Comparison of the Effectiveness in the Application of Competitive and Noncompetitive Internal Control for the Laboratory Developed Polymerase Chain Reaction / 대한수혈학회지

BACKGROUND: A nucleic acid amplification test was adopted to detect transfusion-transmitted infectious agents. In the case of HTLV, however, there was no internal control (IC) because the laboratory developed polymerase chain reaction (laboratory-developed PCR) was used. In this study, noncompetitive IC was constructed for the laboratory-developed PCR of HTLV and the effectiveness was compared with the competitive test that was constructed in a previous study. METHODS: As a competitive IC, plasmid DNA, including the primer recognition sequence for the amplification of the HTLV pX region, was constructed. As a noncompetitive IC, an additional primer was constructed for the amplification of the housekeeping gene, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. The performance of the competitive and noncompetitive IC was verified and compared using 10 HTLV positive samples and 10 negative samples. In addition, the detection limits in the assay adopting competitive IC and noncompetitive IC were compared. RESULTS: In the case of competitive IC applications, all 10 positive samples were positive and all 10 negative samples were negative. In the case of noncompetitive IC applications, however, one positive sample was not detected. The detection limit of the assay using competitive IC was 100 pg and that of the assay using noncompetitive IC was 1 ng. CONCLUSION: Although the manufacturing processes is not required using noncompetitive IC, the adoption of competitive IC is more effective to ensure the assay results because the ability of detection of the assay adopting competitive IC was better than that using noncompetitive IC.

Effect of Ginkgo Biloba Extract on N-Methyl-D-Aspartic Acid Receptor Subunit 2B Expression in a Salicylate-Induced Ototoxicity Model

OBJECTIVES.: Sodium salicylate (SS) is well known for its ototoxic properties that induce functional and morphological changes in the cochlea and brain. Ginkgo biloba extract (GBE) has been widely used for treatment of various neurodegenerative diseases; however, its effects on salicylate-induced ototoxicity remain unclear. Herein, we examined the effects of EGb 761 (EGb), a standard form of GBE, on the plasticity of the N-methyl-D-aspartate receptor subunit 2B (GluN2B) in the inferior colliculus (IC) following SS administration. METHODS.: Seven-week-old Sprague Dawley rats (n=24) were randomly allocated to control, SS, EGb, and EGb+SS groups. The SS group received a single intraperitoneal SS injection (350 mg/kg), the EGb group received EGb orally for 5 consecutive days (40 mg/kg), and the EGb+SS group received EGb for 5 consecutive days, followed by an SS injection. The auditory brainstem responses (ABRs) were assessed at baseline and 2 hours after SS administration. GluN2B expression was examined by Western blot and immunohistochemistry. RESULTS.: There were no significant differences in ABR threshold shifts among the groups. The expression of the GluN2B protein normalized by which of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was significantly lower in the EGb+SS group, as compared to the SS group (P=0.012). Weak and diffused GluN2B immunoreactivity was detected in the IC neural cells of the EGb+SS group, while those of the SS group exhibited strong and diffused GluN2B positivity. CONCLUSION.: EGb may play a role in regulating the GluN2B expression in the IC of salicylate-induced ototoxicity model.

Effects of 1,25-dihydroxyvitamin D₃ on the differentiation of MC3T3-E1 osteoblast-like cells

PURPOSE: The purpose of this study was to evaluate the effects of 1,25-dihydroxyvitamin D₃ on the proliferation, differentiation, and matrix mineralization of MC3T3-E1 osteoblast-like cells in vitro. METHODS: MC3T3-E1 osteoblastic cells and 1,25-dihydroxyvitamin D₃ were prepared. Cytotoxic effects and osteogenic differentiation were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, alkaline phosphatase (ALP) activity assay, ALP staining, alizarin red S staining, and reverse transcription-polymerase chain reaction (RT-PCR) for osteogenic differentiation markers such as ALP, collagen type I (Col-I), osteocalcin (OCN), vitamin D receptor (VDR), and glyceraldehyde 3-phosphate dehydrogenase. RESULTS: The MTT assay showed that 1,25-dihydroxyvitamin D₃ did not inhibit cell growth and that the rate of cell proliferation was higher than in the positive control group at all concentrations. ALP activity was also higher than in the positive control group at low concentrations of 1,25-dihydroxyvitamin D₃ (10−10, 10−12, and 10−14 M). RT-PCR showed that the gene expression levels of ALP, Col-I, OCN, and vitamin D receptor (VDR) were higher at a low concentration of 1,25-dihydroxyvitamin D₃ (10−12 M). Alizarin red S staining after treatment with 1,25-dihydroxyvitamin D₃ (10−12 M) showed no significant differences in the overall degree of calcification. In contrast to the positive control group, formation of bone nodules was induced in the early stages of cell differentiation. CONCLUSIONS: We suggest that 1,25-dihydroxyvitamin D₃ positively affects cell differentiation and matrix mineralization. Therefore, it may function as a stimulating factor in osteoblastic bone formation and can be used as an additive in bone regeneration treatment.

Acetyl Eburicoic Acid from Laetiporus sulphureus var. miniatus Suppresses Inflammation in Murine Macrophage RAW 264.7 Cells

The basidiomycete Laetiporus sulphureus var. miniatus belongs to the Aphyllophorales, Polyporaceae, and grows on the needleleaf tree. The fruiting bodies of Laetiporus species are known to produce N-methylated tyramine derivatives, polysaccharides, and various lanostane triterpenoids. As part of our ongoing effort to discover biologically active compounds from wood-rotting fungi, an anti-inflammatory triterpene, LSM-H7, has been isolated from the fruiting body of L. sulphureus var. miniatus and identified as acetyl eburicoic acid. LSM-H7 dose-dependently inhibited the NO production in RAW 264.7 cells without any cytotoxicity at the tested concentrations. Furthermore it suppressed the production of proinflammatory cytokines, mainly inducible nitric oxide synthase, cyclooxygenase-2, interleukin (IL)-1beta, IL-6 and tumor necrosis factor alpha, when compared with glyceraldehyde 3-phosphate dehydrogenase. These data suggest that LSM-H7 is a crucial component for the anti-inflammatory activity of L. sulphureus var. miniatus.

Phylogenetic Status of an Unrecorded Species of Curvularia, C. spicifera, Based on Current Classification System of Curvularia and Bipolaris Group Using Multi Loci

A seed-borne fungus, Curvularia sp. EML-KWD01, was isolated from an indigenous wheat seed by standard blotter method. This fungus was characterized based on the morphological characteristics and molecular phylogenetic analysis. Phylogenetic status of the fungus was determined using sequences of three loci: rDNA internal transcribed spacer, large ribosomal subunit, and glyceraldehyde 3-phosphate dehydrogenase gene. Multi loci sequencing analysis revealed that this fungus was Curvularia spicifera within Curvularia group 2 of family Pleosporaceae.

Effects of Fixation and Storage of Human Tissue Samples on Nucleic Acid Preservation

BACKGROUND: Because of recent advances in the molecular diagnosis of cancer patients, tissue quality has become more important in daily practice. METHODS: To evaluate the effects of fixative, duration of fixation, decalcification, and storage periods on nucleic acid integrity, DNA and RNA were extracted from gastrointestinal cancer tissue. The yield and purity were analyzed, and polymerase chain reaction (PCR) for glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 60 bp), beta-actin (148 bp), and human growth hormone (hGH; 434 bp) and real-time reverse transcription-PCR for beta-actin (97 bp) were performed. RESULTS: All formalin-fixed paraffin-embedded (FFPE) and methacarn-fixed paraffin-embedded (MFPE) samples tested positive for GAPDH and beta-actin by PCR. hGH was successfully detected in all MFPE samples, but in only 46.7% of the FFPE samples. Prolonged formalin fixation resulted in fewer GAPDH and beta-actin PCR products, and amplification of hGH was not successful. The PCR and reverse transcription-PCR results were significantly affected by the duration of decalcification. The yield, purity, and integrity of mRNA progressively decreased with increased storage periods of paraffin blocks. CONCLUSIONS: Fixation and storage should therefore be standardized in order to improve the quality of molecular pathologic diagnosis.

Metabolism of Reactive Oxygen Species in Neutrophils from Patients with End-Stage Renal Disease / 대한신장학회잡지

BACKGROUND: The present study was aimed to know the cause of impaired bactericidal activity, especially the metabolism of oxygen free radicals in neutrophils from patients with end-stage renal disease (ESRD). METHODS: We measured the amount of superox ide anion, the activity of three antioxidant enzymes, myeloperoxidase, copper ion level, zinc ion level and the amount of malondialdehyde in neutrophils from patients with ESRD before and after hemodialysis. Reverse transcription-polymerase chain reaction (RT-PCR) for superoxide dismutase (SOD) was also done. RESULTS: The malondialdehyde level, the amount of superoxide anion, catalase, and myeloperoxidase levels in the neutrophils from the patients with ESRD were higher than those from healthy controls. SOD activity, hydrogen peroxide level and zinc level were lower in ESRD patients. On the RT-PCR, the relative index, which is defined the ratio of the band densities for SOD to glyceraldehyde 3-phosphate dehydrogenase, was decreased in neutrophils from patients with ESRD. Glutathione peroxidase activity in the neutrophils from ESRD patients did not show any significant change. CONCLUSION: These results indicate that there are some alterations in metabolism of oxygen free radicals including lower levels of hydrogen peroxide which exerting a direct germicidal ability, due to decreased gene expression and mineral levels. And these alterations might be one of the major mechanisms of impaired microbicidal activity in patients with ESRD.

The Developmental Differences of Damage in Rat Brain by Systemic Kainic Acid Injection / 대한해부학회지

Kainic acid[KA], a glutamic acid analogue, has been widely used as a excitotoxin in the study of neurotoxicity due to its ability to induce strong excitation and to increase intracellular calcium concentration of the mature central nervous system[CNS] neurons. However, it is not well known whether KA is also strongly cytotoxic to the neurons of the postnatal brain. We have injected KA into rats at different developmental stages and have investigated the changes in the expression of c-fos[transcriptional factor and a marker of neuronal activity], heat shock proetin 72[hsp 72, a neuronal injury marker], and glial fibrillary acidic protein[GFAP, a neuronal injury marker] mRNAs, which are known to be increased in KA-induced neurotoxicity, and glyceraldehyde 3-phosphate dehydrogenase [GAPDH, a house keeping gene] mRNAs with in situ hybridization histochemistry using specific riboprobes. The expression of c-fos mRNA was first identified in the CA3 area of hippocampus from 6hr after KA treatment in P7 rats. The c-fos mRNA-expressing area and the level of expression was gradually increased from P7 to adult. Hsp 72 mRNA was first expressed in the dentate gyrus and hippocampus from 6hr after KA treatment in P2l rats. In the adult rats, hsp 72 mRNA was broadly expressed in the brain at 2hr after KA treatment. The increase of GFAP mRNA expression was first identified in Pl4 rat brain from 6hrs after KA treatment, and by the development of brain it tends to appear earlier. The expression of GAPDH mRNA, however, did not show changes after KA treatment except for the adult rats showing a slight decrease at 12hr after KA treatment. These results suggest that KA may offer different level of cytotoxicity to the developing neurons by their developmental status and the difference may be correlated with the completion of synaptogenesis and increase of KA receptor.