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

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

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

Xanthohumol (XH), the principal prenylflavonoid of the hop plant (Humulus lupulus L.), dose-dependently inhibited isobutylmethylxanthine (IBMX)-induced melanogenesis in B16 melanoma cells, with little cytotoxicity at the effective concentrations. Decreased melanin content was accompanied by reduced tyrosinase enzyme activity, protein and mRNA expression. The levels of tyrosinase-related protein 1 and 2 mRNAs were decreased by XH. XH also inhibited alpha-melanocyte stimulating hormone- or forskolin-induced increases in melanogenesis, suggesting an action on the cAMP-dependent melanogenic pathway. XH downregulated the protein and mRNA expression of microphthalmia-associated transcription factor (MITF), a master transcriptional regulator of key melanogenic enzymes. These results suggest that XH might act as a hypo-pigmenting agent through the downregulation of MITF in the cAMP-dependent melanogenic pathway.
1-Methyl-3-isobutylxanthine/pharmacology ; Animals ; Cell Line ; Cell Survival/drug effects ; Dose-Response Relationship, Drug ; Down-Regulation ; Drug Antagonism ; Forskolin/pharmacology ; *Humulus ; Intramolecular Oxidoreductases/antagonists & inhibitors/biosynthesis ; Melanins/antagonists & inhibitors/*biosynthesis ; Melanocytes/*drug effects/*metabolism ; Melanoma, Experimental ; Membrane Glycoproteins/antagonists & inhibitors/biosynthesis ; Mice ; Microphthalmia-Associated Transcription Factor/antagonists & inhibitors ; Monophenol Monooxygenase/antagonists & inhibitors/biosynthesis/genetics ; Oxidoreductases/antagonists & inhibitors/biosynthesis ; Propiophenones/*pharmacology ; Signal Transduction/drug effects ; alpha-MSH/metabolism

In this study, the essential oil from lotus flower extract, including petals and stamens, was assessed with regard to its effects on melanogenesis in human melanocytes. The lotus flower essential oil was shown to stimulate melanin synthesis and tyrosinase activity in a dose-dependent manner. The lotus flower essential oil induced the expression of tyrosinase, microphthalmia-associated transcription factor M (MITF-M), and tyrosinase-related proten-2 (TRP-2) proteins, but not tyrosinase mRNA. Moreover, it increased the phosphorylation of ERK and cAMP response element binding protein (CREB). In order to verify the effective components of the lotus flower oil, its lipid composition was assessed. It was found to be comprised of palmitic acid methyl ester (22.66%), linoleic acid methyl ester (11.16%), palmitoleic acid methyl ester (7.55%) and linolenic acid methyl ester (5.16%). Among these components, palmitic acid methyl ester clearly induced melanogenesis as the result of increased tyrosinase expression, thereby indicating that it may play a role in the regulation of melanin content. Thus, our results indicate that lotus flower oil may prove useful in the development of gray hair prevention agents or tanning reagents.
Blotting, Western ; Cell Proliferation ; Cyclic AMP/metabolism ; Cyclic AMP Response Element-Binding Protein/genetics/metabolism ; Flowers/*chemistry ; Gas Chromatography-Mass Spectrometry ; Humans ; Intramolecular Oxidoreductases/genetics/metabolism ; Lotus/*chemistry ; Melanins/*biosynthesis ; Melanocytes/*drug effects/metabolism ; Microphthalmia-Associated Transcription Factor/genetics/metabolism ; Monophenol Monooxygenase/genetics/metabolism ; Phosphorylation ; Plant Oils/*pharmacology ; RNA, Messenger/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Skin/cytology/drug effects/metabolism