• Korean Journal of Microbiology
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• v.45 no.2
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• pp.228-231
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• 2009

We have purified Phytophthora capsici alpha and beta tubulin from Escherchia coli BL21(DE3). The recombinant alpha and beta tubulins were assembled into microtubule in vitro with specific conditions. The metagenome library was isolated from soil in the Mt. Yeo-Ki, Suwon, Korea and manufactured with the method mentioned in experiment contents for in vitro screening of microtubule assembly screening. FRET effect was used for microtubule assembly inhibitor screening with metagenome library. We got 2 metagenome clones from in vitro screening, and these 2 hit clones showed P. capsici growth inhibition activity on the growing pepper plants. These results suggest that new development of potent inhibitor for pepper blight disease and new approach to prevention of pepper blight disease.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.30 no.1
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• pp.129-133
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• 2004

The major advantage of the in vitro test is that it is a rapid, objective and cost-effective screening methodology. In vitro tests can provide a formulation tool to identify new fillers that are optimized by combinations of old ones and they can be used to pre-screen protective formulas prior to in vivo testing in humans. Therefore, the accuracy of in vitro SPF measurement is very important. In this study, improvement of application method of samples was tried to improve the accuracy of in vitro SPF measurement. The outer part of Transpore$\^$(R)/ tape was used to apply samples as the substrates and the standard drying time was set at 15 min. The new method, topical applications at light scan areas, results in more accurate and reliable results. This result suggests that more accurate prediction system can be established for in vivo SPF with in vivo SPF measurement.

• Korean Journal of Plant Resources
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• v.5 no.2
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• pp.95-103
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• 1992

Present anticancer drugs in the clinical side have not showed a conclusive effect of the chemotherapy for cancer patients. In order to find much more efficient antitumor agents fromnatural resources, various screening methods vivo and in vitro have been developed by manyresearchers. The intention of this paper is to provide an outline of some background on the tumorsystem in drug development of natural products, to review some screening programs for theevaluation of antitumor activity and to introduce the practical procedures of some antitumorscreening methods in vivo and in vitro. At the end of this paper, the current literatures related toantitumor natural products from higher plants at our laboratory are described.Key words'anticancer drugs, screening methods.

• Journal of Microbiology and Biotechnology
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• v.15 no.5
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• pp.1022-1027
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• 2005

Based on plasmid display technology by the complexes of fusion protein and the encoding plasmid DNA, an in vitro selection method for high affinity DNA-binding protein was developed and experimentally demonstrated. The GAL4 DNA-binding domain (GAL4 DBD) was selected as a model DNA-binding protein, and enhanced green fluorescent protein (EGFP) was used as an expression reporter for the selection of target proteins. Error prone PCR was conducted to construct a mutant library of the model. Based on the affinity decrease with increased salt concentration, mutants of GAL4 DBD having high affinity were selected from the mutant protein library of protein-encoding plasmid complex by this method. Two mutants of (Lys33Glu, Arg123Lys, Ile127Lys) and (Ser47Pro, Ser85Pro) having high affinity were obtained from the first generation mutants. This method can be used for rapid in vitro selection of high affinity DNA-binding proteins, and has high potential for the screening of high affinity DNA-binding proteins in a sequence-specific manner.

• Korean Journal of Pharmacognosy
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• v.26 no.1
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• pp.51-56
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• 1995

To devise an in vitro screening method for antihepatotoxic activity, $CCl_4-induced$ cytotoxicities in primary cultures rat hepatocytes were examined. When rat hepatocytes were intoxicated with 0.5, 1.0 or 1.5 mM $CCl_4$ for 1.5, 3 or 19hr, in order of LDH>GOT>GPT release form hepatocytes was increased in a dose-dependent manner. Treatment with 1.5 mM $CCl_4$ for 1.5 hr showed maximum increase in activity of LDH, GOT or GPT released in the medium compared with the control. At this experimental condition, well known antihepatotoxic substances, glycyrrhizin and silybin markedly inhibited $CCl_4-induced$ cytotoxicities. These results demonstrated that the screening method using $CCl_4-induced$ injury in primary cultured rat hepatocytes might be suitable in vitro assay for antihepatotoxic activity.

• The Journal of Korean Society of Virology
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• v.27 no.1
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• pp.69-75
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• 1997

To evaluate in vitro anti-HIV efficacies of nucleoside derivatives, MT-4 cell line was infected with HIV-1 and HIV-2 respectively and treated with various compounds and the formerly approved drugs such as AZT, d4T, ddC and ddI. CPE method was used to evaluate their antiviral activity. Most dideoxynucleosides, AZT, d4T, ddC and ddI, showed anti-HIV activities against both viruses but no other compounds including anti-herpesvirus drugs did any. Further experiments were carried out to study their inhibitory mechanism of viral adsorption. The results showed no inhibition of syncytium formation due to an interaction between the gp120 expressed in HIV -infected cell surface and CD4 receptor on the uninfected cell surface in the presence of AZT. AZT showed no activity up to $100\;{\mu}g/ml$. Inhibition of reverse transcriptase (RT) in the presence of AZT-triphosphate was tested by using RT expressed in E. coli and purified and its $IC_{50}$ was 4.5 nM.

• Molecular & Cellular Toxicology
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• v.2 no.2
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• pp.106-113
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• 2006

Phthalate analogues are a plasticizer and solvent used in industry. Phthalates were classified in the category of "suspected" endocrine disruptors. The purpose of our study was to screen and elucidate the endocrine disrupting activity of seven phthalate analogues. E-screen assay was performed in MCF-7 human breast cancer cells with seven phthalate analogues. In this cell proliferation assay, benzyl butyl phthalate (BBP) and dibutyl phthalate (DBP) showed high estrogenic activity. Their relative proliferation efficiencies (RPE) were 109 and 106%, respectively. In vitro estrogen receptor (ER) binding assay, BBP, di-n-octyl phthalate (DOP) and dinonyl phthalate (DNP) showed weak relative binding affinity (RBA: 0.02%) compared to $17{\beta}-estradiol\;(E2)$ (RBA: 100%). In uterotrophic assay, E2 produced a significant increase, whereas four tested phthalate analogues had potential estrogenic effects in vitro did not increased in uterus weight in immature rats. From these results, we demonstrated that phthalate analogues exhibit weak estrogenic activity in vitro assays at high concentrations. Although phthalates induced an increase in MCF-7 cell proliferation by an estrogenic effect, they could not induce a uterus weight increase in vivo. From these, we may suggest that these phthalate analogues are easily metabolized to inactive forms in vivo. Further investigation in other in vitro and in vivo experimental systems might be required.

• Journal of Life Science
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• v.11 no.2
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• pp.94-98
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• 2001

Type I signal peptidase cleaves the signal sequence from the amino terminus of membrane and secreted proteins afters these protein insert across the membrane. This enzyme serves as a potential target for the development of novel antibacterial agents due to its unique physiological and biochemical properties. Despite considerable research, the signal peptidase assay still remains improvement to provide further understanding of the mechanism and high-throughput inhibitor screening of this enzyme. In this paper, three known signal peptidase assays are tested with an E. coli D276A mutant signal peptidase to distinguish the sensitivity of each assays. In vitro assay using the procoat synthesized by in vitro transcription translation shows that the D276A signal peptidase I was inactive while in vivo processing of pro-OmpA expressed in the temperature-sensitive E. coli strain IT41 as well as in vitro assay using pro-OmpA nuclease A substrate show that D276A signal peptidase I has activity like wild-type signal peptidase. These results suggest that in vitro assay using the pro-OmpA nuclease A and in vivo pro-OmpA processing assay are more sensitive monitors than in vitro assay using the pro-coat. In conculsion, caution should be used when interpreting the in vitro results using the procoat.

• Journal of Pharmaceutical Investigation
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• v.34 no.5
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• pp.393-399
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• 2004

For the efficient determination of activity against solid tumors, an in vitro tumor model that resembles the condition of in vivo solid tumors, is required. The purpose of this study was to establish a rapid culture method and viability assay for an in vitro 3-dimensional tumor model, multicellular spheroid (MCS). Among 12 human cancer cell lines, a few cell lines including DLD-1 (human colorectal carcinoma cells) formed fully compact MCS which was adequate for in vitro viability assay. DLD-1 MCS showed steady growth reaching $700\;{\mu}m$ diameter after 11 day culture. DLD-1 cells grown as MCS showed significant increase in $G_0/G_1$ phase compared to the monolayer cells (73.9% vs 45.7%), but necrotic regions or apoptotic cells were not observed. The cells cultured as MCS showed resistance to 5-FU (10.3 fold higher $IC_{50}$) compared to monolayers, however, tirapazamine (a hypotoxin) showed similar activity in both culture systems. In summary, MCS may be a valid in vitro model for activity screening of anticancer agents against human solid tumors and also exploitable for studying molecular markers of drug resistance in human solid tumors.

• Journal of Pharmaceutical Investigation
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• v.30 no.3
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• pp.191-199
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• 2000

Genomics is providing targets faster than we can validate them and combinatorial chemistry is providing new chemical entities faster than we can screen them. Historically, the drug discovery cascade has been established as a sequential process initiated with a potency screening against a selected biological target. In this sequential process, pharmacokinetics was often regarded as a low-throughput activity. Typically, limited pharmacokinetics studies would be conducted prior to acceptance of a compound for safety evaluation and, as a result, compounds often failed to reach a clinical testing due to unfavorable pharmacokinetic characteristics. A new paradigm in drug discovery has emerged in which the entire sample collection is rapidly screened using robotized high-throughput assays at the outset of the program. Higher-throughput pharmacokinetics (HTPK) is being achieved through introduction of new techniques, including automation for sample preparation and new experimental approaches. A number of in vitro and in vivo methods are being developed for the HTPK. In vitro studies, in which many cell lines are used to screen absorption and metabolism, are generally faster than in vivo screening, and, in this sense, in vitro screening is often considered as a real HTPK. Despite the elegance of the in vitro models, however, in vivo screenings are always essential for the final confirmation. Among these in vivo methods, cassette dosing technique, is believed the methods that is applicable in the screening of pharmacokinetics of many compounds at a time. The widespread use of liquid chromatography (LC) interfaced to mass spectrometry (MS) or tandem mass spectrometry (MS/MS) allowed the feasibility of the cassette dosing technique. Another approach to increase the throughput of in vivo screening of pharmacokinetics is to reduce the number of sample analysis. Two common approaches are used for this purpose. First, samples from identical study designs but that contain different drug candidate can be pooled to produce single set of samples, thus, reducing sample to be analyzed. Second, for a single test compound, serial plasma samples can be pooled to produce a single composite sample for analysis. In this review, we validated the issue whether the second method can be applied to practical screening of in vivo pharmacokinetics using data from seven of our previous bioequivalence studies. For a given drug, equally spaced serial plasma samples were pooled to achieve a 'Pooled Concentration' for the drug. An area under the plasma drug concentration-time curve (AUC) was then calculated theoretically using the pooled concentration and the predicted AUC value was statistically compared with the traditionally calculated AUC value. The comparison revealed that the sample pooling method generated reasonably accurate AUC values when compared with those obtained by the traditional approach. It is especially noteworthy that the accuracy was obtained by the analysis of only one sample instead of analyses of a number of samples that necessitates a significant man-power and time. Thus, we propose the sample pooling method as an alternative to in vivo pharmacokinetic approach in the selection potential lead(s) from combinatorial libraries.