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

OBJECTIVETo investigate the mRNA and protein expression levels of apolipoprotein M (apoM) in the human colorectal cancer tissues, and to explore its clinical relevance.

METHODSReal-time PCR was carried out to determine the mRNA expression levels both in cancer tissue and its adjacent normal tissue from 20 patients with colorectal cancer. Immunohistochemistry was also carried out to determine the protein levels in 23 colorectal biopsy samples (7 normal mucosa, 6 inflammatory mucosa and 10 polyp tissues) and 20 cases of colorectal cancer tissues as well as the adjacent normal tissues.

RESULTSReal-time PCR result showed that apoM mRNA level in the colorectal cancer tissues was significantly lower than that in their adjacent normal tissues (0.05±0.01 vs. 0.19±0.05, P<0.05). ApoM mRNA level in colorectal cancer tissues was statistically significant higher in the patients with lymph node metastasis as compared to the patients without lymph node metastasis (P<0.01). The median value of apoM protein in cancer tissues was 5.50, which was significantly lower than that in the adjacent normal tissues (10.5, P<0.05), inflammatory mucosa tissues (9.75, P<0.05), polyp tissues (11.0, P<0.01) and normal mucosa (10.5, P<0.05). No significant association was observed between the apoM protein level and the clinicopathological parameters of patients.

CONCLUSIONSBoth apoM mRNA and protein expression levels in colorectal cancer tissues are significantly decreased in contrast to normal and benign colorectal tissues. The apoM mRNA expression in colorectal cancer tissues is closely associated with nodal metastasis.

Adult ; Aged ; Apolipoproteins ; genetics ; metabolism ; Apolipoproteins M ; Colorectal Neoplasms ; metabolism ; pathology ; Female ; Humans ; Lipocalins ; genetics ; metabolism ; Lymphatic Metastasis ; Male ; Middle Aged ; RNA, Messenger ; genetics

OBJECTIVETo investigate the distribution characters and linkage disequilibrium of apolipoprotein M gene (APOM) polymorphisms in Han population of North China.

METHODSThe single nucleotide polymorphisms (SNPs) in six exons and five introns of APOM gene of 330 normal subjects in Han population of North China were detected by PCR-restriction fragment length polymorphism and DNA sequencing analysis.

RESULTSThree SNPs in Han Chinese were found, including rs805264 of intron 1, rs707922 and rs707921 of intron 5 of APOM gene. The frequency distribution of these SNPs was different among different races and territory. In addition, linkage disequilibrium among the SNPs of rs805264, rs707922 and rs707921 of APOM gene was found and A-T-A and G-G-C were predominant haplotypes.

CONCLUSIONThere is apparent linkage disequilibrium among SNPs of APOM gene in Han population of North China.

Adult ; Aged ; Aged, 80 and over ; Apolipoproteins ; genetics ; Apolipoproteins M ; Asian Continental Ancestry Group ; genetics ; China ; Ethnic Groups ; genetics ; Female ; Humans ; Introns ; genetics ; Linkage Disequilibrium ; Lipocalins ; Male ; Middle Aged ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide

OBJECTIVETo investigate the association between genetic polymorphisms of proximal promoter region of apolipoprotein M (apoM) gene and susceptibility of coronary artery diseases (CAD) in Han Chinese population.

METHODSTwo pairs of primers were designed according to the sequence (GenBank accession nos. EU030444.1) and the PCR products of apoM proximal promoter region were directly sequenced. Two hundred and six patients [165 males, mean age (61.9 ± 9.2) years old] diagnosed with CAD according to the results of angiography (a lesion was classed as being significant when stenosis was more than 50%) were enrolled in the present study, 209 age- and gender-matched patients[157 males, mean age (60.4 ± 9.1) years old] without CAD according to the results of angiography were selected as the control group. The allelic frequencies and genotype distributions of polymorphism in CAD and non-CAD patients were analyzed. Furthermore the wide-type and mutant promoter region of apoM were cloned into the luciferase expression vector pGL3, respectively. Luciferase reporter assay was used to detect the activity of apoM promoter.

RESULTSA new deletion mutation -724delC in apoM promoter was found. The frequency of Del C allele was 8.0% in CAD patients and only 4.1% in the non-CAD controls (OR = 2.054, 95%CI 1.125-3.749, P = 0.017). The mean TC level was lower in groups with wide-type homozygotes compared to the mutant allele carriers [ (6.04 ± 0.90) mmol/L vs. (4.95 ± 1.00)mmol/L, P < 0.01]. -724delC mutant showed obvious decreased luciferase activities (1.13 ± 0.25 vs. 2.11 ± 0.15, P = 0.009).

CONCLUSIONIt is reasonable to speculate that -724delC could affect the activity of the apoM promoter and downregulate apoM expressions, therefore, influence the susceptibility of CAD in this patient cohort.

Aged ; Apolipoproteins ; genetics ; Apolipoproteins M ; Asian Continental Ancestry Group ; genetics ; Coronary Artery Disease ; genetics ; Female ; Genetic Predisposition to Disease ; Humans ; Lipocalins ; genetics ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; Promoter Regions, Genetic

OBJECTIVE: To express and purify the extra cellular full-length human apolipoprotein M(ApoM). METHODS: The ApoM gene fragment was amplified from the human liver cDNA library by PCR. The resulting product was cloned into pGEXT vector and sequenced. Then the confirmed canstatin cDNA was cloned into plasmid E.coli JM109 and then transformed into E.coli DL21(DE3) where it was induced to express protein by IPTG. RESULTS: The ApoM gene was cloned by PCR and a 560 bp DNA fragment was shown on the agarose electrophoresis. The cloned gene was sequenced and demonstrated to have the same sequence as that of human ApoM gene in GenBank. Then ApoM cDNA gene fragment was induced by IPTG, and a 24 kD recombinant ApoM protein was tested on SDS-PAGE. CONCLUSION Human ApoM gene is successfully cloned and its recombinant proteins are expressed.
Apolipoproteins ; biosynthesis ; genetics ; isolation & purification ; Apolipoproteins M ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; metabolism ; Humans ; Lipocalins ; Molecular Sequence Data ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

Our goal is to decipher which DNA sequences are required for tissue-specific expression of epididymal genes. At least 6 epididymis-specific lipocalin genes are known. These are differently regulated and regionalized in the epididymis. Lipocalin 5 (Lcn5 or mE-RABP) and Lipocalin 8 (Lcn8 or mEP17) are homologous genes belonging to the epididymis-specific lipocalin gene cluster. Both the 5 kb promoter fragment of the Lcn5 gene and the 5.3 kb promoter fragment of the Lcn8 gene can direct transgene expression in the epididymis (Lcn5 to the distal caput and Lcn8 to the initial segment), indicating that these promoter fragments contain important cis-regulatory element(s) for epididymis-specific gene expression. To define further the fragments regulating gene expression, the Lcn5 promoter was examined in transgenic mice and immortalized epididymal cell lines. After serial deletion, the 1.8 kb promoter fragment of the Lcn5 gene was sufficient for tissue-specific and region-specific gene expression in transgenic mice. Transient transfection analysis revealed that a transcription factor forkhead box A2 (Foxa2) interacts with androgen receptor and binds to the 100 bp fragment of the Lcn5 promoter between 1.2 kb and 1.3 kb and that Foxa2 expression inhibits androgen-dependent induction of the Lcn5 promoter activity. Immunohistochemistry indicated a restricted expression of Foxa2 in the epididymis where endogenous Lcn5 gene expression is suppressed and that the Foxa2 inhibition of the Lcn5 promoter is consistent with the lack of expression of Lcn5 in the corpus and cauda. Our approach provides a basic strategy for further analysis of the epididymal lipocalin gene regulation and flexible control of epididymal function.
Animals ; Base Sequence ; Carrier Proteins ; genetics ; Epididymis ; physiology ; Hepatocyte Nuclear Factor 3-beta ; genetics ; Humans ; Lipocalins ; Male ; Mice ; Molecular Sequence Data ; Multigene Family ; Promoter Regions, Genetic ; Prostate ; physiology ; Receptors, Retinoic Acid ; genetics ; Retinol-Binding Proteins, Plasma

OBJECTIVE: To investigate the effect of administration of rosiglitazone, an artificial ligand of PPARγ, on the expression and secretion of apolipoprotein (apoM) in fat-fed, streptozotocin-treated rats, an animal model for type 2-like diabetes. METHODS: Healthy male SD rats were divided into 4 groups: a control group (n=7), a high-fat chow group (HF group, n=8), a diabetes mellitus group (DM group, n=7), and a diabetes mellitus group with rosiglitazone intervention group (RSG group, n=7). Fasting blood glucose (FBG), fasting insulin (FINS), triglyceride (TG) and total cholesterol (TC) were measured at the beginning of the study. The diabetic rats model was established by feeding high fat chow and intraperitoneal injection of streprozotocin. Then the randomly selected treatment group was given rosiglitazone by daily gavage for 8 weeks. All the rats were killed at the fifteenth week, at which time blood and tissues (liver, kidney, adipose) were collected and prepared. The levels of FBG, FINS, TG and TC were assayed. The level of apoM in serum was measured by enzyme-linked immunosorbent assay (ELISA). Reverse transcription polymerase chain reaction (RT-PCR) was used to determine apoM mRNA expression in liver, kidney, and adipose tissues. RESULTS: Compared with either control group or HF group, serum apoM concentration in the DM group was reduced significantly (P<0.05); compared with the DM group, however, serum apoM concentrations in RSG group were increased (P<0.05). The expression of apoM mRNA in liver was highest, in kidney medium, and in adipose tissue extremely low (P<0.05). ApoM mRNA expression in liver and kidney was decreased in both DM and HF groups compared to control group (P<0.05). But, as with serum apoM concentration, apoM mRNA in the liver, kidney and adipose tissues of the RSG group were all increased markedly (P<0.05). The level of serum apoM in SD rats correlated negatively with TG (r=-0.466, P=0.011), TC (r=-0.568, P= 0.001), FBS (r =-0.371, P<0.001), and FINS(r=-0.768, P= 0.048 ). CONCLUSION These results suggest that apoM may participate in the glucose and lipid metabolism by the regulation of PPARγ.
Animals ; Apolipoproteins ; blood ; genetics ; metabolism ; Apolipoproteins M ; Diabetes Mellitus, Experimental ; drug therapy ; metabolism ; Dietary Fats ; administration & dosage ; Lipocalins ; blood ; genetics ; metabolism ; Male ; PPAR gamma ; agonists ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Rosiglitazone ; Thiazolidinediones ; therapeutic use

OBJECTIVESIn order to identify whether the hens immunized with recombinant human testis prostaglandin D synthase (rhtL-PGDS) DNA can produce anti-L-PGDS antibody.

METHODSThe serum were got from the hens immunized with recombinant plasmid pGEX-2T/htL-PGDS DNA (100 micrograms) every 2 weeks for 2 times. The exist of anti-L-PGDS antibody and its titer were tested with agarose dual immunodiffusion and ELISA with rhtL-PGDS as antigen.

RESULTSThe serum anti-L-PGDS antibody in hen immunized with pGEX-2T/htL-PGDS DNA were confirmed and its titer tested by ELISA was 1:2,048.

CONCLUSIONSIt is feasible to produce anti-L-PGDS antibody by immunizing hens with recombinant pGEX-2T/htL-PGES DNA.

Animals ; Antibodies ; analysis ; Chickens ; DNA, Recombinant ; administration & dosage ; immunology ; Enzyme-Linked Immunosorbent Assay ; Female ; Genetic Vectors ; Humans ; Immunization ; Intramolecular Oxidoreductases ; genetics ; immunology ; Lipocalins ; Male ; Models, Animal ; Plasmids ; genetics ; Testis ; enzymology ; Vaccines, DNA ; administration & dosage ; immunology

Although the genetic component in the etiology of rheumatoid arthritis (RA) has been consistently suggested, many novel genetic loci remain to uncover. To identify RA risk loci, we performed a genome-wide association study (GWAS) with 100 RA cases and 600 controls using Affymetrix SNP array 5.0. The candidate risk locus (APOM gene) was re-sequenced to discover novel promoter and coding variants in a group of the subjects. Replication was performed with the independent case-control set comprising of 578 RAs and 711 controls. Through GWAS, we identified a novel SNP associated with RA at the APOM gene in the MHC class III region on 6p21.33 (rs805297, odds ratio (OR) = 2.28, P = 5.20 x 10(-7)). Three more polymorphisms were identified at the promoter region of the APOM by the re-sequencing. For the replication, we genotyped the four SNP loci in the independent case-control set. The association of rs805297 identified by GWAS was successfully replicated (OR = 1.40, P = 6.65 x 10(-5)). The association became more significant in the combined analysis of discovery and replication sets (OR = 1.56, P = 2.73 +/- 10(-10)). The individuals with the rs805297 risk allele (A) at the promoter region showed a significantly lower level of APOM expression compared with those with the protective allele (C) homozygote. In the logistic regressions by the phenotype status, the homozygote risk genotype (A/A) consistently showed higher ORs than the heterozygote one (A/C) for the phenotype-positive RAs. These results indicate that APOM promoter polymorphisms are significantly associated with the susceptibility to RA.
Apolipoproteins/*genetics ; Arthritis, Rheumatoid/*genetics ; Case-Control Studies ; DNA/genetics ; Female ; *Genetic Predisposition to Disease ; Genome-Wide Association Study ; Genotype ; Heterozygote ; Homozygote ; Humans ; Lipocalins/*genetics ; Luciferases/metabolism ; Male ; Middle Aged ; Polymorphism, Single Nucleotide/*genetics ; Promoter Regions, Genetic/*genetics ; Real-Time Polymerase Chain Reaction ; Risk Factors