Association of endothelial constitutive nitric oxide synthase gene polymorphism with acute coronary syndrome in Koreans.

OBJECTIVE: To examine the effects of two polymorphisms of the endothelial constitutive nitric oxide synthase (ecNOS) gene, 4a/4b(A:B) located in intron 4 and Glu298Asp(G:T) located in exon 7, on the development of acute coronary syndromes (ACS). METHODS: 164 patients with ACS and 142 control participants were investigated for genotype and conventional risk factors. Genotype was determined by polymerase chain reaction and restriction fragment length polymorphism analysis. RESULTS: Genotype and allele frequencies of the A:B polymorphism in the ACS group (0.15:0.85 for AA+AB:BB, 0.09:0.91 for A:B) differed from those in the control group (0.26:0.74 for AA+AB:BB, 0.15:0.85 for A:B). However, genotype and allele frequencies of the G:T polymorphism in the ACS group (0.22:0.78 for TT+TG:GG, 0.11:0.89 for T:G) were similar to those in the control group (0.17:0.83 for TT+TG:GG, 0.09:0.91 for T:G). Multiple logistic regression analysis showed that the non-BB (AA+AB) and the non-BB+GG genotypes were significant protective factors against ACS (odds ratios 0.49 and 0.34, 95% confidence intervals 0.26 to 0.93 and 0.14 to 0.83, respectively). In addition, linear association analysis showed that the percentage of ACS patients was significantly lower in the genotype group non-BB+GG than in the genotype group BB+non-GG (39.6% v 62.7%, p = 0.01). CONCLUSIONS: The non-BB genotype of the ecNOS 4a/4b gene polymorphism is a protective factor against the development of ACS. The GG genotype of the ecNOS Glu298Asp polymorphism exerts a benefit in addition to the non-BB genotype in the Korean population.

Determination of rat leptin activity in vitro using a novel luciferase reporter assay.

Leptin is small cytokine-like protein that is involved in appetite and body weight regulation. Due to increased interest in using leptin as an anti-obesity reagent, recombinant forms of leptin have been produced for several species, including humans, mice, rats, pigs, dogs, sheep etc. The biological activities of such recombinant proteins were determined using various in vitro or in vivo systems; however so far, no specific assay system for rat leptin is available. Since rats are representative animal models in obesity research, the establishment of a biological assay system for determining rat leptin activity has been eagerly awaited. This study describes the generation of such a system using chinese hamster ovary (CHO)-cells that were transfected with the long form of the rat leptin receptor isoform, OB-Rb, whereby a signal transduces and activators of transcription-sensitive luciferase reporter system is further employed to quantify the leptin-mediated signals. This system is the first rat-specific leptin bioassay system that has been reported. It is expected that this assay will be used to further quantify and determine leptin activity from various biological fluids and sources.

Regulation of phosphatidylinositol-phosphate kinase IIgamma gene transcription by thyroid-stimulating hormone in thyroid cells.

This study was performed to evaluate the effects of thyroid-stimulating hormone (TSH) on phosphatidylinositol-4-phosphate 5-kinase type IIgamma (PIPKIIgamma) gene expression in the thyrocytes of FRTL-5 cells. Although PIPKIIgamma mRNA was expressed constantly in the absence of added TSH, its expression increased remarkably in the presence of 10(-9) M TSH. This increase started within 6 h of the addition of TSH, and reached a maximum at 8 h. The mRNA expression properties of PIPKIIgamma in the cells were identified using inhibitors. Actinomycin D blocked PIPKIIgamma transcription strongly, while cycloheximide did not. In an experiment using 5,6-dichlo-1-beta-d -ribofuranosylbenzimidaxole, the half-life of PIPKIIgamma mRNA was approximately 6 h in the presence or absence of TSH, and it was not affected by the stability of the PIPKIIgamma mRNA. The effects of TSH on PIPKIIgamma gene expression were specific, and other growth factors examined (transferrin, insulin and hydrocortisone) did not alter its expression. It is possible that the mechanism of PIPKIIgamma gene expression is involved in the permissive effect of the TSH-cAMP cascade proper. Our results indicate, for the first time, that the expression of PIPKIIgamma is regulated transcriptionally by TSH in thyrocytes.

Specific detection of cell surface-displayed human melanocortin 4 receptors with antibodies generated in mice.

The melanocortin-4 receptor (MC-4R) is a 7-transmembrane protein, which is involved in the central regulation of appetite and obesity. Despite the great interest in this protein, tools for detecting this molecule (as expressed on the cell surface in its native state) have been unavailable. Radioactive- or otherwise labeled ligands showed low receptor specificity to this particular melanocortin receptor isotype. Also, the antibodies were only available for epitopes that were displayed in the cytoplasm. To produce antibodies that enable the detection of this receptor (as expressed on the cell surface without disruption of the target cells), a candidate epitope was selected from the extracellular domains by a computer-aided analysis of the IC-4R secondary structure. This particular region was then recombinantly expressed in E. coli. Immunization of BALB/c mice with the recombinant proteins induced a specific immune reaction, which resulted in the production of MC-4R-specific antibodies. Enzyme-linked immunosorbent assays confirmed the specificity of these antibodies. To examine whether this tool also reacts with native cell surface-displayed MC-4R, HEK-293 cells were transfected with the human MC-4R cDNA. They were analyzed with these antibodies using Western blot and flow cytometry. Specificity and exclusion of cross-reactivity of these antibodies to other MC receptors were further confirmed by an immunofluorescence analysis of the HEK-293 cells that were transfected with other MC receptor isotypes. It is evident that with the availability of this tool, studies on the cell- and tissue-specificity, as well as the regulation mechanism of the MC-4 receptor, will be largely facilitated.

TSH regulates a gene expression encoding ERp29, an endoplasmic reticulum stress protein, in the thyrocytes of FRTL-5 cells.

This experiment was performed to evaluate the effect of thyroid-stimulating hormone (TSH) on the endoplasmic reticulum resident 29 kDa protein (ERp29) gene expression in the thyrocytes of FRTL-5 cells. Although ERp29 mRNA was constantly expressed, its expression began to increase remarkably from 10(-9) M TSH. On the other hand, the effect of TSH on the abundance of ERp29 mRNA started within 6 h and peaked at 8 h. Actinomycin D strongly blocked this effect while cycloheximide did not. The half-life of ERp29 mRNA was about 4-4.5 h in the presence or absence of TSH that was not affected by the stability of ERp29 mRNA. The effect of TSH on the ERp29 gene expression was specific, while other growth factors (transferrin, insulin and hydrocortisone) did not alter its expression. Our data indicate for the first time that the expression of ERp29 is regulated transcriptionally by TSH in thyrocytes.

Effects of red ginseng saponins and nootropic drugs on impaired acquisition of ethanol-treated rats in passive avoidance performance.

Effects of single and repeated administration of red ginseng total saponins (ROTS) and nootropic drugs were examined on impairment of acquisition induced by single oral administration of 3 g/kg ethanol (EtOH) in a step through test. The inhibitory effect of EtOH on acquisition was significantly reduced following single or repeated RGTS administration. The nootropic drugs, piracetam and N-methyl-D-glucamine, given orally significantly reduced impairment of acquisition induced by EtOH. On the other hand, the inhibitory effect of repeated RGTS on the EtOH-induced amnesia was blocked by the pretreatment of alpha-methyl-p-tyrosine (alpha-MT), an inhibitor of catecholamine synthesis, in a dose-dependent manner but not p-chlorophenylalanine (PCPA), an inhibitor of serotonin synthesis, whereas the inhibitory effect of repeated N-methyl-D-glucamine on the EtOH-induced amnesia was blocked neither by alpha-MT nor PCPA. These results suggest that repeated RGTS and N-methyl-D-glucamine ameliorate the impairing effect of EtOH on acquisition, and the effect of RGTS on EtOH-induced amnesia is dependent on the catecholaminergic but not serotonergic neuronal activity, while RGTS and N-methyl-D-glucamine seem to have a different mechanism on EtOH-induced amnesia.

Production and characterization of monoclonal antibodies directed to the kringle V and protease domains of human apolipoprotein(a).

Production and use of anti-apolipoprotein(a) monoclonal antibodies (MAbs) specific to single copy regions in the polymorphic lipoprotein(a) (Lp(a)) has been emphasized to be important for the standardization of measurements of the coronary heart disease risk factor, Lp(a). Here, mouse MAbs were prepared against the kringle V (V) and protease (P) domains of human apolipoprotein(a) (apo(a)), which domains are present in single copy in the apo(a) molecule. The cDNA for apo(a)VP was cloned from human liver cDNA library, and the V-P recombinant protein overexpressed in Escherichia coli was used as an antigen for the antibody production. Two antibodies named as MAb(a)20 and MAb(a)23 were finally produced, and they were characterized for their binding specificity and epitopes. The specificity of the antibodies was confirmed by an immunoblotting procedure and an enzyme-linked immunoassay (ELISA). It was shown that the antibodies had little, if any, cross-reactivity with human plasminogen, which is relatively abundant in human serum and is highly homologous (85%) with apo(a) in amino acid (aa) sequence. For epitope analysis, 3'-deletional series of apo(a)VP cDNA were constructed, and expression products of them were analyzed for the binding MAb(a)20 and MAb(a)23 do. It has been revealed that distinct epitopes were recognized by the two MAbs: MAb(a)23 (gamma2b, kappa) bound to the V region about 60 aa downstream from the N-terminal, and MAb(a)20 (gamma1, kappa) bound to the P region close to the C-terminal. A one step-sandwich ELISA system for Lp(a) was developed using MAb(a)20 as a capturing antibody and horseradish peroxidase (HRP)-coupled MAb(a)23 as a detecting antibody. The assay was found to be sensitive and useful for detecting Lp(a) in the range of 4-150 microg/dL (80 pM-3 nM).

Generation of self-antigen reactive, anti-urocortin specific antibodies by immunization of recombinantly expressed urocortin fusion proteins.

Urocortin is a recently described 40-meric neuropeptide, which was originally detected in the rat mid-brain and is believed to play a key role in response to stress situations. While its function in the central nervous system is rather well established, the biological role in the periphery is still to be determined. To investigate its distribution and effect on peripheral cells and tissues, in the present study, urocortin was recombinantly expressed and specific antibodies were generated. So far, the immunological detection of urocortin in the rat was largely dependent on antisera generated in rabbits. However, the polyclonal nature of the serum and the remote species origin tend to show cross-reactivities and higher backgrounds. On the other hand, generation of mouse antibodies to rat urocortin was hampered since mouse and rat urocortin sequences are identical, and such antibodies would represent auto-reactive antibodies. Despite such restrictions, the immunization with a combination of various recombinantly expressed urocortin fusion proteins resulted in the successful generation of mouse antiurocortin antisera, whose specificities were confirmed by ELISA and Western blot analysis. To produce the recombinant proteins for immunization, a cDNA encoding the mature urocortin sequence was cloned and expressed in fusion either with the glutathione-S-transferase, the maltose-binding protein, thioredoxin, or a 6X His tag. Depending on the expression system, the solubility and yield of the recombinant proteins greatly varied. Together with the newly generated antibodies, these recombinantly expressed urocortin proteins will serve as valuable tools in further investigations of the biological function of urocortin.

Identification of cytochrome P450 2E1 in rat brain.

We have isolated cDNA fragments that were originated from P450 2E1 in rat brain by PCR analysis. Their size matched up to what we expected based on the reported sequence of rat liver P450 2E1 mRNA. Internal structure of the longest first-round PCR products were investigated by Southern blot analysis and "nested" PCR. Their results confirmed that PCR products actually originated from P450 2E1 mRNA in rat brain. RT-PCR was also carried out using P450 2E1 specific primers and the size of the product was exactly as we expected for P450 2E1. These experimental evidences should clarify the presence of P450 2E1 in rat brain.

Molecular cloning and sequencing of sulfated glycoprotein-2 cDNA from testis of mouse: implications of two different mRNAs of SGP-2.

We cloned a mouse homolog of sulfated glycoprotein-2 (SGP-2) cDNA by screening a mouse testicular cDNA library and its nucleotide sequence was determined. The predicted amino acid sequence of the cDNA shares 93% identity with that of rat SGP-2. The nucleotide sequences of both cDNAs show extensive homology throughout the open reading frames and 3' untranslated regions. The 5' untranslated regions, however, share homology only up to 28 bp upstream from the start codons; the rest of sequences are quite different. DNA sequence homology search to mouse SGP-2 cDNA through the EMBL/GenBank database and a recent study on the genomic organization of rat TRPM-2 gene suggest a possibility that there are at least two different SGP-2 mRNAs as a result of alternative splicing and/or different promoter usage in mouse.

DNA sequence, genomic organization, and chromosomal localization of the mouse peripheral myelin protein zero gene: identification of polymorphic alleles.

We have cloned and characterized the mouse gene, P0, that encodes the predominant protein of peripheral myelin. Similar to the rat gene, the mouse P0 gene is encoded by six exons that span about 7 kb of DNA. The DNA sequence of the mouse gene is highly homologous with the rat gene, including the regions believed to be important in transcriptional control. Furthermore, the P0 protein appears well conserved throughout evolution. The gene was mapped to mouse chromosome 1 by Southern analysis of a Chinese hamster x mouse somatic cell hybrid panel. Several polymorphic restriction enzyme sites were identified within the P0 locus. Recombinant inbred strain mapping has linked the P0 gene to Ly-9/Ly/Sap in a region corresponding to band 1H3.

Presence of a bi-directional S phase-specific transcription regulatory element in the promoter shared by testis-specific TH2A and TH2B histone genes.

During mammalian spermatogenesis, somatic histones are replaced by testis-specific variants. The synthesis of the variants occurs primarily in the germ cells undergoing meiosis in the absence of DNA replication. We have cloned the genes encoding rat somatic and testis-specific H2A (TH2A) histones. The two genes share 300 bp of 5' upstream region with respective H2B genes: somatic H2A with somatic H2B and testis-specific TH2A with testis-specific TH2B gene. The deduced amino acid sequences show that H2A and TH2A histones have eight amino acid differences in the first half of the molecules and three consecutive changes in the C-terminal region. TH2A gene is expressed only in testis. Although synthesis of TH2A and TH2B histones is independent of DNA replication and insensitive to inhibitors of DNA synthesis in testis, the regulatory region shared by the two genes contain a bi-directional S phase-specific transcription regulatory element. In addition, TH2A gene, like TH2B gene, contains the consensus sequence element in the 3' non-coding region which is involved in the S phase-specific stabilization of histone mRNA.

Selenocysteine tRNA[Ser]Sec gene is ubiquitous within the animal kingdom.

Recently, a mammalian tRNA which was previously designated as an opal suppressor seryl-tRNA and phosphoseryl-tRNA was shown to be a selenocysteyl-tRNA (B. J. Lee, P. J. Worland, J. N. Davis, T. C. Stadtman, and D. Hatfield, J. Biol. Chem. 264:9724-9727, 1989). Hence, this tRNA is now designated as selenocysteyl-tRNA[Ser]Sec, and its function is twofold, to serve as (i) a carrier molecule upon which selenocysteine is biosynthesized and (ii) as a donor of selenocysteine, which is the 21st naturally occurring amino acid of protein, to the nascent polypeptide chain in response to specific UGA codons. In the present study, the selenocysteine tRNA gene was sequenced from Xenopus laevis, Drosophila melanogaster, and Caenorhabditis elegans. The tRNA product of this gene was also identified within the seryl-tRNA population of a number of higher and lower animals, and the human tRNA[Ser]Sec gene was used as a probe to identify homologous sequences within genomic DNAs of organisms throughout the animal kingdom. The studies showed that the tRNA[Ser]Sec gene has undergone evolutionary change and that it is ubiquitous in the animal kingdom. Further, we conclude that selenocysteine-containing proteins, as well as the use of UGA as a codon for selenocysteine, are far more widespread in nature than previously thought.

Conserved sequences in both coding and 5' flanking regions of mammalian opal suppressor tRNA genes.

The rabbit genome encodes an opal suppressor tRNA gene. The coding region is strictly conserved between the rabbit gene and the corresponding gene in the human genome. The rabbit opal suppressor gene contains the consensus sequence in the 3' internal control region but like the human and chicken genes, the rabbit 5' internal control region contains two additional nucleotides. The 5' flanking sequences of the rabbit and the human opal suppressor genes contain extensive regions of homology. A subset of these homologies is also present 5' to the chicken opal suppressor gene. Both the rabbit and the human genomes also encode a pseudogene. That of the rabbit lacks the 3' half of the coding region. Neither pseudogene has homologous regions to the 5' flanking regions of the genes. The presence of 5' homologies flanking only the transcribed genes and not the pseudogenes suggests that these regions may be regulatory control elements specifically involved in the expression of the eukaryotic opal suppressor gene. Moreover the strict conservation of coding sequences indicates functional importance for the opal suppressor tRNA genes.