• Journal of Microbiology and Biotechnology
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• v.20 no.10
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• pp.1463-1470
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• 2010

E. coli has long been widely used as a host system for the manufacture of recombinant proteins intended for human therapeutic use. When considering the impurities to be eliminated during the downstream process, residual host cell DNA is a major safety concern. The presence of residual E. coli host cell DNA in the final products is typically determined using a conventional slot blot hybridization assay or total DNA Threshold assay. However, both the former and latter methods are time consuming, expensive, and relatively insensitive. This study thus attempted to develop a more sensitive real-time PCR assay for the specific detection of residual E. coli DNA. This novel method was then compared with the slot blot hybridization assay and total DNA Threshold assay in order to determine its effectiveness and overall capabilities. The novel approach involved the selection of a specific primer pair for amplification of the E. coli 16S rRNA gene in an effort to improve sensitivity, whereas the E. coli host cell DNA quantification took place through the use of SYBR Green I. The detection limit of the real-time PCR assay, under these optimized conditions, was calculated to be 0.042 pg genomic DNA, which was much higher than those of both the slot blot hybridization assay and total DNA Threshold assay, where the detection limits were 2.42 and 3.73 pg genomic DNA, respectively. Hence, the real-time PCR assay can be said to be more reproducible, more accurate, and more precise than either the slot blot hybridization assay or total DNA Threshold assay. The real-time PCR assay may thus be a promising new tool for the quantitative detection and clearance validation of residual E. coli host cell DNA during the manufacturingprocess for recombinant therapeutics.

• Biomolecules & Therapeutics
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• v.3 no.4
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• pp.245-250
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• 1995

The carbon tetrachloride($CCl_4$) has been demonstrated to have a hepatotoxic effect in human or many other species. To investigate the enzyme induction of mixed function oxygenases in liver of male Sprague-Dawley rats a single 0.1, 0.5 mι/kg dose of carbon tetrachloride were given. At 24 hr after a single dose of 0.1 mι CC1$_4$/kg weight, methanol extract of Hedera rhombea leaves was administered with 100, 500 mg/kg weight. Assays of 7-ethoxyresorufin-Ο-deethylation(EROD),7-benzyloxyresorufin-Ο-deathylation(BROD),4-nitro-phenol-UDP-glucuronosyltransferase(UDPGT), Western blot and RNA slot blot were used as representatives of the activities of cytochrome P-450 enzymes. The change of the activity of CYP1A1 form measured by EROD assay and Western analysis using 1-7-1 monoclonal antibody was not observed. The activity CYP2B1 form by BROD assay and using 2-66-3 monoclonal antibody was remarkably increased. Elevated level of CYP2B1 mRNA was shown by slot hybridization with 2B1-specific probe. Administration of methanol extract of Hedera rhombea leaves reversed the enzyme activity and the level of mRNA, which suggest the chemoprotective effect of methanol extracts of Hedera rhombea leaves to carbon tetrachloride hepatotoxlcity.

• Journal of the Korean Society of Food Science and Nutrition
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• v.25 no.6
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• pp.1024-1030
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• 1996

Capsaicin (8-methyl-N-vanillyl-6-nonenamide: CAP) is a mai or ingredient of hot pepper that has been used as a spicy food additive, preservative, and medicine. In this study, we evaluated the effect of dietary CAP on the selected proto-oncogene(c-jun, c-myc, H-ras, erbB, p53) expressions in various tissues of mice. Male ICR mice were divided into four groups and fed the experimental diets containing CAP at the levels of 0, 5, 20 and 100ppm for four weeks. Steady state RNA levels in various tissues were measured by slot blot hybridization assay. C-jun expression level was enhanced in stomach tissue from mice fed 20ppm CAP and significantly reduced from mice fed 100ppm CAP. The c-jun expression levels were differentially altered in organ-specific manner, Tumor suppressor gene p53 expression level appeared to be slightly increased in the liver from mice fed 20ppm CAP. These results suggested that dietary CAP differentially modulates c-jun and p53 expression in various organs.

• Tuberculosis and Respiratory Diseases
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• v.51 no.6
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• pp.517-529
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• 2001

Background : Surfactant protein C(SP-C) is a hydrophobic 5,000 dalton molecule. SP-C has the primary roles in accelerating surface spreading of a surfactant phospholipid. The filter hybridization and solution hybridization assays are both rapid and sensitive and can be used to measure the RNAs complementary to any cloned DNA sequence. Methods : The authors measured the SP-C mRNA levels quantitatively using solution hybridization and filter hybridization assays to obtain a standard curve equation to quantify the mRNA of unknown samples comparatively. Results : 1. The minimum level of the specimens by solution hybridization was 3 pg for SP-C mRNA. 2. The standard curve equation of the solution hybridization assay between the counts per minute(Y) and the SP-C mRNA transcript input(X) was Y=6.46 X+244. The correlation coefficient was 0.99. 3. The minimum detection level of specimens by filter hybridization was 0.1 ng for SP-C mRNA. 4. The standard curve equation of the filter hybridization assay between the counts per minute(Y) and SP-C mRNA transcript input(X) is Y=2541.6 X+252.7. The correlation coefficient was 0.99. Conclusions : A comparison of CPM/filter in the linear range allowed an accurate and reproducible estimation of the SP-C mRNA copy number. Filter hybridization and solution hybridization assays are both rapid and sensitive and can be used to measure the RNAs complementary to any cloned DNA sequence. It is ideally suited to situations where accurate quantitation of multiple samples is required.

• The Korean Journal of Pharmacology
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• v.30 no.1
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• pp.19-28
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• 1994

The effects of gonadoropin-releasing hormone (GnRH) and ovarian steroid hormones on the release of luteinizing hormone (LH) and its subunit mRNA levels were investigated in anterior pituitary cells in culture. LH concentration was measured by a specific radioimmunoassay and mRNA levels of u and $LH{\beta}$ subunits by RNA slot blot hybridization assay. GnRH stimulated LH release in a dose-dependent manner from cultured pituitary cells. However, the basal LH release in the absence of GnRH was not changed during the course of 24h culture, strongly suggesting that release of LH is directly controlled by GnRH. The treatment of the pituitary cells with GnRH increased $LH{\beta}$ subunit mRNA levels in a dose-dependent manner, reaching the maximum with $2\;{\times}\;10^{-10}M$ GnRH while no significant increase in ${\alpha}$ subunit mRNA levels was observed after GnRH treatment. Estradiol did not augment GnRH-induced LH release while progesterone augmented GnRH-induced LH release in a dose-dependent manner at the level of pituitary. However, estradiol and progesterone increased basal and GnRH-induced $LH{\beta}$ subunit mRNA levels in a dose-dependent manner. The treatment of estrogen antagonist, LYI17018 blocked the effect of estradiol on GnRH-induced $LH{\beta}$ subunit mRNA levels in a dose-dependent manner while progesterone antagonist, Ru486 tended to block the effect of progesterone on GnRH-induced $LH{\beta}$ subunit mRNA levels. It is therefore suggested that GnRH Playa a major role in LH release and subunit biosynthesis by influencing the steady state $LH{\beta}$ subunit mRNA loves and ovarian steroid hormones modulate subunit biosynthesis via directly acting on pituitary gonadotropes.