• BMB Reports
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• 제34권6호
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• pp.502-508
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• 2001

A cDNA, encoding the human growth hormone (hGH), was synthesized based on the known 191 amino acid sequence. Its codon usage was optimized for a high level expression in Escherichia coli. Unique restriction sites were incorporated throughout the gene to facilitate mutagenesis in further studies. To minimize an initiation translation problem, a 624-bp cassette that contained a ribosome binding site and a start codon were fused to the hGH-coding sequence that was flanked between the EcoRI and HindIII sites. The whole fragment was synthesized by an overlapped extension of eight long synthetic oligonucleotides. The four-short duplexes of DNA, which were first formed by annealing and filling-in with a Klenow fragment, were assembled to form a complete hGH gene. The hGH was cloned and expressed successfully using a pET17b plasmid that contained the T7 promoter. Recombinant hGH yielded as much as 20% of the total cellular proteins. However, the majority of the protein was in the form of insoluble inclusion bodies. N-terminal amino acid sequencing also showed that the hGH produced in E. coli contained formyl-methionine. This study provides a useful model for synthesis of the gene of interest and production of recombinant proteins in E. coli.

• 대한의생명과학회지
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• 제8권4호
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• pp.257-268
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• 2002

Chronic infection and inflammation have recently been implicated as important etiologic agents of atherosclerosis. Several agents have been suggested as possible candidates including cytomegalovirus (CMV), herpes simplex vims type 1 (HSV-1), Epstein-Barr virus (EBV), Chlamydia pneumoniae, and Helicobacter pylori. We evaluated the relationship between cytornegalovirus infection and atherosclerosis by in situ hybridization and polymerase chain reaction (PCR). We examined 23 subjects with atherosclerosis and 10 matched control subjects without atherosclerosis. CMV was detected by in situ hybridization in 60.9% (14/23) of aorta and 42.9% (9/21) of coronary arteries in subjects with atherosclerosis. It was also detected by PCR in 65.2% (15/23) of aorta and 52.4% (11/21) of coronary arteries. CMV was detected on areas showing early or advanced atheromatous changes. Cells morphologically identical to smooth muscle cells, endothelial cells, lymphocytes, fibroblasts, and Schwann cells were positively reacted with the CMV probe. However. none of the cells to which the probe hybridized contained inclusion bodies, thus strongly suggesting that the arterial wall may be a site of CMV latency. This result Indicates that CMV may potentially play a direct or indirect role in the pathogenesis of human atherosclerosis.

• BMB Reports
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• 제39권6호
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• pp.717-721
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• 2006

In vitro analyses of type I signal peptidase activities require protein precursors as substrates. Usually, these pre-proteins are expressed in vitro and cleavage of the signal sequence is followed by SDS polyacrylamide gel electrophoresis coupled with autoradiography. Radioactive amino acids have to be incorporated in the expressed protein, since the amount of the in vitro expressed protein is usually very low and processing of the signal peptide cannot be followed by SDS polyacrylamide gel electrophoresis alone. Here we describe a rapid and simple method to express large amounts of a protein precursor in E. coli. We have analyzed the effect of ionophors as well as of azide on the accumulation of expressed protein precursors. Azide blocks the function of SecA and the ionophors dissipate the electrochemical gradient across the cytoplasmic membrane of E. coli. Addition of azide ions resulted in the formation of inclusion bodies, highly enriched with pre-apo-plastocyanine. Plastocyanine is a soluble copper protein, which can be found in the periplasmic space of cyanobacteria as well as in the thylakoid lumen of cyanobacteria and chloroplasts, and the pre-protein contains a cleavable signal sequence at its N-terminus. After purification of cyanobacterial pre-apo-plastocyanine, its signal sequence can be cleaved off by the E. coli signal peptidase, and protein processing was followed on Coomassie stained SDS polyacrylamide gels. We are optimistic that the presented method can be further developed and applied.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권5호
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• pp.414-419
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• 2006

When the alginate lyase gene (aly) from Pseudoalteromonas elyakovii was expressed in E. coli, most of the gene product was organized as aggregated insoluble particles known as inclusion bodies. To examine the effects of chaperones on soluble and nonaggregated form of alginate lyase in E. coli, we constructed plasm ids designed to permit the coexpression of aly and the DnaK/DnaJ/GrpE or GroEL/ES chaperones. The results indicate that coexpression of aly with the DnaK/DnaJ/GrpE chaperone together had a marked effect on the yield alginate lyase as a soluble and active form of the enzyme. It is speculated this result occurs through facilitation of the correct folding of the protein. The optimal concentration of L-arabinose required for the induction of the DnaK/DnaJ/GrpE chaperone was found to be 0.05mg/mL. An analysis of the protein bands on SDS-PAGE gel indicated that at least 37% of total alginate lyase was produced in the soluble fraction when the DnaK/DnaJ/GrpE chaperone was coexpressed.

• Journal of Microbiology and Biotechnology
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• 제17권4호
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• pp.632-637
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• 2007

A cytoplasmic polyhedrosis virus (CPV) was isolated from the larvae of Thaumetopoea pityocampa and shown to cause an infection of midgut cells. This viral infection revealed several important diagnostic symptoms, including discoloration of the posterior midgut, reduced feeding, and extended development time of the larvae. The virus infection is lethal to Thaumetopoea pityocampa, and with the increasing doses kills the larvae within 4-5 days post infection. Electron microscopy studies showed typical cytoplasmic polyhedral inclusion bodies that are icosahedral, and ranged from 2.4 to $5.3{\mu}m$ in diameter. Electrophoretic analysis of the RNA genome showed that the virus has a genome composed of 10 equimolar RNA segments with the sizes of 3,907, 3,716, 3,628, 3,249, 2,726, 1,914, 1,815, 1,256, 1,058, and 899 bp, respectively. Based on morphology and nucleic acid analysis, this virus was named Thaumetopoea pityocampa cytoplasmic polyhedrosis virus (TpCPV), and belongs to the genus Cypovirus, family Reoviridae.

• Journal of Microbiology and Biotechnology
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• 제9권3호
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• pp.303-310
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• 1999

A cost-effective mass production method for a strong antimicrobial peptide, buforin II, which was isolated from the stomach of Bufo bufo gargarizans, has been developed. This method is based on the neutralization of the positive charge of buforin II by fusion with a cysteine-rich acidic peptide (CAP) to avoid any lethal effect on the host. The neutralized fusion peptide was multimerized and expressed in Escherichia coli as tandem repeats to increase the production yield. Multimers of the CAP-buforin II fusion peptide were successfully expressed at high levels in E. coli as inclusion bodies. More than 100mg of pure buforin II was obtained per 11 of E. coli culture after cleaving the multimeric polypeptide with CNBr. The buforin II obtained from the recombinant E. coli had antimicrobial activity identical to that of natural buforin II. The proposed expression system can provide a cost-effective mass production method for both antimicrobial peptides and other host-lethal basic proteins.

• 한국식물병리학회지
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• 제14권6호
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• pp.625-629
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• 1998

Turnip mosaic potyviruses (TuMV) were isolated from Rorippa indica and Armoracia lapathifolia showing mosaic symptoms in field. Identification of the TuMVs were carried out by host reactions of indicator plants, electron micrograph, serological properties and reverse transcription-poly-merase chain reaction (RT-PCR). Both viruses systemically infected Chenopodium quinoa, Nicotiana clevelandii, Brassica rapa, B. campestris subsp. pekinensis, B. juncea and Raphanus sativus, and developed local infection on inoculated leaves of C. quinoa, C. amaranticola, C. album, N. tabacum cv. Xanthi nc and Gomphrena grobosa. However, the viruses did not infect on N. glutinosa, Cucumis sativus and Vigna unguiculata. The filamentous particles, about 720 nm in length, and inclusion bodies were observed from the infected leaf tissues by dipping on electron microscopy. Crude sap of leaf infected with the viruses was reacted positively with an antiserum of TuMV in agar gel double diffusion. For detection of the viruses, RT-PCR was carried out with TuMV--specfic oligonucleotide primer. The RT-PCR products, a 1,092 bp DNA fragment, were obtained from naturally infected leaves of R. indica and A. lapathifolia. In inoculation test to seven cruciferous weeds with TuMV, infection occurred in Arabis glabra, Barbarea orthoceras, Capsella bursa-pastoris, Draba nomorosa var. hebecarpa, Rorippa cantoniensis and Thlaspi arvense.

• 대한수의학회지
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• 제60권3호
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• pp.155-161
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• 2020

From 2006 to 2009, 50 pigs suspected of enteritis associated with porcine circovirus type 2 (PCV-2) (EAPC) were collected. Gross and histopathologic examinations and immunohistochemistry (IHC) were performed on the small intestine of 50 pigs. The pigs with EAPC were concentrated in grower pigs (68%), and diarrhea (84%) was the most common clinical sign. Grossly, the walls of the small intestine were thickened, and mesenteric lymph nodes were enlarged. The histopathologic features in the small intestine with EAPC were characterized by lymphoid depletion, histiocytic infiltration, and formation of basophilic grape-like inclusion bodies and multinucleated giant cells in gut-associated lymphatic tissue (GALT) and lamina propria. According to IHC, PCV-2 antigens were more intense and distributed widely in GALT (98%) than lamina propria (82%) of the small intestine. Co-infection with other enteric pathogens was detected in 26 pigs (52%). Twenty-four pigs (48%) were confirmed to be a PCV-2 single infection. Collectively, infected PCV-2 in the small intestine may induce an immunosuppressive status of individuals and then allow secondary co-infections in the digestive system. This study shows that PCV-2 can induce diarrhea and enteric lesions in pigs without a co-infection of other enteric pathogens.

• 자원환경지질
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• 제15권1호
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• pp.1-16
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• 1982

Many skarn type iron ore deposits are distributed in Kimhae-Mulgeum area of Gyeongnam Province. Integrated field, mineralogic, geochemical and fluid inclusion studies were undertaken to illustrate the character and origin of the ores in this area. The iron ore deposits in this area are NS or NNE trending fracture filling magnetite veins which are developed in andesitic rocks near the contact with late Cretaceous micrographic granite bodies. Symmetrically zoned skarns are commonly developed in the magnetite veins of this area. Zoning of skarn from center to margin of the vein are as follows; garnet quartz skarn-epidote skarn-epidote orthoclase skarn-altered andesitic rocks. Major ore mineral is magnetite and small amount of hematite, pyrite, pyrrhotite, chalcopyrite and sphalerite are associated. Vein paragenesis reveals four depositional stages; 1) skarn stage, 2) iron sulfide and oxide stage, 3) skarn stage, 4) sulfide stage Minute halite-bearing polyphase inclusions and liquid inclusions are contained in quartz. Filling temperatures range from $257^{\circ}$ to $370^{\circ}C$.

• 한국동물위생학회지
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• 제33권1호
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• pp.7-12
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• 2010

There are several immunosuppressive viral diseases in chickens such as avian adenovirus (AAV), chicken anemia virus (CAV), infectious bursal disease (IBD) and Marek's disease (MD). In this study, we have investigated two broiler chicken farms suffered from high mortality in Jeonbuk in July to August 2009. Clinically high fever and growth retardation were observed in the diseased chicken. In necropsy, the hemorrhages in thigh leg and thymus, hemorrhages and enlargement of liver, kidney and proventriculus, and yellowish fluid in heart were seen. Histologically, necrotic foci and basophilic intranuclear inclusion bodies of hepatocytes, hemorrhages and infiltrated lymphocytes in kidney and proventriculus were observed. By using polymerase chain reaction (PCR), the genes of avian adenovirus, CAV and ND virus were detected in specimens. We suggested that these coinfection cases with high mortality were due to primarily infection of immunosuppressive diseases such as avian adenovirus, CAV, followed by secondary infection of Newcastle disease (ND) virus.