Isolation of a Δ5 desaturase gene from Euglena gracilis and functional dissection of its HPGG and HDASH motifs.

Delta (Δ) 5 desaturase is a key enzyme for the biosynthesis of health-beneficial long chain polyunsaturated fatty acids such as arachidonic acid (ARA, C20:4n-6), eicosapentaenoic acid (C20:5n-3) and docosahexaenoic acid (C22:6n-3) via the "desaturation and elongation" pathways. A full length Δ5 desaturase gene from Euglena gracilis (EgΔ5D) was isolated by cloning the products of polymerase chain reaction with degenerate oligonucleotides as primers, followed by 5' and 3' rapid amplification of cDNA ends. The whole coding region of EgΔ5D was 1,350 nucleotides in length and encoded a polypeptide of 449 amino acids. BlastP search showed that EgΔ5D has about 39 % identity with a Δ5 desaturase of Phaeodactylum tricornutum. In a genetically modified dihomo-gamma-linoleic acid (DGLA, C20:3n-6) producing Yarrowia lipolytica strain, EgΔ5D had strong Δ5 desaturase activity with DGLA to ARA conversion of more than 24 %. Functional dissection of its HPGG and HDASH motifs demonstrated that both motifs were important, but not necessary in the exact form as encoded for the enzyme activity of EgΔ5D. A double mutant EgΔ5D-34G158G with altered sequences within both HPGG and HDASH motifs was generated and exhibited Δ5 desaturase activity similar to the wild type EgΔ5D. Codon optimization of the N-terminal region of EgΔ5D-34G158G and substitution of the arginine with serine at residue 347 improved substrate conversion to 27.6 %.

[An epidemiological analysis of hepatitis A virus serologic markers during the recent four years in Korea].

BACKGROUND: To establish effective preventive measures for hepatitis A virus (HAV) infection, a nationwide epidemiologic study on seroprevalence of anti-HAV and the disease prevalence is needed. The aim of this study was to analyze the recent sero-epidemiological changes of hepatitis A markers in Korea. METHODS: The results of 11,068 anti-HAV total and 32,360 anti-HAV IgM tests by electro-chemiluminescence immunoassay (ECLIA) that had been requested in recent four years (2005-2008) to a reference medical laboratory from 1,699 institutions nationwide were retrospectively analyzed according to the distribution of year, sex, and age groups. RESULTS: The overall positive rate of anti-HAV total was 62.8%. The overall positive rate of anti-HAV IgM was 11.0%, showing a significantly increasing trend by year: 7.7%, 10.9%, 8.9%, and 14.3% in 2005, 2006, 2007, and 2008, respectively (P<0.0001). The positive rate of anti-HAV IgM was higher in male than in female subjects (11.8% vs 10.0%, P<0.0001), and 81.8% (2,916/3,566) of the anti-HAV IgM positive results were observed in the age groups of 21-40 yr. The annual positive rates of anti-HAV total and anti-HAV IgM showed significantly decreasing and increasing trends, respectively, in the age groups of > or = 21 yr. CONCLUSION: In accordance with a decreasing sero-positivity of anti-HAV total, the prevalence of acute hepatitis A virus infection has been considerably increased during the recent four years in the age groups of > or = 21 yr. The results of this study could be used effectively as a basic data for establishing effective preventive measures for hepatitis A including vaccination in these susceptible age groups.

The p300 acetylase is critical for ligand-activated farnesoid X receptor (FXR) induction of SHP.

The primary bile acid receptor farnesoid X receptor (FXR) maintains lipid and glucose homeostasis by regulating expression of numerous bile acid-responsive genes, including an orphan nuclear receptor and metabolic regulator SHP. Using SHP as a model gene, we studied how FXR activity is regulated by p300 acetylase. FXR interaction with p300 and their recruitment to the SHP promoter and acetylated histone levels at the promoter were increased by FXR agonists in mouse liver and HepG2 cells. In contrast, p300 recruitment and acetylated histones at the promoter were not detected in FXR-null mice. p300 directly interacted with and acetylated FXR in vitro. Overexpression of p300 wild type increased, whereas a catalytically inactive p300 mutant decreased, acetylated FXR levels and FXR transactivation in cells. While similar results were observed with a related acetylase, CBP, GCN5 did not enhance FXR transactivation, and its recruitment to the promoter was not increased by FXR agonists, suggesting functional specificity of acetylases in FXR signaling. Down-regulation of p300 by siRNA decreased acetylated FXR and acetylated histone levels, and occupancy of FXR at the promoter, resulting in substantial inhibition of SHP expression. These results indicate that p300 acts as a critical coactivator of FXR induction of SHP by acetylating histones at the promoter and FXR itself. Surprisingly, p300 down-regulation altered expression of other metabolic FXR target genes involved in lipoprotein and glucose metabolism, such that beneficial lipid and glucose profiles would be expected. These unexpected findings suggest that inhibition of hepatic p300 activity may be beneficial for treating metabolic diseases.

Clinical and cytogenetic findings on 31,615 mid-trimester amniocenteses.

BACKGROUND: Since amniocentesis made prenatal diagnosis feasible in 1967, the method has become a popular tool in obstetric practices. In Korea, the demand for genetic counseling and prenatal tests has increased markedly because the number and proportion of pregnancies in women aged 35 yr and older have increased over a 20-yr period. Here we report clinical and cytogenetic findings on 31,615 mid-trimester amniocenteses. METHODS: To investigate the changes in the annual number of amniocentesis, distribution of indications and age, and cytogenetic findings and abnormality rate according to indications, this study retrospectively analyzed 31,615 cases of mid-trimester amniocentesis performed at Seoul Clinical Laboratories, an independent medical laboratory center, during the past 13 yr (1994-2007). RESULTS: The annual number of amniocenteses has increased substantially since 1994. Among the 31,615 amniocentesis cases, the maternal age between 30 and 34 yr was the most common age group (35.4%). Among clinical indications, abnormal maternal serum screening results have been the most common indication for amniocentesis since 1994. Chromosomal abnormalities were detected in 973 cases (3.1%). Down syndrome was the most common abnormality found (36.9%, 359/973). In sex chromosomal abnormalities, 53 cases of Turner syndromes, 32 cases of Klinefelter syndromes, 20 cases of triple X syndromes, and 15 cases of 47,XYY were diagnosed. Of structural rearrangements, reciprocal translocations between two autosomes were the most common (15.5%, 151/973). Abnormal ltrasonographic findings showed the highest positive predictive value (5.9%) among the clinical indications. CONCLUSIONS: The present study could be used for the establishment of a database for genetic counseling. The discovery of an abnormality provides the option of termination or continuation in the pregnancy, a more suitable obstetric management in Korea.

Her-2/neu status in breast cancer metastases to the central nervous system.

CONTEXT: Breast cancer is lethal when it metastasizes; one frequent site for spread is the central nervous system (CNS). Approximately 15% to 30% of breast cancers overexpress the protein HER-2/neu at the primary site, but there are few data on whether metastases from these tumors overexpress HER-2/neu and might be responsive to the potentially toxic anti-HER-2/neu immunotherapy (trastuzumab [Herceptin]) used in patients with disseminated disease. OBJECTIVE: To assess CNS breast cancer metastases for HER-2/neu protein overexpression by immunohistochemistry and gene amplification by fluorescence in situ hybridization (FISH) and to compare the status in primary and metastatic sites in the same patient, whenever possible. DESIGN: Central nervous system breast cancer metastases (n = 53) from 33 patients and corresponding primary breast cancer specimens in a subset of these patients (n = 12) were retrospectively identified in surgical pathology and autopsy databases. Fluorescence in situ hybridization analysis using PathVysion probes for HER-2/neu and chromosome enumeration probe 17 (CEP 17) and immunohistochemistry using the c-Erb-B2 antibody (Dako A0485) were compared. Immunohistochemical sections were evaluated by both visual and image analysis techniques. RESULTS: Of 31 cases assessable by FISH, 26% showed gene amplification. One hundred percent concordance for HER-2/neu status was detected between the primary and CNS metastatic lesions in 10 of 10 patients analyzed by FISH; lesser concordance was noted in 12 cases compared by immunohistochemistry. In 9 patients with multiple CNS metastases, FISH showed concordance among different lesions within the same patient. CONCLUSIONS: When FISH is the detection method, CNS metastases accurately reflect the HER-2/neu status of the primary tumor. Central nervous system metastases from breast cancer received as surgical specimens can therefore be used to assess HER-2/neu status in patients in whom the primary tumor is unavailable for analysis.