• Journal of Microbiology and Biotechnology
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• v.19 no.5
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• pp.474-481
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• 2009

A novel microorganism, designated as LG12, was isolated from soil based on its ability to use acrylic acid as the sole carbon source. An electron microscopic analysis of its morphological characteristics and phylogenetic classification by 16S rRNA homology showed that the LG12 strain belongs to Rhodococcus erythropolis. R. erythropolis LG12 was able to metabolize a high concentration of acrylic acid (up to 40 g/l). In addition, R. erythropolis LG12 exhibited the highest acrylic acid-degrading activity among the tested microorganisms, including R. rhodochrous, R. equi, R. rubber, Candida rugosa, and Bacillus cereus. The effect of the culture conditions of R. erythropo/is LG12 on the production of 3-hydroxypropionic acid (3HP) from acrylic acid was also examined. To enhance the production of 3HP, acrylic acid-assimilating activity was induced by adding 1 mM acrylic acid to the culture medium when the cell density reached an $OD_{600}$ of 5. Further cultivation of R. erythropo/is LG 12 with 40 g/l of acrylic acid resulted in the production of 17.5 g/l of 3HP with a molar conversion yield of 44% and productivity of 0.22 g/l/h at $30^{\circ}C$ after 72 h.

• Journal of Periodontal and Implant Science
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• v.24 no.3
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• pp.472-482
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• 1994

Cytokines expressed specifically in leukocytes subsets and in activated cells, which are involved in chemotaxis and activation of leukocytes, are recently defined as chemokines. Macrophage inflammatory $protein-1{\alpha}(MIP-1{\alpha})$ and $MIP-1{\beta}$ are members of C-C chemokine subfamily which produces wide immunomodulatory, proinflammatory, and hematopoietic modulatory actions. We have studied their gene expression by using Northern blot analysis in various blood cells such as cytolytic T lymphocyte(CTL), helper T lymphocyte(HTL), macrophage, and B lymphocyte. Resting CTL line CTLL-R8 expressed $MIP-1{\alpha}$ mRNA which was downregulated by ConA stimulation. Both of resting and ConA stimulated HTL line Hut78 and Jurkat did not express $MIP-1{\alpha}$ mRNA. There was detectable $MIP-1{\alpha}$ transcript in HTL hybridoma 2B4.11 which was a little upstimulated by ConA stimulation. B cell line 230, and macrophage cell line RAW264.7 and WR19M.1 showed distinct $MIP-1{\alpha}$ message which were induced after LPS stimulation. Expression pattern of $MIP-1{\beta}$ in all cell lines or cell were almost identical to that of $MIP-1{\alpha}$. Also strategies employed to identify and characterize the biological functions was preceded by receptor cloning to trace the shorcut to the final goal of cytokine research. For the cloning of $MIP-1{\alpha}$ receptor(R), we used synthetic oligonucleotides of transmembrane(T) conserved sequences of already cloned human(h) IL-8-R, and performed reverse transcription-polymerase chain reaction(RT-PCR) amplification using murine(m) macrophage cell line mRNA. Among 5RT-PCR products, we isolated a homologous cDNA with hIL-8-R which were shown to be putative mIL-8-R cDNA.

• Journal of Life Science
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• v.17 no.12
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• pp.1641-1648
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• 2007

Yippee-like proteins, which have been identified as the homolog of Drosophila yippee protein containing a zinc-finger domain, are known to be highly conserved among eukaryotes. However, their functional roles are still poorly understood. Recently we initiated ordered differential display (ODD)-polymerase chain reaction (PCR) to isolate genes of which expressions are altered following activation of human T cells. On the ODD-PCR image, one PCR-product detected in unstimulated T cells was not detectable at the time when the activated T cells traversed near $G_1/S$ boundary following activation by immobilized anti-CD3. Cloning and nucleotide sequence analysis revealed that the PCR-product was yippee-like 5 (YPEL5) gene, which was known as a human homolog of the Drosophila yippee gene. Northern blot analysis confirmed the amount of ${\sim}2.2$ kb YPEL5 mRNA expression detectable in unstimulated T cells was sustained until 1.5 hr after activation and then rapidly declined to undetectable level by 5 hr. Ectopic expression of YPEL5 gene in human cervix epitheloid carcinoma HeLa cells caused a significant reduction in cell proliferation to the level of 47% of the control. Expression of GFP-YPEL5 fusion protein in HeLa cells showed its nuclear localization. These results demonstrated that the expression level of human YPEL5 mRNA was negatively regulated in the early stage of T cell activation, and suggested that YPEL5 might exert an inhibitory effect on the cell proliferation as a nuclear protein.

• Applied Biological Chemistry
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• v.34 no.4
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• pp.386-392
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• 1991

Whole cells of Methylosinus trichosporium OB3b, the obligate methylotroph, were used to produce propylene oxide from propane. This strain has methane monooxygenase, which catalyzes the conversion methane to methanol and can catalyze also the conversion propane to propylene oxide. Optimal condition for the production of propylene oxide was investigated in resting-whole cell system. The optimal pH and temperature was 7.0 and $35^{\circ}C$ respectively. The end product, propylene oxide, didn't inhibit the production of propylene oxide and was not further metabolized in reaction mixture. The addition of methane metabolites (methanol, formaldehde and formic acid) to the reaction mixture stimulated formation of propylene oxide by $3{\sim}4$ times, and methanol was the most effective especially. Under the optimal conditions, the 14.2 mM of propylene oxide was produced after incubation of 60 min. and the conversion ratio of propane to propylene oxide was approximately 8%.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.3
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• pp.183-190
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• 2001

Objective: Recently, microdissection of tissue sections has been used increasingly for the isolation of morphologically identified homogeneous cell populations, thus overcoming the obstacle of tissue complexity for the analysis cell-specific expression of macromolecules. The aim of the present study was to establish the minimal conditions required for the RNA extraction and amplification from the cells captured by the laser captured microdissection. Methods : Mouse ovaries were fixed and cut into serial sections (7 im thickness). Oocytes were captured by laser captured microdissection (LCM) method by using PixCell $II^{TM}$ system. The frozen sections were fixed in 70% ethanol and stained with hematoxylin and eosin, while the paraffin sections were stained with Multiple stain. Sections were dehydrated in graded alcohols followed by xylene and air-dried for 20 min prior to LCM. All reactions were performed in ribonuclease free solutions to prevent RNA degradation. After LCM, total RNA extraction from the captured oocytes was performed using the guanidinium isothiocyanate (GITC) solution, and subsequently evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) for glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). Results: With the frozen sections, detection of the GAPDH mRNA expression in the number of captured 25 oocytes were not repeatable, but the expression was always detectable from 50 oocytes. With 25 oocytes, at least 27 PCR cycles were required, whereas with 50 oocytes, 21 cycles were enough to detect GA PDH expression. Amount of the primary cDNA required for RT-PCR was reduced down to at least 0.25 $\grave{i}$ l with 50 oocytes, thus the resting 19.75 il cDNA can be used for the testing other interested gene expression. Tissue-to-slide, tissue-to-tissue forces were very high in the paraffin sections, thus the greater number of cell procurement was required than the frozen sections. Conclusion: We have described a method for analyzing gene expression at the RNA level with the homogeneously microdissected cells from the small amount of tissues with complexity. We found that LCM coupled with RT-PCR could detect housekeeping gene expression in 50 oocytes captured. This technique can be easily applied for the study of gene expression with the small amount of tissues.

• The Korean Journal of Physiology and Pharmacology
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• v.6 no.5
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• pp.255-260
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• 2002

The effects of intracellular and extracellular pH on the inwardly rectifying $K^+$ (IRK) channel of the bovine aortic endothelial cells (BAECs) were examined using whole-cell patch-clamp technique. The IRK current, efficiently blocked by $Ba^{2+}\;(200{\mu}M),$ is the most prominent membrane current in BAECs, which mainly determines the resting membrane potential. The expression of Kir2.1 was observed in BAECs using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Intracellular alkalinization, elicited by the extracellular substitution of NaCl with $NH_4Cl$ (30 mM), significantly augmented the amplitude of IRK current. On the contrary, the amplitude of IRK current was attenuated by the Na-acetate (30 mM)-induced intracellular acidification. The changes in extracellular pH also closely modulated the amplitude of IRK current, which was decreased to $40.2{\pm}1.3%$ of control upon switching the extracellular pH to 4.0 from 7.4. The extracellular pH value for half-maximal inhibition (pK) of IRK current was 5.11. These results demonstrate that the activity of IRK channel in BAECs, probably Kir2.1, was suppressed by proton at both sides of plasma membrane.

• Journal of Korean Neurosurgical Society
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• v.30 no.6
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• pp.688-698
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• 2001

Objectives : Parkinson's disease is a well-known neurodegenerative disease characterized by dopaminergic cell death in the substantia nigra. The reactive gliosis by activated astrocytes and microglias is no more regarded as a simple sequel of neuronal cell death. Microglial activation takes place in a stereotypic pattern with graded morphologic and functional(resting, activated and phagocytic) changes. In Parkinson's disease animal model, the degree of microglial activation along the nigro-striatal dopaminergic tract has not been studied intensively. The purpose of this study was to elucidate the characteristics of microglial reaction and to grade its degree of activation at substantia nigra and corpus striatum using 6-hydroxydopamine induced rat model of Parkinson's disease. Methods : Using Sprague-Dawley rat, parkinsonian model was made by 6-hydroxydopamine(OHDA) induced destruction of medial and lateral substantia nigra(SN). The rat was sacrificed 3-, 5-, 7-, 14- and 21-day-after operation. For control group, we injected saline with same manner and sacrificed 3-day after operation. With immunohistochemistry, we examined dopaminergic neuronal cells and microglial expression using tyrosine hydroxylase (TH) and OX-42 antibodies, respectively. Also we performed in situ hybridization for osteopontin, a possible marker of subset in activated microglia. Results : 1) In lesioned side of substantia nigra and corpus striatum, the TH immunoreactivity was markedly decreased in whole experimental groups. 2) Using optical densitometry, microglia induced immunoreactivity of OX-42 was counted at SN and corpus striatum. At SN, it was increased significantly on the lesioned side in control and all time-dependent experimental groups. At striatum, it was increased significantly in post lesion 3-day group only(p <0.05). Compared to control group, immunoreactivity of OX-42 on lesioned side was increased in groups, except post lesion 21-day group, at SN. Only post lesion 3-day group showed significance at striatum(p <0.05). Compared to SN region, immunoreactivity of OX-42 was much weaker in striatum. 3) Microscopically, the microglias showed typically different activation pattern. At SN, numerous phagocytic microglias were found at pars compacta and reticularis of lesion side. At striatum, no phagocytic form was found and the intensity of staining was much weaker. 4) At SN, the immunoreactivity of osteopontin showed definite laterality and it was markedly increased at pars compacta of lesion side with relatively short duration time. At striatum, however, it was not detected by in situ hybridization technique. Conclusion : The nigral 6-OHDA induced rat model of Parkinson's disease revealed several characteristic patterns of microglial reaction. At SN, microglias was activated shortly after direct neuronal damage and maintained for about three weeks. In contrast, despite of sufficient dopaminergic insufficiency at striatum, activation of microglias was trivial, and distinguished 3 day later. Antegrade slow neuronal degeneration is major pathophysiology in striatal dopaminergic deficiency. So, the acuteness of neuronal damage and consequential degree of neuronal degeneration may be important factor for microglial activation in neurodegenerative diseases such as Parkinson's disease. Additionally, osteopontin may be a possible marker for several subsets of activated microglia, possibly the phagocytic form.