• Proceedings of the Korean Society of Plant Pathology Conference
• /
• 2003.10a
• /
• pp.75.2-75
• /
• 2003

To develop an effective protection strategy against tomato bushy stunt virus (TBSV), tobacco plants expressing single-chain Fv antibodies (scFv), were established. A previous had shown that the replication activity of viral replicase was inhibited by the selected scFvs. Moreover, no systemic symptom was found after virus inoculation on leaves of wt N. benthamiana infiltrated with an Agrobacterium suspension resulting i3l expression of the scFvs. However, control plants showed systemic symptoms. In this study the localization of the scFvs within two transgenic plant lines, (CP28H3, CP-P55) was demonstrated using immunogold labelling. The gold particles, indicating the presence of scFv, were mostly found In the cytoplasm of the plant cells including chloroplasts and in the cell walls. However, they were hardly found in the vacuole, nucleoplasm and intercellular spaces. Gold particles often accumulated in either the cytosol or chloroplasts showing a specific labeling, There was no difference in type of gold labeling between both transgenic lines. The localization of the scFv in the cytoplasm further conforms the inhibition of the RNA-dependent RNA polymerase (RdRp) by the selected scFv because it is known that the RdRp is localized to membraneous cytosolic structures.

• Parasites, Hosts and Diseases
• /
• v.39 no.2
• /
• pp.119-132
• /
• 2001

An immunoelectron microscopy employing immunogold labeling method was performed to detect tissue origin of Dl fraction (DIA) among 5 antigenic protein fractions partially purified by DEAE- anion exchange chromatography from water- soluble crude antigen (PIWA) of adult Paragonimus iloktsuenensis. Immune reactions of adult worm tissues with rabbit serum immunoglobulin immunized with crude antigen (PI-Ig) and D1 antigen (D1-Ig), as well as rat serum immunoglobulin infected with P. iloktsuenensis were observed. DlA showed strong antigenicity in the intestinal epithelium of the worms during the early infection period of 2-4 weeks after infection. The vitellaria also showed stronger antigenicity than the other tissue sites in immune reaction of tissues against all immunoglobulins from 4 to 33 weeks after vitelline development. Therefore, it is suggested that DlA was mainly originated from the intestinal epithelial tissues before the development of vitelline gland of the parasites. Immune-reactivity of two immunoglobulins (PI-Ig, Dl-Ig) was significantly different in intestinal epithelial cytoplasmic protrusions (CP) and intestinal epithelial secretory granules (SG). In the experimental group with Dl-Ig, gold particles were labeled significantly in CP than in SG when compared to the PI-Ig group. Thus, the major antigenic materials in Dl antigen having a strong antigenicity in the early infection period was considered to be originated from the intestinal epithelial tissue .

• BMB Reports
• /
• v.38 no.1
• /
• pp.65-70
• /
• 2005

Choristoneura fumiferana granulovirus (ChfuGV) infection results two types of enveloped virions: Occlusion-derived virus (ODV) and budded virus (BV). Structural proteins ODVP-6E/ODV-E56 and p74 are two major conserved ODV-associated proteins that may be involved in the initiation of viral infection cycle in susceptible host insect larvae. This study presents the characterization of ChfuGV odvp-6e/odv-e56 and p74 transcription and translation as well as immunolocalization of these proteins in the occluded ChfuGV virion. Our results revealed that the transcription of odvp-6e/odv-e56 and p74 genes, both, start at 24 hours post infection (h p.i.). Using monospecific polyclonal antibodies made against ODVP-6E/ODV-E56 and p74 we demonstrated that these proteins are both expressed late in infection (24 h p.i.). Immunogold labeling using antisera against ODVP-6E/ODV-E56 and p74 proteins demonstrated that ODVP-6E/ODV-E56 and p74 proteins are both associated with the ODV envelop of ChfuGV.

• Journal of Microbiology and Biotechnology
• /
• v.15 no.6
• /
• pp.1229-1239
• /
• 2005

Human immunodeficiency virus-like particles (HIVVLPs) with native conformations similar to that of the wild-type virion could be valid candidates for vaccine development. To this end, we used a Semliki Forest Virus (SFV) expression system to produce HIV- VLPs containing high quantities of native envelope proteins. Here, we described a single SFV replicon containing the HIV gagpol and env genes under the control of separate subgenomic promoters. Mature VLPs incorporating the Gag and Env proteins were detected in the supernatant of replicon-expressing cells by Western blot analysis. The HIV-VLPs showed the expected molecular density (1.14-1.18 g/ml) on a $20-60\%$ sucrose gradient; the particles were 100-120 nm in diameter and Env proteins were observed on their surfaces by immunogold electron microscopy. RT-PCR analysis of VLP-associated RNAs in mature HIV-VLPs revealed two SF V-derived RNA species (full-length and subgenomic). Immunization studies in Balb/c mice showed that these HIV-VLPs were capable of inducing both HIV-specific antibodies and cell-mediated immune responses. Taken together, our results indicate that the SFV replicon system is useful for the production of HIV-VLPs, which may be valuable candidates for an HIV vaccine.

• Journal of Microbiology and Biotechnology
• /
• v.19 no.5
• /
• pp.495-501
• /
• 2009

A new spore display method is presented that enables recombinant proteins to be displayed on the surface of Bacillus spores via fusion with InhA, an exosporium component of Bacillus thuringiensis. The green fluorescent protein and $\beta$-galactosidase as model proteins were fused to the C-terminal region of InhA, respectively. The surface expression of the proteins on the spores was confirmed by flow cytometry, confocal laser scanning microscopy, measurement of the enzyme activity, and an immunogold electron microscopy analysis. InhA-mediated anchoring of foreign proteins in the exosporium of Bacillus spores can provide a new method of microbial display, thereby broadening the potential for novel applications of microbial display.

• The Korean Journal of Physiology and Pharmacology
• /
• v.9 no.3
• /
• pp.159-164
• /
• 2005

Effects of antioxidants on the established nephropathy were investigated. The experimental nephropathy was induced in rats by intravenous injection of adriamycin (2 mg/kg). Six weeks later, when proteinuria was apparent, the rats were supplemented with N-acetylcysteine (NAC, 1 g/kg/day) in drinking water for additional 6 weeks. Glomerulosclerosis score and tubulointerstitial injury index were determined by light microscopy. Expression of transforming growth factor (TGF) ${\beta}1$ and laminin ${\beta}1$ was determined in the renal cortex by reverse transcription-polymerase chain reaction, Western blotting, immunohistochemistry, and immunogold electron microscopy. The adriamycin-induced proteinuria as well as the glomerulosclerosis and tubulointerstitial injury was ameliorated by the treatment with NAC. Adriamycin increased the expression of TGF ${\beta}1$ mRNA and protein, which was ameliorated by NAC. Although the expression of laminin ${\beta}1$ mRNA was increased, adriamycin did not significantly alter that of its protein. These results indicate that antioxidants ameliorate the established nephropathy in association with normalization of overexpressed TGF ${\beta}1$.

• Applied Microscopy
• /
• v.24 no.4
• /
• pp.41-51
• /
• 1994

The topology of the enzyme has been investigated by biochemical studies including chemical labeling and cross linking. Thirteen subunits(polypeptides) of the cytochrome-c-oxidase have localistic characteristics of existing in the matrix side or cytoplasmic side in the mitochondria. In order to observe the distribution of the enzyme subunit on the mitochondria membrane, immunogold-labeling methods were employed. Antibody was obtained from the serum of immunized rabbit with enzyme subunit antigen which was obtained from cytochrome-c-oxidase of the beef heart muscle mitochondria. Beef heart muscle tissue as a tissue antigen was stained with immunized rabbit IgG and protein A gold complex. Electron microscopy has identified the existance of cytochrome-c-oxidase subunit $Mt_I,\;Mt_{II}\;and\;Mt_{III}$ on the membrane of cristae and outer chamber of mitochondria and the subunit $C_{IV}$ on the membrane of cristae and matrix of mitochondria. Particularly, the subunit $C_{IV}$ was also observed to exist in the sarcoplasm of muscle tissue.

• Korean Journal Plant Pathology
• /
• v.14 no.3
• /
• pp.229-239
• /
• 1998

Antibodies were raised against fusion proteins of the N-terminus and a region containing the GDNQ (Gly-Asp-Asn-Gln) polymerase motif of the L (polymerase) protein of sonchus yellow net virus (SYNV). Immunoblot analyses using these antibodies revealed the presence of the L protein in purified SYNV preparations and in nuclear extracts from infected tobacco. The serological analyses and detection in a polyacrylamide gels suggested that the L protein is present in at least a 20 fold lower abundance than the G, N, M1 and M2 proteins, and has size corresponding to a molecular weight of over 200 kDa as predicted from nucleotide sequence data. Electron microscopy with gold-labelled antibodies was used to localize the N, M2, and G proteins of SYNV in thin sections of infected tissue. When sections of SYNV-infected tissue were treated with antisera against total SYNV proteins and N protein, gold label could be detected in both the viroplasms and in virus particles. With the anti-M2 protein antiserum, the gold label was strongly localized in the viroplasms but only limited labelling of the virus particle sonly. Limited labelling of the L protein was observed in the viroplasms and the virus particles, presumably because of the low abundance of L protein in the tissues.

• Journal of Microbiology
• /
• v.39 no.4
• /
• pp.293-299
• /
• 2001

The nucleocapsid(NP) protein of Newcastle disease virus (NDV) and its derivative (NP$\sub$cfus)containing the myc region and six histidine residues fused to its C-terminus were pcpressed aboundantly in Escherichia coli. The proteins were purified by sucrose gradient centrifugation. Both the NP and NP$\sub$cfus/ proteins self-assem- bled into ring-like particles stacked together to from nucleocapsid-like structure which are heterogeneous in length with a diameter of 20${\pm}$2 nm and central holow of 5${\pm}$1 nm. Only a very small amount of the monomers in the particles was linked by inter-molecular disulfide bonds. Fusion of the C-terminal end to 29 amino acids inclusive of the myc epitope and His tag did not impair ring assembly buy inhibited the formation of the long herringbone structures. Immunogold lableing of the particles with the anti-myc antibody showed that the C-terminus of the NP$\sub$cfus/ protein is exposed on the surface of these ring-like particles.

• Animal cells and systems
• /
• v.5 no.3
• /
• pp.263-266
• /
• 2001

Metallothionein (MT) is induced in the regenerating rat liver. We have investigated expression of MT gene by RT PCR as well as specific localization of MT by immunocytochemistry in regenerating rat liver after partial hepatectomy (PH). MT mRNA level started to increase from 1 h and reached the peak at 8 h after PH. The level decreased gradually by 24 h, and became similar to that of control group. In the immunocytochemical study, in all groups treated with primary antibody, immunogold particles indicating the presence of MT were evenly distributed throughout both cytoplasm and nucleus of the rat hepatocytes. Within the nucleus, the gold particles appeared to be intensely localized in the areas of euchromatin and nucleolus. Within the cytoplasm, gold particles did not seem to adhere to mitochondria or Iysosomes, but were freely distributed. However, rough endoplasmic reticulum was the obvious compartment on which the gold particles were localized. Time course of MT immunoreactivity revealed that distribution of gold particles in hepatocytes increased gradually by 24 h, and decreased at 48 h after PH. Briefly, PH resulted in the sharpest increase in the expression of MT mRNA at 8 h and in the immunoreactivity of MT at 24 h, respectively. It is suggested that the increase of MT mRNA expression, the intensity of immunoreactivity and the specific localization of MT may be associated with the compensatory cell proliferation followed by PH.