• Proceedings of the Korean Society for Applied Microbiology Conference
• /
• 2002.10a
• /
• pp.18-25
• /
• 2002

The pikromycin biosynthetic system in Streptomyces venezuleae is unique for its ability to produce two groups of antibiotics that include the 12-membered ring macrolides methymycin and neomethymycin, and the 14-membered ring macrolides narbomycin and pikromycin. The metabolic pathway also contains two post polyketide-modification enzymes, a glycosyltransferase and P450 hydroxylase that have unusually broad substrate specificities. In order to explore further the substrate flexibility of these enzymes a series of hybrid polyketide synthases were constructed and their metabolic products characterized. The plasmid-based replacement of the multifunctional protein subunits of the pikromycin PKS in S. venezuelae by the corresponding subunits from heterologous modular PKSs resulted in recombinant strains that produce both 12- and 14-membered ring macrolactones with predicted structural alterations. In all cases, novel macrolactones were produced and further modified by the DesVII glycosyltransferase and PikC hydroxylase leading to biologically active macrolide structures. These results demonstrate that hybrid PKSs in S. venezuelae can produce a multiplicity of new macrolactones that are modified further by the highly flexible DesVII glycosyltransferase and PikC hydroxylase tailoring enzymes. This work demonstrates the unique capacity of the S. venezuelae pikromycin pathway to expand the toolbox of combinatorial biosynthesis and to accelerate the creation of novel biologically active natural products. The polyketide backbone of rifamycin B is assembled through successive condensation and ${\beta}$-carbonyl processing of the extender units by the modular rifamycin PKS. The eighth module, in the RifD protein, contains nonfunctional DH domain and functional KR domain, which specify the reduction of the ${\beta}$-carbonyl group resulting in the C-21 bydroxyl of rifamycin B. A four amino acid substitution and one amino acid deletion were introduced in the putative NADPH binding motif in the proposed KR domain encoded by rifD. This strategy of mutation was based on the amino acid sequences of the corresponding motif of the KR domain of module 3 in the RifA protein, which is believed dysfunctional, so as to introduce a minimum alteration and retain the reading frame intact, yet ensure loss of function. The resulting strain produces linear polyketides, from tetraketide to octaketide, which are also produced by a rifD disrupted mutant as a consequence of premature termination of polyketide assembly. Much of the structural diversity within the polyketide superfamily of natural products is due to the ability of PKSs to vary the reduction level of every other alternate carbon atom in the backbone. Thus, the ability to introduce heterologous reductive segments such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) into modules that naturally lack these activities would increase the power of the combinatorial biosynthetic toolbox. The dehydratase domain of module 7 of the rifamycin PKS, which is predicted to be nonfunctional in view of the sequence of the apparent active site, was replaced with its functional homolog from module 7 of rapamycin-producing polyketide synthase. The resulting mutant strain behaved like a rifC disrupted mutant, i.e., it accumulated the heptaketide intermediate and its precursors. This result points out a major difficulty we have encountered with all the Amycolatopsis mediterranei strain containing hybrid polyketide synthases: all the engineered strains prepared so far accumulate a plethora of products derived from the polyketide chain assembly intermediates as major products instead of just analogs of rifamycin B or its ansamycin precursors.

• Tuberculosis and Respiratory Diseases
• /
• v.50 no.1
• /
• pp.29-41
• /
• 2001

Background : The appearance of multiple-drug-resistant Mycobacterium tuberculosis strains has been seriously compromising successful control of tuberculosis. Rifampin-resistance, caused by mutations in the rpoB gene, can be indicative of multiple-drug-resistance, and its detection is of great importance. The present study aimed to develop an oligonucleotide chip for accurate and convenient screening of drug-resistance. Methods : In order to detect point mutations in the rpoB gene, an oligonucleotide chip was prepared by immobilizing specific probe DNA to a microscopic slide glass by a chemical reaction. The probe DNA that was selected from the 81 bp core region of the rpoB gene was designed to have mutation sites at the center. A total of 17 mutant probes related to rifampin-resistance including 8 rifabutin-sensitive mutant probes were used in this study. For accurate determination, wild type probes were prepared for each mutation position with an equal length, which enabled a direct comparison of the hybridization intensities between the mutant and wild type. Results : Mycobacterial genomic DNA from clinical samples was tested with the oligonucleotide chip and the results were compared with those of the drug-susceptibility test in addition to sequencing and INNO-LiPA Rif. TB kit test in some cases. Out of 15 samples, the oligonucleotide chip results of 13 samples showed good agreement with the rifabutin-sensitivity results. The two samples with conflicting result also showed a discrepancy between the other tests, suggesting such possibilities as existence of mixed strains and difference in drug-sensitivity. Further verification of these samples in addition to more case studies are required before the final evaluation of the oligonucleotide chip can be made. Conlcusion : An oligonucleotide chip was developed for the detection of rpoB gene mutations related to drugsusceptibility. The results to date show the potential for using the oligonucleotide chip for accurate and convenient screening of drug-resistance to provide useful information in antituberculosis drug therapy.

• Journal of the Korean Society of Combustion
• /
• v.12 no.3
• /
• pp.33-39
• /
• 2007

The present study is mainly motivated to investigate the vaporization, auto-ignition and combustion processes in high-pressure engine conditions. In order to realistically simulate the dimethyl ether (DME) spray dynamics and vaporization characteristics in high-pressure and high-temperature environment, the high-pressure vaporization model is utilized. The interaction between chemistry and turbulence is treated by employing the Representative Interaction Flamelet (RIF) model. The detailed chemistry of 336 elementary steps and 78 chemical species is used for the DME/air reaction. Numerical results indicate that the RIF approach, together with the high-pressure vaporization model, successfully predicts the essential feature of ignition and spray combustion processes.

• Journal of ILASS-Korea
• /
• v.10 no.4
• /
• pp.16-25
• /
• 2005

The present study is mainly motivated to investigate the vaporization, auto-ignition and combustion processes in high-pressure engine conditions. In order to realistically simulate the dimethyl ether (DME) spray dynamics and vaporization characteristics in high-pressure and high-temperature environment, the high-pressure vaporization model is utilized. The interaction between chemistry and turbulence is treated by employing the Representative Interaction Flamelet(RIF) model. The detailed chemistry of 336 elementary steps and 78 chemical species is used for the DME/air reaction. Numerical results indicate that the RIF approach, together with the high-pressure vaporization model, successfully predicts the essential feature of ignition and spray combustion processes.

• Journal of ILASS-Korea
• /
• v.13 no.1
• /
• pp.28-33
• /
• 2008

The present study is mainly motivated to investigate the vaporization, auto-ignition and combustion processes in high-pressure diesel engines. In order to realistically simulate the dimethyl ether (DME) fueled diesel engine, the high pressure vaporization model is utilized and the interaction between turbulence and chemistry is treated by employing the Representative Interactive Flamelet (RIF) model. The detailed chemisty consisted of 336 elementary reaction steps and 78 species is used for DME/air reaction. Numerical results indicate that the RIF model with high pressure vaporization model successfully predicts the essential feature of the combustion processes and pollutants formations in the DME fueled diesel engines.

• Tuberculosis and Respiratory Diseases
• /
• v.87 no.3
• /
• pp.302-308
• /
• 2024

In cases where pulmonary tuberculosis (PTB) is not microbiologically diagnosed via sputum specimens, bronchoscopy has been the conventional method to enhance diagnostic rates. Although the additional benefit of bronchoscopy in diagnosing PTB is well-known, its overall effectiveness remains suboptimal. This review introduces several strategies for improving PTB diagnosis via bronchoscopy. First, it discusses how bronchoalveolar lavage or an increased number of bronchial washings can increase specimen abundance. Second, it explores how thin or ultrathin bronchoscopes can achieve specimen acquisition closer to tuberculosis (TB) lesions. Third, it highlights the importance of conducting more sensitive TB-polymerase chain reaction tests on bronchoscopic specimens, including the Xpert MTB/RIF assay and the Xpert MTB/RIF Ultra assay. Finally, it surveys the implementation of endobronchial ultrasound with a guide sheath for tuberculomas, collection of post-bronchoscopy sputum, and reduced use of lidocaine for local anesthesia. A strategic combination of these approaches may enhance the diagnostic rates in PTB patients undergoing bronchoscopy.

• BMB Reports
• /
• v.32 no.2
• /
• pp.112-118
• /
• 1999

The presence of hypoxic cells in solid tumors has long been considered a problem in cancer treatment such as in radiation therapy or treatment with some anticancer drugs. It has been suggested that hypoxic cells are involved in the development of a more aggressive phenotype and contribute to metastasis. In this study, as an attempt to understand how tumor cells adapt to hypoxic stress, we investigated the regulation of the hypoxia-induced expression of proteins that control essential processes of tumor cell survival and angiogenesis. We first examined whether hypoxia induces stress protein gene expression of murine solid tumor RIF cells. We also examined hypoxia-induced changes in angiogenic gene expression in these cells. Finally, we investigated the association of the elevated levels of stress proteins with the regulation of hypoxia-induced angiogenic gene expression. Results demonstrated that hypoxia induced the expression of the erythropoietin (EPO) gene and at least two major members of stress proteins, heat shock protein 70 (HSP70) and 25 (HSP25) in RIF tumor cells. Evidence that the expression of EPO gene was greatly potentiated in TR cells suggested that the elevated levels of HSPs may play an important role in the regulation of the hypoxia-induced EPO gene expression. One of the RIF variant cell lines, TR, displays elevated levels of HSPs constitutively. Taken together, our results suggest that a hypoxic tumor microenvironment may promote the survival and malignant progression of the tumor cells by temporarily increasing the level of stress proteins and expressing angiogenic genes. We suspect that stress proteins may be associated with the increase of the angiogenic potential of tumor cells under hypoxia.

• Nuclear Engineering and Technology
• /
• v.38 no.3
• /
• pp.285-292
• /
• 2006

Thermoresistant (TR) clones of radiation-induced fibrosarcoma (RIF) cells have been reported to show an adaptive response to 1cGy of low dose radiation, and HSP25 and inducible HSP70 are involved in this process. In this study, to further elucidate the mechanism by which HSP25 and inducible HSP70 regulate the adaptive response, HSP25 or inducible HSP70 overexpressed RIF cells were irradiated with 1cGy and the cell cycle was analyzed. HSP25 or inducible HSP70 overexpressed cells together with TR cells showed increased G1 phase after 1cGy irradiation, while RIF cells did not. $[^3H]-Thymidine$ and BrdU incorporation also indicated that both HSP25 and inducible HSP70 are involved in G1 arrest after 1cGy irradiation. Molecular analysis revealed upregulation of p27Cip/Kip protein in HSP25 and inducible HSP70 overexpressed cells, and cotransfection of p27Cip/Kip antisense abolished the induction of the adaptive response and 1cGy-mediated G1 arrest. The above results indicate that induction of an adaptive response by HSP25 and inducible HSP70 is mediated by upregulation of p27Cip/Kip protein, resulting in low dose radiation-induced G1 arrest.

• Radiation Oncology Journal
• /
• v.28 no.4
• /
• pp.219-223
• /
• 2010

Purpose: To test the radiosensitizing effect of the newly synthesized novel histone deacetylase inhibitor, SK-7041 in vivo. Materials and Method: The RIF-l cell line was implanted into the back of a 6-week-old female C3H mouse, intradermally, The mice were grouped into control, drug, radiation (RT), and RT+drug group. SK-7041, 4 mg/kg was administered intraperitoneally for six cycles every 12 hours for mice in the drug and RT+drug group, An identical volume of phosphate buffered saline (PBS) was administered at the same frequency to mice in the control and RT groups. A single 5 Gy fraction was delivered to mice in RT and RT+drug group 6 hours after the fourth delivery. The volume of the implanted tumor was measured every 2~3 days to formulate the growth delay curve. Results: For the control, drug, RT, and RT +drug groups, the average duration for implanted tumor to reach a volume of $1,500mm^3$ was 10 days, 10 days, 9 days, and 12 days, respectively. Moreover, the tumor volume on D14 was $276.7mm^3$, $279.9mm^3$, $292.5mm^3$, and $185.5mm^3$, respectively (p=0.0004). The difference for the change in slope for the control and drug versus the RT and RT+drug groups were borderline significant (p=0.0650). Conclusion: The results of this study indicate that SK-7041 has a radiosensitizing effect for the RIF-1 cell line in vivo at a low concentration and this effect may be synergistic. Implementing this result to clinical trial is warranted.

• Tuberculosis and Respiratory Diseases
• /
• v.82 no.1
• /
• pp.88-89
• /
• 2019