligo (as an sign of autoimmunity) were observed until 3 months after challenge with 10(5) B16F10 tumor cells. The results showed that Ad encoding AdmTrp2 induced weak tumor immune response. Similar immunization with AdhTrp-2 elicited stronger protective immunity. CTL activity and IFN-gamma-produced CD8+T cells were directly proportional to dose of AdhTrp2 or AdmTrp2. Moreover, AdhTrp2 group showed tumor rejection in 100% of challenged mice till the end of 3rd month while 60% of mice immunized with AdmTrp2 were protected against tumor. In the whole process of this experiment, no vitiligo was observed in mice immunized either with AdhTrp2 or AdmTrp2. It is concluded that anti-melanoma responses induced by genetic vaccination expressing xenoantigens breaks immune tolerance effectively and is able to elicit strong antigen-specific cytotoxic T cell response without vitiligo.
Adenoviridae/metabolism ; Antineoplastic Agents/*pharmacology ; Cell Line, Tumor ; Cytokines/metabolism ; Immune System ; Immune Tolerance ; Interferon-gamma/metabolism ; Intramolecular Oxidoreductases/*biosynthesis ; Intramolecular Oxidoreductases/*genetics ; Mice, Inbred C57BL ; T-Lymphocytes, Cytotoxic/*metabolism ; Vitiligo/*metabolism

To investigate the effect of a microsomal prostaglandin E synthase-1 (mPGES-1) inhibitor MK886 on cell cycle of the human acute myeloid leukemia HL-60 cells. HL-60 cells were treated with different concentration of MK886 (10, 25, 50 µmol/L) for 24 h. Flow cytometry, Western blot and ELISA were used to measure cell cycle, cyclin D1, mPGES-1, PGE(2), Akt, P-Akt and C-MYC. The results indicated that after treated with MK886, the percentage of HL-60 cells decreased in G(0)/G(1) phase and increased in S phase, and expressions of mPGES-1, cyclin D1, P-Akt and C-MYC and synthesis of PGE(2) decreased significantly. It is concluded that MK886 can arrest HL-60 cells in G(0)/G(1) phase, the mechanism of which is possibly associated to inhibition of mPGES-1 expression, reduction of PGE(2) synthesis, suppression of Akt phosphorylation and C-MYC expression, down-regulation of cyclin D1 expression.
Cell Cycle ; drug effects ; HL-60 Cells ; Humans ; Indoles ; pharmacology ; Intramolecular Oxidoreductases ; antagonists & inhibitors ; Leukemia ; metabolism ; pathology ; Prostaglandin-E Synthases

OBJECTIVESTo express human testis Lipocalin-type prostaglandin D synthase in Pichia Pastoris for further research on biological function and clinical applications.

METHODSHuman testis L-PGDS gene coding region was amplified from plasmid pGEX-2T/htL-PGDS by PCR with a deletion of the signal peptide sequence. The DNA fragment was inserted into pPIC9 to construct yeast expression plasmid followed by transformation of the yeast GS115 strain with electroporation. The recombinant his-tag protein was induced to express by methanol.

RESULTSThe sequence of the amplified DNA fragment was identical to that of human testis L-PGDS previously reported. The recombinant protein was found with a molecular mass of 27,000 on SDS-PAGE, which was identical to that of native L-PGDS.

CONCLUSIONSSecretory expression of human L-PGDS was obtained in Pichia Pastoris.

Cloning, Molecular ; Gene Expression ; Humans ; Intramolecular Oxidoreductases ; biosynthesis ; genetics ; Lipocalins ; Male ; Pichia ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; secretion

OBJECTIVE: To determine the effect of tetramethylpyrazine (TMP) on the expression of migration inhibitory factor (MIF) in acute spinal cord injury (ASCI) in rats. METHODS: Allen's weight-drop method was used to establish a rat model of ASCI at T10. A total of 110 adult SD rats were divided into a sham operation group (group S, n=10), a control group (group C, n=50), and a TMP group (group T, n=50). Spinal cord functionality was measured by a modified Rivilin loxotic plate degree, BBB score, and combined behavioral score (CBS) at 1, 3, 5, 7, 14 and 21 d postoperatively. The injured spinal cord tissue samples were harvested at 1, 3, 6, 12 h and 1, 3, 5, 7, 14, 21 d postoperatively (n=5 at each time point) and used to prepare continuous histological sections, in which the expression of MIF was analyzed by immunohistochemistry. RESULTS: The degree in group T measured by modified Rivlin loxotic plate test after the ASCI was significantly higher than that in group C at 7, 14, and 21 d (P<0.05). BBB score in group T was significantly higher than that in group C at 5, 7, 14, and 21 d after the ASCI (P<0.05). CBS score in group C was significantly higher than that in group T at 5, 7, 14, and 21 d after the ASCI (P<0.05). The significantly low number of MIF positive cells was shown in group T when compared with that in group C at 12 h and 1, 3, 5, 7 d after the ASCI (P<0.05). As time passed, there was negative correlation between modified Rivlin loxotic plate degree and MIF expression and also between BBB score and MIF, and there was positive correlation between CBB score and MIF expression. CONCLUSION TMP has protective effect after the ASCI, and may promote the repair of injured spinal cord tissues. TMP may decrease the MIF expression in cells after the ASCI.
Animals ; Immunohistochemistry ; Intramolecular Oxidoreductases ; metabolism ; Macrophage Migration-Inhibitory Factors ; metabolism ; Pyrazines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; metabolism

OBJECTIVETo investigate the effect of lipopolysaccharide of Porphyromonas gingivalis (Pg-LPS) on the bio-thythetic pathway of prostaglandin E2 (PGE2) and its difference from lipopolysaccharide of Escherichia coli (Ec-LPS).

METHODSPurified Pg-LPS and Ec-LPS were used to stimulate a human monocytic cell strain THP-1. PGE2 concentration was determined by an enzyme immunoassay kit. The release of tritium labeled arachidonic acid (AA) was detected by a liquid scintillation counter. Reverse transcription polymerase chain reaction and western blot were used to analyse the expression of cytosolic phospholipase A2 (cPLA2) enzyme, cyclooxygenase-2 (COX-2), and microsomal prostaglandin E synthase-1 (mPGES-1).

RESULTSThe effect of Pg-LPS on induction of PGE2 and release of AA was significantly weaker than that of Ec-LPS (P < 0.05).Increased secretion of PGE2 was observed after stimulation with Pg-LPS for 6 h, which peak at 24 h at (221.40 +/- 29.46) ng/L; or with Ec-LPS for 1-48 h, at (161.80 +/- 17.31) approximately (379.80 +/- 37.35) ng/L. The highest levels of COX-2 and mPGES-1 were shown after 16 h treatment by Pg-LPS, or after 8 h and 16 h by Ec-LPS respectively.cPLA2 inhibitor AACOCF3 could lower the level of LPS-induced release of AA, while it did not influence the production of PGE2. COX-2 inhibitor NS-398 could remarkably reduce the concentration of PGE2.

CONCLUSIONSPg-LPS showed delayed and weaker effect on PGE2 biosynthetic pathway than Ec-LPS. Pg-LPS-induced PGE2 synthesis was mainly due to enhanced expression of COX-2 and mPGES-1, whereas cPLA2 played an insignificant role.

Cell Line ; Cyclooxygenase 2 ; metabolism ; Dinoprostone ; biosynthesis ; Humans ; Intramolecular Oxidoreductases ; metabolism ; Lipopolysaccharides ; pharmacology ; Monocytes ; drug effects ; metabolism ; Porphyromonas gingivalis ; Prostaglandin-E Synthases

OBJECTIVETo investigate the inhibitory effect and underlying mechanism of total saponins from Paris polyphylla var. yunnanensis on the proliferation of salivary adenoid cystic carcinoma ACC-83 cells.

METHODSIn vitro cell culture was performed. The proliferation of ACC-83 cells treated with different concentrations (5, 10, 20, 40, 60, 80, 100 μg·mL⁻¹) of total saponins from Paris polyphylla var. yunnanensis was observed using CCK-8 assay. Meanwhile, the apoptosis of ACC-83 cells treated with different concentrations (25, 50, 100 μg·mL⁻¹) of the total saponins was observed using flow cytometry. The expression levels of macrophage migration inhibitory factor (MIF) and CD74 were measured using Western blot and reverse transcription-polymerase chain reaction.

RESULTSThe total saponins from Paris polyphylla var. yunnanensis induced apoptosis and expressed dose-effect relationship. ACC-83 cells expressed MIF and CD74, and the total saponins suppressed MIF and CD74 expression in ACC-83 cells.

CONCLUSIONSThe total saponins from Paris polyphylla var. yunnanensis can significantly inhibit the proliferation, suppress MIF and CD74 expression, and promote apoptosis in ACC-83 cells. This study provides a theoretical basis for the treatment of salivary adenoid cystic carcinoma using Paris polyphylla var. yunnanensis.
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Carcinoma, Adenoid Cystic ; drug therapy ; Humans ; Intramolecular Oxidoreductases ; Liliaceae ; Macrophage Migration-Inhibitory Factors ; Rhizome ; Salivary Gland Neoplasms ; drug therapy ; Saponins ; therapeutic use

Objective To investigate the effect of 15-Deoxy-△(12,14)-prostaglandin J2 (15 d-PGJ2) on the expression of macrophage migration inhibitory factor (MIF) and its underlying mechanism in J774A.1. Methods The murine monocyte/macrophage cell line J774A.1 were divided into six groups:lipopolysaccharide (LPS) group,incubated with 1 μg/ml LPS for 1 h;normal control group,incubated with PBS for 1 h;negative control group,incubated with 5 μmol/L 15 d-PGJ2 for 1 h;15 d-PGJ2 group,incubated with 5 μmol/L 15 d-PGJ2 for 1 h followed by 1 μg/ml LPS for 1 h;GW9662 group,incubated with 5 μmol/L 15 d-PGJ2 for 1 h following GW9662 10 μmol/L for 1 h,and then incubated with 1 μg/ml LPS for 1 h;and Vehicle group,control of GW9662,GW9662 was replaced by its solvent DMSO. The expression of MIF was detected via immunofluorescence and agarose gel electrophoresis. RT-qPCR and Western blotting were used to test whether 15 d-PGJ2 could regulate mRNA and protein expression of MIF in J774A.1 upon LPS challenge. The effect of peroxisome proliferator-activated receptor-γ (PPAR-γ) antagonist GW9662 on the regulation of MIF by 15 d-PGJ2 was observed. The effects of 15 d-PGJ2 on the nuclear translocation of PPAR-γ upon LPS challenge were detected via high content screening analysis. Results MIF DNA and protein expressions were detected in J774A.1. MIF mRNA expression was up-regulated (1.75±0.09,P=0.037) when challenged with LPS and 15 d-PGJ2 inhibited its upregulation (0.84±0.08,P=0.026) in J774A.1. The protein level was consistent with the mRNA level. PPAR-γ antagonist GW9662 reversed the effect of 15 d-PGJ2 (mRNA,1.48±0.06,P=0.016;protein,1.28). Furthermore,nuclear translocation of PPAR-γ was regulated by 15 d-PGJ2 in J774A.1 upon LPS challenge(1.39±0.02 vs. 1.01±0.03,P=0.003). Conclusion 15 d-PGJ2 may down-regulate the MIF expression in J774A.1 in a PPAR-γ-dependent manner.
Anilides ; pharmacology ; Animals ; Cell Line ; Intramolecular Oxidoreductases ; metabolism ; Lipopolysaccharides ; Macrophage Migration-Inhibitory Factors ; metabolism ; Mice ; Monocytes ; drug effects ; PPAR gamma ; antagonists & inhibitors ; Prostaglandin D2 ; analogs & derivatives ; pharmacology

To determine the precise chromosomal localization of tyrosine related protein-1 and -2 (TRP-1 and TRP-2) genes by fluorescence in situ hybridization, we used DNAs isolated from human bacterial artificial chromosome clones. They contain genomic sequences with approximately 120 kb inserts for TRP-1 and TRP-2. The TRP-1 and TRP-2 genes were assigned to human chromosome bands 9p23 and 13q32.1, respectively. These results confirmed the previously mapped location for the TRP-1 gene and more precisely located the TRP-2 gene, which had previously been mapped to chromosome 13q31-q32.
Chromosomes, Human, Pair 13/genetics* ; Chromosomes, Human, Pair 9/genetics* ; Gene Library ; Human ; In Situ Hybridization, Fluorescence ; Intramolecular Oxidoreductases/genetics* ; Proteins/genetics*

BACKGROUND: Prostaglandins (PGs) play pathogenic and protective roles in inflammatory diseases. The novel concept of PGs as immune modulators is being documented by several investigators. By establishing an in vitro experimental model containing human follicular dendritic cell-like cells, HK cells, we reported that HK cells produce prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2) and that these PGs regulate biological functions of T and B cells. METHODS: To investigate the respective contribution of cyclooxygenase-1 (COX-1) and COX-2 to PGE2 and PGI2 production in HK cells, we performed siRNA technology to knock down COX enzymes and examined the effect on PG production. RESULTS: Both PGE2 and PGI2 productions were almost completely inhibited by the depletion of COX-2. In contrast, COX-1 knockdown did not significantly affect PG production induced by lipopolysaccharide (LPS). CONCLUSION: The current results suggest that mPGES-1 and PGIS are coupled with COX-2 but not with COX-1 in human follicular dendritic cell (FDC) and may help understand the potential effects of selective COX inhibitors on the humoral immunity.
Cyclooxygenase 1 ; Cyclooxygenase 2 ; Dendritic Cells, Follicular ; Dinoprostone ; Epoprostenol ; Humans ; Immunity, Humoral ; Intramolecular Oxidoreductases ; Models, Theoretical ; Prostaglandins ; Research Personnel ; RNA, Small Interfering ; Stromal Cells