Seong-Karp Hong

Anti-HIV-1 efficacy of extracts from medicinal plants

The anti-HIV-1 activities of butanol, hexane, chloroform and water extracts from four widely used folk medicinal plants (Sophora flavescens, Tulipa edulis, Herba ephedra, and Pachyma hoelen Rumph) were evaluated in this study. The hexane extract of Pachyma hoelen Rumph, PH-4, showed effective inhibition against HIV-1. The 50% effective concentration (EC50) of PH-4 was 37.3 μg/ml in the p24 antigen assay and 36.8% in the HIV-1 recombinant RT activity test (at 200 μg/ml). In addition, the PH-4 showed the protective effect on the infected MT-4 cells, with a 58.2% rate of protection. The 50% cytotoxic concentration (CC50) of PH-4 was 100.6 μg/ml. These results suggest that PH-4 from Pachyma hoelen Rumph might be the candidate for the chemotherapy agent against HIV-1 infection with further study.

Detection of Mycobacterium kansasii Using DNA-DNA Hybridization with rpoB Probe

A microtiter well plate DNA hybridization method using Mycobacterium kansasii-specific rpoB DNA probe (kanp) were evaluated for the detection of M. kansasii from culture isolates. Among the 201 isolates tested by this method, 27 strains show positive results for M. kansasii, but the other 174 isolates were negative results for M. kansasii. This result was consistent with partial rpoB sequence analysis of M. kansasii and the result of biochemical tests. The negative strains by this DNA-DNA hybridization method were identified as Mycobacterium tuberculosis (159 strains), Mycobacterium avim (5 strains), Mycobacterium intracellulare (8 strains), and Mycobacterium flavescens (2 strain) by rpoB DNA sequence analysis. Due to high sensitivity and specificity of this test result, we suggest that DNA-DNA hybridization method using rpoB DNA probes of M. kansasii could be used for the rapid and convenient detection of M. kansasii.

PCR-linked reverse DNA hybridization using oligonucleotide-specific probes of rpoB for identification of Mycobacterium avium and Mycobacterium intracellulare

A PCR-linked reverse DNA hybridization method using two different specific rpoB DNA probes (Avp and Intp) of Mycobacterium avium and Mycobacterium intracellulare, respectively, were evaluated for the differentiation and identification of M. avium and M. intracellulare culture isolates. Among the 504 culture isolates tested by this method, 48 strains showed positive results for M. avium and 60 strains showed positive results for M. intracellulare. The other 396 culture isolates showed negative results for both M. avium and M. intracellulare. These results were consistent with those obtained from partial rpoB (306 bp) sequence analysis and biochemical tests. The negative strains obtained by this DNA hybridization method were identified as M. tuberculosis (366 strains), M. peregrinum (11 strains), M. abscessus (9 strains), M. fortuitum (8 strains), and M. flavescens (2 strains) by rpoB DNA sequence analysis. Due to the high sensitive and specific result obtained by this assay, we suggest that this PCR-linked reverse DNA hybridization method using two different specific rpoB DNA probes of M. avium and M. intracellulare would be used for the rapid and precise method for differentiation and identification of M. avium and M. intracellulare.

Coculture of Schwann Cells and Neuronal Cells for Myelination in Rat

For in vitro myelination system, Schwann cells and neuronal cells of rat were cocultured. Schwann cells and neuronal cells, respectively, were obtained from dorsal root ganglion of rat embryos (E15). This method includes four steps: first step of suspension of the embryonic dorsal root ganglion cells, second step of addition of anti-mitotic cocktail, third step of purification of dorsal root cells, and fourth step of addition of Schwann cells to dorsal root ganglion cells. We made a highly purified population of myelination in a short period through this procedure and identified myelination basic protein using antibody of myelination basic protein.

Facile synthesis, crystal structure, DFT calculation and biological activities of 4-(2-fluorophenyl)-3-(3-methoxybenzyl)-1H-1,2,4-triazol-5(4H)-one (5)

BACKGROUND In the past few decades, design, synthesis, and characterization of novel heterocyclic compounds with auspicious biological profile received the considerable attention of the scientific community. Among them, the small and simple organic molecular backbone like triazole moiety have a broad spectrum of applications in the medicinal as well as diagnostic areas. OBJECTIVE The objective of present study was synthesis, characterization, and exploration of biological profile of 4-(2-fluorophenyl)-3-(3-methoxybenzyl)-1H-1,2,4-triazole-5(4H)-one (5). The tautomeric interconversion of the molecule was observed by the single crystal XRD and DFT analysis. METHODS N-(2-fluorophenyl)-2-[2-(3-methoxyphenyl)acetyl]hydrazine carboxamide (4) was synthesized by the condensation of 2-(3-methoxyphenyl)acetohydrazide (3) with 1-fluoro-2- isocyanatobenzene. The dehydrocyclization of compound (4) yielded target compound (5) by refluxing in 2 N aqueous sodium hydroxide solutions. The target molecule was characterized by FTIR, 1H NMR, 13C NMR, single crystal X-ray diffraction analysis and DFT calculation. The enzymatic assay measurements were carried out by using a microplate reader (OPTI Max, Tunable Microplate Reader; Wavelength range: 340-850 nm; for 96-well plates) while DFT calculation was performed by Gaussian 09 package. RESULTS The XRD result and DFT calculations showed that molecule 5 predominantly exists in thione conformation and crystallized in the triclinic system of P-1 space group. Furthermore, for the practical applicability of synthesized compound 5, the in vitro acetylcholinesterase as well as α-glucosidase inhibition activities were performed and found moderate enzyme inhibition potential comparable with that of reference inhibitors. CONCLUSION This study might be helpful for future design and development of potent enzyme inhibitor to control Alzheimers as well as diabetic disease. The DFT and single crystal XRD analysis data might be helpful for understanding the mechanism of drug binding and its mode of action.

Induction of Demyelination by Infection of Semliki Forest Virus

Schwann cells and neuronal cells from dorsal root ganglion (DRG) in embryos of rat were cultured in vitro respectively. The purified neuronal cells with anti-mitotic agents and purified Schwann cells were co-cultured and then accomplished myelination processing. Infection of Semliki forest virus into this myelinated coculture system was performed and then accomplished demyelination. We identified myelination and demyelination processing using antibody of neuropeptide Y. Many viruses such as Theiler’s virus, mouse hepatitis virus (MHV), corona, measles, herpes viruses, and Semliki Forest virus are known as cause of inducing demyelination (meaning destruction of myelination) in nervous system of mice. Especially Semliki Forest virus infection induces a demyelinating encephalomyelitis in the central nervous system of mice. Mice and rats are used as an important model for the study of myelination and demyelination research in vitro and in vivo. Generally adult mammalian DRG neuron cells can survive and regenerate in culture. In vitro myelination had been established by co-culturing with pure populations of primary Schwann cells and primary neuronal cells. These pure populations of primary Schwann cells and primary neuronal cells were driven from DRG of rat embryos. This method produced highly purified populations of Schwann cells and neuronal cells faster than any other conventional method. From this research, we constructed a population of myelinated cells with co-culture of neuronal cells and Schwann cells from DRG. After this myelinated cells were infected with Semliki Forest virus and processing of demyelination was progressed. We could identify and distinguish myelination and demyelination processing using antibody of neuropeptide Y which represented as myelinated cells. Cultures were incubated at 37 °C, with 5% CO2. 24 h later, 50 mL of NGF stock solution (40 mM of 5-fluorodeoxyuridine and uridine, 1 mM Arabinofur-anosyl Cytidine (Ara C, Sigma-Aldich, Saint Louis, MO) in NG medium) was added into each well to make the final concentration of 5-fluorodeoxyuridine/uridine 20 mM. Cultures were incubated at 37 °C in a 5% CO2 incubator. 6 After 72 hours, 2/3 of the NGF stock solution was changed to NG medium and the cultures were re-fed every 2 days with NG medium. After three medium changes, the neurons were ready for the Schwann cell addition. Schwann cells were digested and washed once with DMEM (Invitrogen, Carlsbad, CA) containing 10% FBS, and then resuspended in C medium (MEM, 10% FBS, 2mM l-glutamine, 0.4% glucose, and 50 ng/mL 2.5 S NGF). 100 mL (approximately 200,000 cells) of Schwann cells was added to each of the DRG cultures in C media. After 3 days, the co-cultures were supplemented with 50 mg/mL of ascorbic acid to initiate basal lamina formation and myelination. Myelination was allowed to proceed for up to 21 days. The process of myelination was observed and recorded under phase contrast microscope. To observe the formation of the myelin, the DRG neuron/Schwann co-cultures (14 days after initiation myelin formation) were then labeled with primary antibodies, which included monoclonal antibody against neuropeptide Y. After three washes with PBS, the coverslips were further incubated with Alexa Fluor 488 mouse antirabbit IgG and Alexa Fluor 594 goat anti-mouse IgG (Invitrogen, Carlsbad, CA) for 60 min at room temperature. After a final wash in PBS, the slides were mounted with mounting fluid (DAKO Ltd., Carpenteria, CA) and visualized under a fluorescence microscope (Olympus, Tokyo, Japan). The images were digitally recorded and processed with Image-Pro Plus (Media Cybernetics, Atlanta, GA). Processing for co-culture of Schwann cells and neuronal cells for myelination from DRG of rat embryos was described. This procedure contains following four steps: first step of suspension of the embryonic dorsal root ganglion cells, second step of addition of anti-mitotic cocktail, third step of purification of dorsal root cells, and fourth step of addition of Schwann cells to dorsal root ganglion cells. As a result of this study, for formation of myelination, Schwann cells and neuronal cells, respectively, were prepared and cultured from DRG of rat embryos (E 16 day) (Figure 1). Figure 1. Purification of populations of Schwann cells and neuronal cells, respectively, from DRG of rat embryo (E 16 day) (A: DRG cells; B: neuronal cells; C: Schwann cells). To identify and distinguish myelination and demyelination processing, population of cells were labeled with monoclonal antibody against neuropeptide Y and observed by fluorescent microscope. Population of myelinated cells represents fluorescent spots due to monoclonal antibody against neuropeptide Y which binds myelinated proteins. On the other hand, population of *To whom correspondence should be addressed. E-mail: cschoi@kdu.ac.kr, karp@mokwon.ac.kr Communication Hyun Joo Kim, Chang-Shik Choi, and Seong-Karp Hong This Journal is

Generation of Demyelination through Use of M. leprae-specific phenolic glycolipid-1 (PGL-1)

For myelination, Schwann cells and neuron cells from dorsal root ganglion (DRG) of rat embryos (E16) were cultured in vitro system. The purified DRG cells with anti-mitotic agents and purified Schwann cells were cocultured and then accomplished myelination processing. Treatment of M. leprae-specific phenolic glycolipid-1 (PGL-1) into this coculture system was performed and then accomplished demyelination. Therefore, we identified demyelination processing using antibody of myelin basic protein (MBP). The study of Schwann cell, Neuronal cell, and myelination has been facilitated by the availability to isolate and establish pure population of primary Schwann cells. Moreover, mice serve as an important model for the study of Schwann cell research. The specialized source of neurons from nonneuronal cells were provided in dorsal root ganglia. 1 Adult mammalian DRG neuron cells can survive and regenerate in culture. 2,3,4 There are several researches on purified populations of these neurons. Coculture of purified DRG neurons and Schwann cells can be used in myelin formation. The most widely used method for preparing primary Schwann cell culture uses DRG as the primary source of Schwann cells. The procedure is very simple and produces a highly purified population of Schwann cells in a short time. The method has also been used to prepare Schwann cells from mouse embryos. In this study, we performed a purified population of myelination by coculture of DRG neuronal cells and Schwann cells. The purified DRG cells with anti-mitotic agents and purified Schwann cells were cocultured and then accomplished myelination processing. Treatment of M. leprae-specific phenolic glycolipid-1 (PGL-1) into this coculture system was performed and then accomplished demyelination. Therefore, we identified demyelination processing using antibody of myelin basic protein (MBP). Cultures were incubated at 37 ℃, with 5% CO2. 24 h later, 150 mL of NGF stock solution (40 mM of 5-fluorodeoxyuridine and uridine, 1 mM Arabinofur-anosyl Cytidine (Ara C, Sigma-Aldich, Saint Louis, MO) in NG medium) was added into each well to make the final concentration of 5-fluorodeoxyuridine/uridine 20 mM. Cultures were incubated at 37 ℃ in a 5% CO2 incubator. 5 After 72 hours, 2/3 of the NGF stock solution was changed to NG medium and the cultures were re-fed every 2 days with NG medium. After three

Inducible Vesicular Stomatitis Virus (VSV) L Cell Line for Packaging of Recombinant VSV

Recently, recombinant vesicular stomatitis viruses (VSV) have been developed as high-level expression vectors which serve as effective vaccine vectors in animals. An ideal approach for VSV vector production would be the development of stable packaging cell lines that can produce vector particles without transfection step. In this report, we describe generation of an inducible cell line that expresses the VSV polymerase gene (L) under the control of the reverse tetracycline-controlled transactivator (rtTA) system as a first step to make VSV-based packaging cell lines. Integrated polymerase (L) gene was controlled by an rtetR-dependent promoter in the rtTA-producing BHK cell line. When the cell lines were cultured in the presence of tet (tetracycline) or tetracycline derivative doxycycline, the recombinant VSV and wild type VSV were replicated, whereas in the absence of tet or tetracycline derivative doxycycline, the recombinant VSV was not replicated. Viral supernatants were harvested, diluted, and monitored by plaque assay for the presence of infectious VSV. Plaques of VSV containing an additional sequence encoding the EGFP protein allowed rapid detection of infection. Our results suggest wide applications of other surrogate viruses based on VSV. The availability of stable packaging cell lines represents a step toward the use of a VSV vector delivery system that can allow scale-up production of vector-stocks for gene therapy.