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

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

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 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*

OBJECTIVETo investigate the changes in the expressions of inducible cyclooxygenase type 2 (COX-2) and membrane associated prostaglandin E-1(mPGES-1) in human carotid atherosclerotic plaques and to explore possible mechanisms of inflammatory process involved in plaque stability.

METHODSThe mRNA and protein levels of COX-2 and mPGES-1 were compared between minimally and grossly atherosclerotic arterial tissues. COX-2 and mPGES-1 gene expression were established by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) in 10 mesenchymal artery controls and 24 atherosclerotic specimens. Presence of COX-2 and mPGES-1 protein was assessed by Western blotting.

RESULTSImmunohistochemical staining showed that the COX-2 and mPGES-1 immunoreactive substances were present in the cytoplasm of smooth muscle cell. Compared with the control group, immunostaining positive cells increased in carotid atherosclerotic plaque group. COX-2 and mPGES-1 gene expression was significantly elevated in atherosclerotic plaques (P< 0.05, respectively). The increased mRNA and protein levels of COX-2 and mPGES-1 were correlated in atherosclerotic tissue (P< 0.05). The mRNA and protein levels of COX-2 and mPGES-1 related to degree of pathological damage in atherosclerotic tissue (P< 0.05). COX-2 and mPGES-1 were not found in the control group (mesenteric vascular walls).

CONCLUSIONCOX-2 and mPGES-1 expression in plaques is significantly higher than that in the control group. These findings suggests that COX-2 and mPGES-1 might play a role in pathogenesis of atheroscleros and modulation of inflammatory process involved in plaque stability, and COX-2 may have proinflammatory enzyme properties.

Aged ; Atherosclerosis ; genetics ; metabolism ; Blotting, Western ; Carotid Artery Diseases ; genetics ; metabolism ; Cyclooxygenase 2 ; genetics ; metabolism ; Female ; Gene Expression ; Humans ; Immunohistochemistry ; Intramolecular Oxidoreductases ; genetics ; metabolism ; Male ; Middle Aged ; Prostaglandin-E Synthases ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the association between the polymorphism of prostacyclin synthase gene (CYP8A1) and myocardial infarction (MI) in Uigur population.

METHODSTotally 210 patients with MI and 206 healthy control subjects were detected by polymerase chain reaction and restriction fragment length polymorphism. The serum 6-keto-PGF(1alpha) was detected with radioimmunoassay kit in all subjects.

RESULTSThe frequencies of CC, AC and AA were 0.024 (5/210), 0.124 (26/210) and 0.852 (179/210) in MI group while ones those 0.010 (2/206), 0.073 (15/206) and 0.917 (189/206) in the controls. There was no significant difference in frequencies of CC, AC and AA genotypes between controls and MI cases (chi(2) = 0.782, P > 0.05), but the frequency of CC + AC genotype in MI group [0.14 (31/210)] was higher than that in the controls [0.083 (17/206)] giving significant difference (chi(2) = 4.321, P = 0.031). The C allele frequency in the MI group [0.086 (36/420)] was higher than that in the controls [0.046 (19/412)] showing significant statistical difference (chi(2) = 5.284, P = 0.021). There was significant difference (t = 6.255, P < 0.01) in serum 6-keto-PGF(1alpha) level between MI group [(17.40 +/- 4.56) pg/ml] and control group [(20.34 +/- 5.02) pg/ml]. In the cases and control group, the serum 6-keto-PGF(1alpha) level of the persons with CC + AC genotype [(14.30 +/- 3.31) pg/ml, (18.31 +/- 4.62) pg/ml] was lower than those of AA genotypes [(19.34 +/- 5.51) pg/ml, (25.10 +/- 5.00) pg/ml], and the statistical significance was also observed (t' = 6.934, P < 0.05; t = 5.393, P < 0.01). Logistic regression analysis showed that the C allele of the CYP8A1 gene was an independent risk factor for MI (OR = 1.77; 95% CI: 1.06 - 2.05).

CONCLUSIONThe C allele of CYP8A1 might be a risk factor of MI in Uigur population, and be resulting from the decrease of serum 6-keto-PGF(1alpha) level for gene variation.

Aged ; Alleles ; Asian Continental Ancestry Group ; genetics ; Cytochrome P-450 Enzyme System ; genetics ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Intramolecular Oxidoreductases ; genetics ; Male ; Middle Aged ; Myocardial Infarction ; ethnology ; genetics ; Polymorphism, Genetic ; Population Groups

OBJECTIVETo investigate the association between the polymorphisms and haplotypes of prostacyclin synthase gene with MI in Uigur patients in Xinjiang.

METHODS210 patients with MI and 206 healthy control subjects were genotyped for 3 SNPs of the human prostacyclin synthase gene by polymerase chain reaction and restriction fragment length polymorphism.

RESULTSThe genotype distributions of the control group and MI group were in the Hardy-Weinberg equilibrium (both P > 0.05). The frequency of CC of rs5629 in MI group was significantly higher than that in controls (71.42% vs. 61.65%, P = 0.035). The frequency of A-C-T haplotype was significantly higher in the control group than that in the MI patients (4.01% vs. 0.60%, P = 0.001). The frequency of C-T-T haplotype was significantly higher in the MI patients than that in the controls (7.40% vs. 3.31%, P = 0.011). Logistic regression analysis showed that, after adjusting hypertension, hyperlipemia and smoking, the CC genotype of rs5629 (P = 0.021, OR = 1.665, 95%CI: 1.024 - 2.156) and the C-T-T haplotype (P = 0.011, OR = 1.876, 95%CI: 1.410 - 3.171) was the independent risk factors for MI.

CONCLUSIONThe CC genotype of rs5629 and the C-T-T haplotype of prostacyclin synthase gene are associated with MI but the A-C-T haplotype of prostacyclin synthase gene might be a protective factor of MI in Uigur population of Xinjiang.

Aged ; Asian Continental Ancestry Group ; genetics ; Cytochrome P-450 Enzyme System ; genetics ; Female ; Gene Frequency ; Genotype ; Haplotypes ; Humans ; Intramolecular Oxidoreductases ; genetics ; Male ; Middle Aged ; Myocardial Infarction ; ethnology ; genetics ; Polymorphism, Single Nucleotide

OBJECTIVETo investigate the gene expression of MAPEG in the cortex of concanavalin A (Con A)-induced mouse immune inflammatory model and the effect of cyclosporine A (Cs A).

METHODSMale Balb/c mouse immune inflammation model was developed by intravenous injection of Con A (20 mg/kg). Cs A (150 mg/kg) was intravenously infected prior to Con A administration. The MAPEG expressions were determined by RT-PCR.

RESULTmGST1, mGST3, LTC(4)S, FLAP and mPGES-1 were detected by RT-PCR but not mGST2. Eight hours after Con A treatment, mGST1 level was up-regulated to 1.2 approximately 1.5 folds of control with or without Cs A treatment. mGST3ìLTC(4)S, FLAP and mPGES-1 mRNA levels were not influenced by Con A administration.

CONCLUSIONImmune mechanism may be not involved in mGST1 up-regulation in this model and Con A does not alter arachidonic acid metabolism in cortex.

5-Lipoxygenase-Activating Proteins ; Animals ; Brain ; metabolism ; Carrier Proteins ; genetics ; metabolism ; Concanavalin A ; toxicity ; Cyclosporine ; pharmacology ; Eicosanoids ; metabolism ; Glutathione ; metabolism ; Glutathione Transferase ; genetics ; metabolism ; Intramolecular Oxidoreductases ; genetics ; metabolism ; Male ; Membrane Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Prostaglandin-E Synthases

Glycyrrhizin (GR), triterpenoid saponin composed of one glycyrrhetinic acid (GA) and two glucuronic acids, is a main constituent of the hydrophilic fraction of licorice (Glycyrrhiza glabra) extracts and is known to have a wide range of pharmacological actions. In this study, we investigated the mechanism of GR effect on melanogenesis in B16 murine melanoma cells. The cellular levels of tyrosinase mRNA, protein, enzyme activities and melanin contents were increased by GR in a dose dependent manner. Expression of tyrosinase-related protein-2 (TRP-2) mRNA was also increased by GR, however, no significant change was observed on TRP-1. No cytotoxicity was observed at the effective concentration range of GR. GA showed no effect on melanogenesis at the equivalent nontoxic concentrations, indicating that glycoside structure is important in the stimulatory effect of GR on melanogenesis. These results indicate that GR-induced stimulation of melanogenesis is likely to occur through the transcriptional activation.
Animal ; Blotting, Western ; Glycyrrhetinic Acid/pharmacology ; Glycyrrhizic Acid/*pharmacology ; Intramolecular Oxidoreductases/genetics/metabolism ; Melanins/*biosynthesis ; Melanoma, Experimental/enzymology/*metabolism ; Mice ; Monophenol Monooxygenase/genetics/metabolism ; Proteins/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the effect of ADMA on macrophage migration inhibitory factor (MIF) expression and tumor necrosis factor-α (TNF-α) and IL-8 secretion in THP-1 monocyte-derived macrophages. METHIDS: THP-1 monocytes were induced to differentiate into macrophages by a 24-h incubation with 160 nmol/L PMA. The THP-1 monocyte-derived macrophages were exposed to different concentrations of ADMA for 24 h, and the changes in MIF mRNA and protein expressions were analyzed with RT-PCR and Western blotting, respectively. Enzyme-linked immunosorbent assay was used to detect the levels of TNF-α and IL-8 in the supernatant of THP-1-derived macrophages following ADMA treatments.

RESULTSADMA obviously up-regulated MIF mRNA and protein expressions in THP-1-derived macrophages in a concentration- dependent manner. Exposure of the cells to 15 µmol/L ADMA for 24 h showed the most potent effect in up-regulating MIF mRNA and protein expressions. ADMA treatment also resulted in a dose-dependent increase of the levels of TNF-α and IL-8 in the culture supernatant of the macrophages, and the peak levels occurred following the treatment with 15 µmol/L ADMA.

CONCLUSIONADMA can up-regulate MIF expression and induce TNF-α and IL-8 secretion in THP-1 monocyte-derived macrophages.

Arginine ; analogs & derivatives ; pharmacology ; Cell Differentiation ; Cell Line ; Humans ; Interleukin-8 ; secretion ; Intramolecular Oxidoreductases ; genetics ; metabolism ; Macrophage Migration-Inhibitory Factors ; genetics ; metabolism ; Macrophages ; cytology ; metabolism ; Monocytes ; cytology ; Phenanthrenes ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; secretion