• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.138.2-139
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• 2003

Grapevine leafroll-associated 1 virus (GLRaV-1) and Grapevine leafroll-associated 3 virus (GLRaV-3), member of the genus Ampelovirus, are important viral disease of grapevine in the world. these viruses transmitted only dicotyledonous host by vectors such as mealybugs and there is no suitable herbaceous host for virus. The diseased leaves turn yellowish or reddish depending on cultivars and viruses. Viruses are existed at low concentration and ununiformly distribution in grapevine. Using small-scale double-stranded RNA (dsRNA) extraction method, reverse transcription and polymerase chain reaction (RT-PCR) product of 1Kb long which encoded of coat protein (CP) gene for both viruses was successfully amplified with a specific primers. The RT-PCR product was cloned into the plasmid vector and its nucleotide sequences were determined from selected recombinant cDNA clones. Sequence analysis revealed that the CP of GLRaV-1 consisted of 969 nucleotide, which encoded 323 amino acid residues and CP of GLRaV-3 consisted of 942 nucleotide, which encoded 314 amino acid residues. The CP of GLRaV-1 and GLRaV-3 has 93.8％ and 98.7％ amino acid sequence identities, respectively.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.191-193
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• 2000

A whole-cell biocatalyst was constructed by immobilizing an enzyme on the surface of the yeast Saccharomyces cerevisiae. The gene encoding Bacillus macerans cyclodextrin glucanotransferase(CGTase) was fused with the AGA2 gene encoding a small peptide disulfide-linked to the aga1, a cell wall protein of a-agglutinin. The plasmid was introduced S. cerevisiae and expressed in the medium consisting of 10g/L yeast extract, 20g/L peptone, and 20g/L galactose. The activity was detected with the formation of cyclodextrin(CD) from 10g/L soluble starch. Surface display of CGTase was also verified with the halo-test, flow cytometry, and immunofluorescence microscopy. The recombinant S. cerevisiae produced ${\alpha}-cyclodextrin$ more efficiently than the free CGTase by simultaneous fermentation and cyclization as yeast consumes glucose and maltose which are inhibitors for CD synthesis.

• Biomolecules & Therapeutics
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• v.18 no.1
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• pp.1-15
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• 2010

Progress in the development of DNA vaccines and their delivery strategies has been made since their initial concept as a next generation vaccine. Since DNA vaccine includes non-infectious DNA parts of pathogens, it can't cause disease yet it closely mimic the natural process of infection and immune responses. Despite their early promising results of controlling infectious diseases and cancer in small animal models, DNA vaccines failed to display a level of immunogenicity required for combating these diseases in humans, possibly due to their lower protein expression levels. However, increasing evidence has shown that DNA vaccines are clinically well-tolerated and safe. Furthermore, one notable advantage of DNA vaccines includes convenient utilities of plasmid DNAs coding for antigens. For instance, any emerging pathogens could be prevented easily and timely by allowing the simple exchange of antigen-encoding genes. In this review, newly developed DNA vaccine strategies, including electroporation, which has emerged as a potent method for DNA delivery, targeting infectious diseases and cancer will be discussed with a focus on any on-going DNA vaccine trials or progress made pre-clinically and in clinics.

• Archives of Pharmacal Research
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• v.27 no.7
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• pp.797-805
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• 2004

LPD vectors are non-viral vehicles for gene delivery comprised of polycation-condensed plasmid DNA and liposomes. Here, we described a novel anionic LPD formulation containing protamine-DNA complexes and pH sensitive liposomes composed of DOPE and cholesteryl hemisuccinate (Chems). Central composite design (CCD) was employed to optimize stable LPD formulation with small particle size. A three factor, five-level CCD design was used for the optimization procedure, with the weight ratio of protamine/DNA ($X_1$), the weight ratio of Chems/DNA ($X_2$) and the molar ratio of Chems/DOPE in the anionic liposomes ($X_3$) as the independent variables. LPD size ($Y_1$) and LPD protection efficiency against nuclease ($Y_2$) were response variables. Zeta potential determination was utilized to define the experimental design region. Based on experimental design, responses for the 15 formulations were obtained. Mathematical equations and response surface plots were used to relate the dependent and independent variables. The mathematical model predicted optimized $X_1-X_3$ levels that achieve the desired particle size and the protection efficiency against nuclease. According to these levels, an optimized LPD formulation was prepared, resulting in a particle size of 185.3 nm and protection efficiency of 80.22％.

• Korean Journal of Microbiology
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• v.31 no.3
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• pp.179-183
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• 1993

A restricted facultatively methanol-oxidizing bacterium, Methylovorus sp. strain SS1, was isolate dfrom soil samples from Kuala Lumpur, Malaysia, through methanol-enrichment culture technique. The isolate was nonmotile Gram-negative rod and did not have complex internal membrane system. The colonies were small, pale-yellow, and raised convex with entire margin. The cell did not produce any spores and capsular materials. The cell was obligately aerobic and exhibited catalase, but no oxidase, activity. Plasmid, carotenoid pigment, and poly-.betha.-hydroxybutyric acid were not found. The guanine plus cytosine content of the DNA was 55%. The isolate was found to grow only on methanol methylamine, or glucose. Growth factors were not required. Cells growing on methanol was found to produce extracellular polysaccharides containing glucose, lactose, and fructose. Growth was optimal (t$_{d}$= 1.7) with 0.5%(v/v) methanol at 40.deg.C and pH 6.5. No Growth was observed at over 60.deg.C. Cell-free extracts of the methanol grown cells exhibited the phenazine methosulfate-linked methanol dehydrogenase activity Methanol was found to be assimilate dthrough the ribulose monophosphate pathway.y.

• Journal of Animal Science and Technology
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• v.66 no.1
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• pp.232-236
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• 2024

Ligilactobacillus is a genus of Gram-positive lactobacilli commonly found in the intestinal tracts of vertebrates. It has been granted a Qualified Presumption of Safety (QPS) status from the European Food Safety Authority (EFSA). One specific strain, Ligilactobacillus salivarius B4311, was isolated from fecal samples of broiler chickens from a farm associated with Chung-Ang University (Anseong, Korea). This strain was observed to have inhibitory effects against Listeria monocytogenes. In this paper, we present the complete genome sequence of Lig. salivarius B4311. The whole genome of strain B4311 comprises 2,071,255 bp assembled into 3 contigs representing a chromosome, repA-type megaplasmid, and small plasmid. The genome contains 1,963 protein-coding sequences, 22 rRNA genes, and 78 tRNA genes, with a guanine + cytosine (GC) content of 33.1%. The megaplasmid of strain B4311 was found to contain the bacteriocin gene cluster for salivaricin P, a two-peptide bacteriocin belonging to class IIb.

• Korean Chemical Engineering Research
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• v.43 no.2
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• pp.313-317
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• 2005

Synthetic gene carriers such as poly-cationic polymers easily form complexes with plasmid DNA which contains negative charge. Chitosan is a polysaccharide that demonstrates much potential as a gene delivery system. The ability of depolymerized chitosan to condense DNA was determined using electrophoresis. Dynamic laser scattering and scanning electron microscopy were used to examine the size and the morphology of the chitosan/DNA complex. Parameters such as chitosan molecular weight and charge density influenced the complex size and the DNA amount condensed with chitosan. The cell viabilities in the presence of chitosan ranged between 84-108% of the control in all experiments. Gene expression efficacy using chitosan/DNA complex was enhanced in 293 cells relative to that using naked DNA, although it was lower than that using lipofecamine. Transfection efficacy using low molecular weight chitosan (Mw=8,517) was higher than those of the control and the other chitosan (MW=4,078). The low molecular weight chitosan (MW=8,517) with a high charge density (18.32 mV) fulfilled the requirements for a suitable model gene delivery system with respect to the condensing ability of DNA, complex formation, and transfection efficacy.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.11
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• pp.1529-1540
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• 2011

Silencing of a specific gene using RNAi (RNA interference) is a valuable tool for functional analysis of a target gene. However, information on RNAi for analysis of gene function in farm animals is relatively nil. In the present study, we have validated the interfering effects of siRNA (small interfering RNA) using both quantitative and qualitative gene silencing in buffalo granulosa cells. Qualitative gene knockdown was validated using a fluorescent vector, enhanced green fluorescence protein (EGFP) and fluorescently labeled siRNA (Cy3) duplex. While quantitatively, siRNA targeted against the luciferase and CYP19 mRNA was used to validate the technique. CYP19 gene, a candidate fertility gene, was selected as a model to demonstrate the technique optimization. However, to sustain the expression of CYP19 gene in culture conditions using serum is difficult because granulosa cells have the tendency to luteinize in presence of serum. Therefore, serum free culture conditions were optimized for transfection and were found to be more suitable for the maintenance of CYP19 gene transcripts in comparison to culture conditions with serum. Decline in fluorescence intensity of green fluorescent protein (EGFP) was observed following co-transfection with plasmid generating siRNA targeted against EGFP gene. Quantitative decrease in luminescence was seen when co-transfected with siRNA against the luciferase gene. A significant suppressive effect on the mRNA levels of CYP19 gene at 100 nM siRNA concentration was observed. Also, measurement of estradiol levels using ELISA (enzyme-linked immunosorbent assay) showed a significant decline in comparison to control. In conclusion, the present study validated gene silencing using RNAi in cultured buffalo granulosa cells which can be used as an effective tool for functional analysis of target genes.

• BMB Reports
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• v.38 no.2
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• pp.225-231
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• 2005

Acid-labile subunit (ALS) is a component of the 150-kDa insulin-like growth factor-binding protein-3 (IGFBP-3) complex, which, by sequestering the majority of IGFs-I and -II and thereby prolonging the half-life of them in plasma, serves as a circulating reservoir of IGFs in mammalian species. A pGEX-2T plasmid and a baculovirus expression constructs harboring a coding sequence for glutathione-S transferase (GST)-porcine ALS (pALS) fusion protein were expressed in BL21(DE3) E. coli and Sf9 insect cells, respectively. The expressed protein was purified by glutathione or Ni-NTN affinity chromatography, followed by cleavage of the fusion protein using Factor Xa. In addition, pALS and hIGFBP-3 were also produced in small amounts in the Xenopus oocyte expression system which does not require any purification procedure. A 65-kDa pALS polypeptide was obtained following the prokaryotic expression and the enzymatic digestion, but biochemical characterization of this polypeptide was precluded because of an extremely low expression efficiency. The baculovirus-as well as Xenopus-expressed pALS exhibited the expected molecular mass of 85 kDa which was reduced into 75 and 65 kDa following deglycosylation of Asn-linked carbohydrates by Endo-F glycosidase, indicating that the expressed pALS was properly glycosylated. Moreover, irrespective of the source of pALS, the recombinant pALS and hIGFBP-3 formed a 130-kDa binary complex which could be immunoprecipitated by anti-hIGFBP-3 antibodies. Collectively, results indicate that an authentic pALS protein can be produced by the current expression systems.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.6
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• pp.2813-2818
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• 2012

Background: Considerable evidence suggests that metadherin (MTDH) is a potentially crucial mediator of tumor malignancy and an important therapeutic target for simultaneously enhancing chemotherapy efficacy and reducing metastasis risk. Inhibition of MTDH expression by RNA interference has been shown in several previous research, but silencing MTDH expression by microRNA (miRNA) interference in breast cancer has not been established. In the present study, we investigated the role of MTDH-miRNA in down-regulation of proliferation, motility and migration of breast carcinoma cells. Methods: Expression vectors of recombinant plasmids expressing artificial MTDH miRNA were constructed and transfected to knockdown MTDH expression in MDA-MB-231 breast cancer cells. Expression of MTDH mRNA and protein was detected by RT-PCR and Western blot, respectively. MTT assays were conducted to determine proliferation, and wound healing assays and transwell migration experiments for cell motility and migration. Results: Transfection of recombinant a plasmid of pcDNA-MTDH-miR-4 significantly suppressed the MTDH mRNA and protein levels more than 69% in MDA-MB-231 breast cancer cells. This knockdown significantly inhibited proliferation, motility and migration as compared with controls. Conclusions: MTDH-miRNA may play an important role in down-regulating proliferation, motility and migration in breast cancer cells, and should be considered as a potential small molecule inhibitor therapeutic targeting strategy for the future.