• IMMUNE NETWORK
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• v.10 no.2
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• pp.35-45
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• 2010

Background: Expression of recombinant antibodies and their derivatives fused with other functional molecules such as alkaline phosphatase in Escherichia coli is important in the development of molecular diagnostic reagents for biomedical research. Methods: We investigated the possibility of applying a well-known Fos-Jun zipper to dimerize $V_H$ and $V_L$ fragments originated from the Fab clone (SP 112) that recognizes pyruvate dehydrogenase complex-E2 (PDC-E2), and demonstrated that the functional zipFv-112 and its alkaline phosphatase fusion molecules (zipFv-AP) can be produced in the cytoplasm of Origami(DE3) trxB gor mutant E. coli strain. Results: The zipFv-AP fusion molecules exhibited higher antigen-binding signals than the zipFv up to a 10-fold under the same experimental conditions. However, conformation of the zipFv-AP seemed to be influenced by the location of an AP domain at the C-terminus of $V_H$ or $V_L$ domain [zipFv-112(H-AP) or zipFv-112(L-AP)], and inclusion of an AraC DNA binding domain at the C-terminus of VH of the zipFv-112(L-AP), termed zipFv-112(H-AD/L-AP), was also beneficial. Cytoplasmic co-expression of disulfide-binding isomerase C (DsbC) helped proper folding of the zipFv-112(H-AD/L-AP) but not significantly. Conclusion: We believe that our zipFv constructs may serve as an excellent antibody format bi-functional antibody fragments that can be produced stably in the cytoplasm of E. coli.

• KSBB Journal
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• v.11 no.1
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• pp.37-45
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• 1996

Marine bacterial strain, highly effective agar degrading, was isolated from south sea of Korea and was identified as Pseudomonas sp. This strain was named Halophilic Pseudomonas sp. W7 and accumulated an extracellular agarase which showed a high level of enzyme activity in the presence of agar and agarose. This extracellular agarase was purified by anion-exchange chromatography and gel filtration. Purified agarase showed a single protein band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated to be about 89KDa. The agarase gene was cloned into Escherichia coli JM83 using the plasmid vector pUC19. DNA fragments(3.7, 3.0Kb) of Hind III-digested chromosomal DNA of Pseudomonas sp. W7 was inserted into the Hind III site of pUC19. Selected transformants, E. coli JM83/pSWl 000000and E. coli JM83/pSW3, produced agarase and this agarase was accumulated In the cytoplasmic space.

• Korean Journal of Veterinary Research
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• v.39 no.3
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• pp.583-592
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• 1999

We have developed the in situ hybridization(ISH) technique for rapid diagnosis of canine distemper(CD) which is the major infectious disease in dogs. In our experiment, we rapidly detected distribution of the specific canine distemper viral genome without disrupting morphology of tissues or cells. Two oligonucleotide probes for ISH were synthesized chemically and labelled 5' end with nonisotopic biotin by DNA synthesizer. The whole procedures of ISH was completed within 1~2 hours using the Microcapillary action system. On histological study, typical cytoplasmic or intranuclear inclusion bodies were observed in the trachea, bronchiole, brain, and urinary bladder with the presence of prominent red positive signals on ISH, indicating specific CDV genome from the paraffin-embedded tissues of infected 13 cases. The results showed ISH can be used as a rapid and effective diagnostic method for diagnosis of CD.

• Korean Journal of Veterinary Research
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• v.22 no.2
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• pp.221-232
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• 1982

This paper dealt with the pathological and clinical findings on the experimentally induced rodenticide (fluoroacetate, zinc phosphide, thallium sulfate, coumarin) and NaCN poisoning of ruminants (Holstein cattle and/or Korean native goat) for the purpose of the diagnosis in the accidental rodenticide poisoning of cattle. The results observed are summarized as follows: Fluoroacetate poisoning (cattle and goat): in the clinical signs, there were depression, convulsion, dyspnea, groan, grinding of the teeth, vomiting, opisthotonus and post-mortem tympany. In the macroscopical findings, the blood was more or less poor coagulative and dark red, bloody fluid with foam in the trachea, hyperemia and hemorrhage of tracheal mucosa and lung, cloudy swelling and hyperemia of kidney, epicardial hemorrhage(cattle), and hyperemia of abomasum, intestine and brain were observed. In the microscopical findings, there were pulmonary edema and hemorrhage, necrosis of convoluted tubular epithelium and interstitial hemorrhage of kidney, focal coagulative necrosis of myocardium, hemorrhage of pancreas and spleen, dilatation of Virchow-Robin space and hyperemia of brain, and necrosis with desquamation of mucosal epithelia of abomasum and upper small intestine. In the histological lesions of the liver, lobular peripheral hyperemia, centrilobular necrosis and cytoplasmic inclusion bodies of the hetatic cells were observed. The cytoplasmic inclusion body of the hepatic cells was not seen in the affected goat, but hydropic degeneration of the hepatic cells was marked. Zinc phosphide poisoning (cattle and goat): clinically, the affected animals died in recumbent position after ataxia, dyspnea and convulsion. In the macroscopical findings, hyperemia and hemorrhage of lung, cloudy swelling and hyperemia of liver and kidney, hemorrhage of spleen (cattle), and catarrh of abomasum and small intestine were observed. In the microscopical findings, necrosis of the convoluted tubular epithelium and hyperemia of kidney, hemorrhage of spleen, hyperemia of lung, hyperemia or hemorrhage of heart, cloudy. swelling and fatty changes of hepatic cells, dilatation of hepatic central vein, hyperemia of brain, and catarrh of abomasal and small intestinal mucosae were observed. Thallium sulfate poisoning (cattle): in the macroscopical findings dark red color of blood, hyperemia and hemorrhage of lung, bloody fluid with foam in the tracheal mucosa, petechiae of tracheal mucosa, cloudy swelling and hemorrhage of liver, necrotic lesions and hemorrhage of renal cortex and epicardial hemorrhage were observed. In the microscopical findings, severe hemorrhages of the lung, cloudy swelling and necrosis of hepatic cells, hyperemia and hemorrhage of liver, focal coagulative necrosis of mycordium, necrosis of the convoluted tubular epithelium and hyperemia of kidney, hyperemia and hemorrhage of spleen and dilatation of Virchow-Robin apace in brain were observed. Coumarin poisoning (goat): the poisoned animals died in the state of groan and depression. In the macroscopical findings, poor coagulation of blood, hemorrhage of lung, cloudy swelling and severe hemorrhages of liver, cloudy swelling and hemorrhage of kidney, abomasal hemorrhage, catarrh of small intestine, and hyperemia and hemorrhage of the other organs were observed, In the microscopical findings, hyperemia and hemorrhage of lung and kidney, cloudy swelling of the convoluted tubular epithelium of kidney, severe hepatic hyperemia, cloudy swelling and hydropic degeneration of heptatic cell, and hyperemia and hemorrhage of brain and spleen were observed. NaCN poisoning (cattle and goat): clinically, there were convulsion, severe dyspnea, paresis of hind limb, depression and then rigor of four limbs. In the macroscopical findings, bright red color of blood, hyperemia and bright and red tinge of lung cloudy swelling of kidney and liver, and hyperemia of abomasum were observed. In the microscopical findings, cloudy swelling and hydropic degeneration of hepatic cell, hyperemia and edema of lung, necrosis and degeneration of the convoluted tubular epithelium and hemorrhage in kidney, dilatation of Virchow-Robin space of brain and hemorrhage of spleen were observed.

• Korean Journal of Veterinary Service
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• v.20 no.1
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• pp.55-67
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• 1997

The morphological development of lungs in fetuses of 60, 90 and 120 days of gestation and neonates of Korean native goats was investigated by light, scanning and transmission electron mictroscope. The results were summarized as follows ; 1. Gross findings ; In the 60-days-old fetus, the lung was developed and differentiated into six lobes. 2. Light microscopic findings : The gland-like bronchioles were formed in loose mesenchyme at 60 days of gestation and the bronchial wall contained smooth muscles. The loose mesenchyme had been replaced by compact parenchymal tissue at 90 days of gestation and the cartilage plates appeared in bronchial wall which contained blood vessels, submucosal glands and smooth muscles. The lung parenchyma consisted of a fine network of alveoli at 120 days of gestation and the bronchial wall contained well-developed blood vessels, submucosal glands, cartilage plates and smooth muscles. In neonates, the lung tissue was similar to the mature lung tissue and the bronchial wall contained well developed cartilage plates. 3. Scanning electron microscopic findings : The epithelial cells lining the tubules were composed of cuboidal or columnar at 60 days of gestation and the epithelial cells lining the large airways were often ciliated : some were covered with stubby microvilli. The epithelial cells lining the canals were cuboidal at 90 days of gestation and the epithelial cells lining the bronchioles were ciliated cells or nonciliated(clara) cells, The clara cells contained row microvilli. The alvealor development of this stage was rapidly progressed ; the subdivision of canals by alveolar crests and assosiated wall attenuation resulted alveoli at 120 days of gestation and the respiratory bronchioles were lined by ciliated or nonciliated epithelial cells. In neonates, the epithelial cells lining the alveolar walls were mainly covered with pneumocyte type I ; Some were covered with pneu-mocyte type II. 4. Transmission electron microscopic findings : The epithelial cells lining the tubules were adhered with tight junction at apical borders of the adjacent cells at 60 days of gestation, which contained few organells and glycogen. The epithelial cells lining the canals were composed mostly of cuboidal cell at 90 days of gestation and the epithelial cells lining of the bronchioles were ciliated of nonciliated cell, which contained few organelles and abundant glycogen. The epithelial cells lining the alveolar walls were composed of pneumocyte type I and a few pneumocyte type II at 120 days of gestation. The epithelial cells lining of the bronchioles were ciliated or nonciliated cells. In neonates, pneumocyte type I was observed as flat and thin cytoplasmic extension in shape. Otherwise, pneumocyte type II was observed as cuboidal type with apical microvilli and contained osmiophillic lamellar inclusion bodies. Putting these various experiment results together, the lung development was slowly progressed at early stage, which was rapidly progressed in the late stage of gestation.

• Korean Journal Plant Pathology
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• v.3 no.4
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• pp.291-298
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• 1987

Samples showing mosaic symptom of cowpea (Vigna sesquipedalis) with vein banding, chlorotic spot, vein yellow were collected from Chinju areas in Korea, Two viruses were distinguishable by stability in sap, host range, and relations with cells and tissues were examined under an electron microscope, Blackeye cowpea mosaic(BICMV) was sap-transmissible to 7 plant species in 2 families, Of the plants, only leguminous species were systemically infected. This virus was inactivated by heating at $50-65^{\circ}C$ for 10 min, by diluting at $10^{-4}-10^{-5}$, and aging at room temperature for 1-6 days. Preparations examined under the electron microscope by direct negative staining method(DN -method) always showed particles of flexuous filament bout 750nm in length and cytopasmic inclusions. Cytoplasmic inclusions and virus particles were also confirmed to present in the cytoplasm of a mesophyll cell by ultrathin sections of BICMV infected cowpea leaves. Cucumber mosaic virus (CMV) was transmitted by sap- inoculation on inoculated leaves of Chenopodium amaranticolor, C. quinoa producing local lesions, but non-inoculated upper leaves of Nicotiana glutinosa, Cucurbita pepo and Vigna sesquipedalis producting systemic mosaic symptoms. Electron microscopic examination of virus preparation by direct negative staining showed spherical particles of about 30nm in diameter. In ultrathin sections of CMV infected tissues, virus particles of crystalline array were found in the vacuole and a large number of virus particles were found in the cytoplasm and the plasmodesmata of mesophyll cells.

• Research in Plant Disease
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• v.8 no.2
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• pp.92-100
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• 2002

For the survey of viruses infected in peanut cultivated in Korea, peanut seeds and leaves showing viral symptoms were collected from their growing areas. Typical symptoms on virus infected peanut leaves including mosaic, mottle with necrosis, yellowing, stripe or vein banding and stunts were observed. Two viruses isolated from the naturally infected peanuts were identified as Bean common mosaic virus(BCMV-PSt) and Peanut mottle virus(PeMoV) by their host range, immunosorbent elcetron microscopy(ISEM), direct immuno staining assay(DISA), RT-PCR, and intracellural symptoms. Direct negative staining method by electron microscope showed filamentous particles of about 780 m in length as well as inclusion bodies. In ultrathin sections of BCMV-PSt and PeMoV infected tissues, cytoplasmic cylindrical inclusions as well as filamentous virus particles were observed in the cytoplasm of parenchyma cells. ISEM revealed filamentous particles strongly decorated with antiserums of BCMV-PSt and PeMoV Peanut seeds were stained with BCMV-PSt and PeMoV antisera indicating the possibility of seed transmission far these viruses. Seedlings germinated from peanut seeds which reacted with antiserums of BCMV-PSt by DISA showed mild mottle or stripe symptoms while mosaic and necrotic mottle symptoms were observed for PeMoV-positive seedlings. Filamentous particles were strongly decorated with each antiserum under ISEM observation. BCMV-PSt coat protein gene of about 1.2 Kbp was amplified by RT-PCR. Altogether these results indicate that BCMV-PSt is the most prevalent virus infecting peanut in Korea.

• KSBB Journal
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• v.23 no.3
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• pp.205-212
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• 2008

The efficient soluble expression of human epidermal growth factor (hEGF) was achieved by using functional fusion partners in cytoplasm and periplasm of Escherichia coli (E. coli). hEGF was over-expressed in inactive inclusion body form in cytoplasm of E. coli due to improper disulfide bond formation and hydrophobic interaction, yielding about 5.9 mg/L in flask culture. Six functional fusion partners were introduced by linking to N-terminal part of hEGF gene for the high-level expression of soluble and active hEGF in cytoplasm and peri plasm region. Three fusion partners for cytoplasmic expression such as acidic tail of synuclein (ATS), thioredoxin (Trx) and lipase, and three fusion partners for periplasmic expression such as periplasmic cystein oxidoreductases (DsbA and DsbC) and maltose binding protein (MBP) were investigated. hEGF fused with ATS and DsbA showed over 90% of solubility in cytoplasm and periplasm, respectively. Especially DsbA was found to be an efficient fusion partner for soluble and high-level expression of hEGF, yielding about 18.1 mg/L and three-fold higher level compared to that of insoluble non-fusion hEGF in cytoplasm. Thus, heterologous proteins containing complex disulfide bond and many hydrophobic amino acids can effectively be produced as an active form in E. coli by introducing a suitable peptide or protein.

• Journal of Sericultural and Entomological Science
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• v.25 no.2
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• pp.1-31
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• 1984

Flacherie, as one of the most prevalent silkworm diseases, causes severe economic damage to sericultural industry and its pathogens have been proved to be flacherie virus (FV) and densonucleosis virus (DNV). Multiplications of the viruses in the larvae of the silkworm, Bombyx mori, were studied by the sucrose density gradient centrifugation and electron microscopy. The quantitative and qualitative changes of nucleic acids and proteins were investigated from the midgut and hemolymph in the silkworm larvae infected separately with FV and DNV. The histopathological changes of epithelial cells of infected midgut also were examined by an electron microscope. 1. Purified fractions of FV or DNV in a sucrose density gradient centrifugation yielded one homogenous and sharp peak without a shoulder, suggesting no heterogenous materials in the preparation. Electron microscopy also revealed that FV and DNV were spherical particles, 27nm and 21nm in diameter, respectively. 2. Silkworm larvae showed a decrease in body weight on the 6th day and in midgut weight on the 3rd day after inoculation with FV or DNV. 3. DNA content was higher in the midgut when infected with FV or DNV, but the hemolymph of the infected larvae showed no difference during first 6 days after inoculation, after which DNA concentration declined rapidly. 4. RNA synthesis of silkworm larvae infected separately with FV and DNV was stimulated in the midgut, but RNA content was reduced in the hemolymph at the early stage of virus multiplication. At the late stage of virus multiplication, however, it was extremely reduced in both midgut and hemolymph. 5. The concentration of protein in the midgut and hemolymph of silkworm larvae infected separately with FV and DNV showed no difference from that of the healthy larvae at the early stage of virus multiplication, but it was significantly reduced at the late stage of virus multiplication. 6. There was no difference in the electrophoretic patterns of RNAs extracted from the midgut of healthy or virus-infected larvae. 7. The electrophoresis of proteins extracted from the midgut infected with FV or DNV, when carried out on the 1st and 5th day after virus inoculation, showed no difference from that of the healthy larvae. But, there was an additional band with medium motility in the proteins on the 8th day after virus inoculation, while a band with low mobility shown in the proteins of healthy larvae disappeared in the infected larvae. However, a band with high mobility in the healthy larvae was separated into two fractions in the infected larvae. 8. The electrophoretic pattern of hemolymph proteins of the silkworm larvae infected separately with FV and DNV was similar to that of the healthy larvae, but the concentration of hemolymph proteins in the infected larvae was lower than that of the healthy larvae at the late stage. 9. Two types of inclusion bodies were shown by the double staining of pyronin-methyl green in the columnar cell of the midgut on the 8th day after FV inoculation. 10. Electron microscopy of the infected midgut revealed that the 'cytoplasmic wall' of the goblet cell thickened on the 5th day after FV inoculation and several types of the cytopathogenic structures, such as virus$.$specific vesicles, virus particles, linear structures, tubular structures, and high electron-dense matrices were observed in the cytoplasm of the goblet cell. The virus particles were also observed in the microvilli and the structures similar to spherical virus particles were observed around the virus-specific vesicles, suggesting the virus assembly in the cytoplasm. 11. Fluorescence micrograph of the infected midgut stained with acridine orange showed that the nucleus, the site of DNV multiplication in the columnar cell, enlarged on the 5th day after virus inoculation. 12. Electron microscopic examination of DNV infected midgut revealed that the nucleolus of the columnar cell was broken into granules and those granules dispersed into apical region of the nucleus on the 5th day after virus inoculation. On the 8th day after inoculation, it was also observed that the nucleus of the columnar cell was full with the high electron-dense virogenic stroma which were similar to virus particles. These facts suggest that the virogenic stroma were the sites of virus assembly in the process of DNV multiplication.