• The Journal of Korean Society of Virology
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• v.26 no.1
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• pp.77-90
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• 1996

Nucleocapsid protein (NP)which exists in the particle of hantavirus and surrounds the viral RNA genome is one of the major structural proteins and plays role of antigen to elicit the antibody detected predorminantly right after infection of the virus in the patients of hemorragic fever with renal syndrome (HFRS)or experimental animals. NP is important target antigen in serological diagnostic system of HFRS utilizing whole antigens from the native virus particle, such as IFA, ELISA and Western blotting. Therefore, the preparation of this protein in the level of higher quantity and purity is desirasble for developed dianosis of the disease. The purpose of this study is the cloning of NP gene which exists in the S genome segment of Maaji (MAA) virus and expression of the gene to obtain qualified, genetically engineered NP to be utilized as an immunodiagnostic antigen. First of all, for the purpose of amplifing the MAA-NP gene by PCR, the specific primers were built from the known nucleotide sequence of Hantaan viral NP gene. The viral cDNA of the NP gene was synthesized by using the primers and RNase $H^-$ AMV reverse transcriptase. Thereafter, using this cDNA as a template, the NP gene was amplified specifically by Taq DNA polymrerase. The pT7blue (R)T-overhang vector systems were used for cloning of the amplified NP gene. The expression system was consisted of BL21 (DE3)pLysS and pET16b as a host and a plasmid repectively. Into Ndel site of pET16b, NP gene was ligated with cohesive end for the expression. Insertion of NP gene in the plasmid was confirmed by PCR and mini prep methods. For expression, IPTG was used and the expressed protein was characterized by Western blotting. The MAA-NP was expressed as the form of inclusion body (insoluble fraction)and the protein purified by affinity and metal chealating columns reacted specifically with the sera from patients of HFRS as to be tested by ELISA and Western blotting.

• Korean Journal of Ecology and Environment
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• v.36 no.3 s.104
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• pp.369-374
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• 2003

Nonylphenol (NP), an well known aquatic contaminant, has been known to induce abnormalities in various aquatic animals. In an effort to develop proteome in the study of aquatic contamination of NP and its impact on the amphibia, protein changes in liver tissues of Korean red bellied frog, Bombina orientalis was investigated following the NP exposure. NP was administered intraperitoneally to male B. orientalis at 10 mg/kg body weight. At 48 and 96h after the treatment, the frog livers were sampled, and the protein fraction was separated using two dimensional gel electrophoresis (2D/E) and visualized with Coomassie brilluant blue staining. The 2D/E Images of the tissue from the animals treated with NP showed marked changes of protein spots (about 20% of total protein spots). Analysis of the 50-60 separated spots allowed identification of the major protein changes in the overall pattern for the stressor (NP) by time (0,48 and 96 h). At 48h after treatment, 8 spots were increased and 12 spots were reduced. Then, at 96h after treatment, 10 spots were increased and 8 spots were reduced. In total, approximately 29% of liver proteins showed the altered expression following the NP treatment. It is suggested that protein expression was repressed by blocking of certain metabolisms at 48 hand induced by the synthesis of new proteins for adaptation at 96 h following NP exposure. This application for 2D/E analysis may show promise in searching biomarkers for environmental proteomics in amphibians.

• The Korean Journal of Physiology
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• v.6 no.2
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• pp.57-63
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• 1972

In an attempt to better understand the radioprotective effect of reduced glutathione(GSH), and to observe a possible radioprotective effect of Ginseng extract, whole body X-irradiation of 1,200 r was administered to the mouse either independently or immediately following the injection of GSH or Ginseng extract to the mouse intraperitoneally. The non-protein sulfhydryl (NP-SH) and non-protein disulfide (NP-SS) levels of the liver, and NP-SH level of NP-SH of the blood of the mouse were measured at 30, 60 and 120 minutes, and results were compared with the normal. The results thus obtained are summarized as follows; 1) The normal values of NP-SH and NP-SS of the mouse liver were $5.90{\pm}0.46\;{\mu}\;mol/gm\;wet\;wt.,\;and\;3.02{\pm}0.42\;{\mu}\;mol/ml$ wet wt., respectively, and the normal value of NP-SH of NP-SH of the mouse blood was $3.98{\pm}1.29\;{\mu}\;mol/ml$ 2) The injection of both GSH and Ginseng extract produced the highest values of NP-SH in the liver at 30 minutes, but a gradual decrease to the normal was observed thereafter. When X-irradiation alone was applied, the liver NP-SH value was lower than the normal at 60 minutes post-irradiation and thereafter. When Ginseng extract was injected immediately prior to X-irradiation, the liver NP-SH was lower than the normal throughout the experiment with the lowest value at 60 minutes. However, the combination of GSH and X-irradiation produced higher than the normal values throughout the entire experiment. 3) The liver NP-SS value was most significantly elevated at 30 minutes after the injection of GSH, hut the recovery to the normal was observed thereafter. The injection of Ginseng extract produced slightly higher liver NP-SS values at 30 and 60 minutes, but the value at 120 minutes was similar to the normal. The single application of X-irradiation resulted in the lower then normal liver NP-SS values throughout the entire experiment. When GSH was injected price to X-irradiation, the liver NP-SS values were higher than the normal at 30 and 60 minutes followed b the recovery to the normal at 120 minutes. The combination of Ginseng extract and X-irradiation showed generally lower liver NP-SS values throughout the experiment. 4) The blood NP-SH showed the higher than the normal values in all the experimental groups except when GSH was injected prior to X-irradiation alone produced e significantly elevated blood NP-SS value at 30 minutes post-irradiation.

• Journal of fish pathology
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• v.8 no.1
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• pp.31-36
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• 1995

Hc nuclear polyhedrosis virus DNA genome was digested with EcoRI endonuclease, these partial fragments were recombined into the pUC8 plasmid vector and transformed in E. coli JM 83 cell. The genome DNA has 24 EcoRI fragments and 12 fragments of them were subcloned. The four recombinants were named as eNP-O, eNP-Q, eNP-R and eNP-S. The expression of foregin gene of the recombinants was investigated by analysing protein patterns on the SDS-PAGE. The eNP-O, eNP-Q and eNP-R were expressed a different weight of protein as comparision with potypeptide bands of E. coli JM 83 host cell.

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

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.8
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• pp.1121-1127
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• 2002

Sixteen crossbred (Bos indicus${\times}$Bos taurus) calves were randomly distributed in two groups (NP and LP) of eight calves each to study the effect of restricted (75%) protein supply on growth and immuno-biochemical response as an indicator of production and health of under-nourished animals during 3 to 9 months of age. The normal requirement of protein was provided to group NP and a less of 25% to group LP through calculated amount of concentrate and roughage in their daily ration. Assessment was made for weekly change in live weight, periodic alteration in blood metabolites and immunological status at six months of age in calves. An initial (during 3 to 6 months of age) depression (p<0.05) in growth was seen in low protein fed group (LP) compared to NP, which became non-significant in the later period of life (6 to 9 months of age). There was no significant effect on haemoglobin, total protein, albumin and globulin concentration except that of urea, which was decreased significantly (p<0.05) in animals fed on low protein diet ($19.83{\pm}1.25$ vs $25.93{\pm}1.29mg/dl$). The treatment effect that was seen in different periods of life was not uniform for other parameters except for urea, which showed a regular depression in LP compared to NP. The assessment of immunological status by indirect haemagglutination (IHA) test against Pasteurella multocida (P52 strain) was considerably (p<0.05) reduced in animals on LP ration compared to those on NP. It is thus argued that with poor nutrition (low protein) and state of compromised immunological response the production and health of the animals will be adversely affected.

• The Korean Journal of Physiology
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• v.5 no.2
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• pp.55-62
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• 1971

In an attempt to better understand the effects of whole body X-irradiation on the levels of non-protein sulfhydryl (NP-SH), non-protein disulfide (NP-SS) and oxygen consumption rate $(QO_2)$ of the mouse duodenum, and to clarify the possible radioprotective action of reduced glutathione (GSH), a whole body X-irradiation of 1,000r was given to albino mouse either singularly or immediately after injecting GSH intraperitoneally to mouse 1 mg per gm of body weight. NP-SH was measured by Ellman's method, NP-SS was measured by the electrolytic reduction method described by Dohan and Woodward, and $(QO_2)$ by the Warburg's standard manometric method. The experiment was performed at 1, 6, 12 and 24 hours post-irradiation, and the comparison was made with the control. The results thus obtained are summarized as follows: 1) Comparing with the intrinsic NP-SH level of $3.31{\pm}0.27{\mu}\;mol/gm$ wet weight in the duodenum of the normal mouse, either whale body X-irradiation or injection of GSH alone produced no significant change in NP-SH from the normal. However, when GSH was injected prior to X-irradiation, markedly elevated NP-SH levels were observed throughout the entire experiment with the highest value of $4.70{\pm}0.10$ at 6 experimental hours. 2) The normal value of NP-SS in the mouse duodenum was $1.57{\pm}0.17{\mu}\;mol/gm$ wet weight, while in the group where injection of GSH and X-irradiation were combined, NP-SS increased to $2.36{\pm}0.33$ at 12 hours and $2.15{\pm}0.53$ at 24 hours, showing the intermediate value between the GSH injection group and X·irradiation group. 3) The normal value of $(QO_2)$ was $4.16{\pm}0.73{\mu}l\;O_2/hr./gm$ D.W., and no noticeable change was observed comparing with the GSH injection group. However, in the group where X·irradiation alone was given, $(QO_2)$ of the duodenum increased significantly throughout the entire experiment with the highest value of $6.35{\pm}1.07$ at 6 experimental hours. When GSH was injected before X-irradiation was given, the levels of $(QO_2)$ were in the middle of the GSH injection group and X-irradiation group. 4) The above results suggest that GSH may be effective as a radioprotector in terms of NP-SH, NP-SS and $(QO_2)$ of the mouse duodenum.

• Journal of Microbiology
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• v.43 no.6
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• pp.537-545
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• 2005

Even though neutralizing antibodies against the Hantaan virus (HTNV) has been proven to be critical against viral infections, the cellular immune responses to HTNV are also assumed to be important for viral clearance. In this report, we have examined the cellular and humoral immune responses against the HTNV nucleocapsid protein (NP) elicited by virus infection or DNA vaccination. To examine the cellular immune response against HTNV NP, we used $H-2K^b$ restricted T-cell epitopes of NP. The NP-specific $CD8^+$ T cell response was analyzed using a $^{51}Cr-release$ assay, intracellular cytokine staining assay, enzyme-linked immunospot assay and tetramer binding assay in C57BL/6 mice infected with HTNV. Using these methods, we found that HTNV infection elicited a strong NP-specific $CD8^+$ T cell response at eight days after infection. We also found that several different methods to check the NP-specific $CD8^+$ T cell response showed a very high correlation among analysis. In the case of DNA vaccination by plasmid encoding nucleocapsid gene, the NP-specific antibody response was elicited $2\~4$ weeks after immunization and maximized at $6\~8$ weeks. NP-specific $CD8^+$ T cell response reached its peak 3 weeks after immunization. In a challenge test with the recombinant vaccinia virus expressing NP (rVV-HTNV-N), the rVV-HTNV-N titers in DNA vaccinated mice were decreased about 100-fold compared to the negative control mice.

• Rapid Communication in Photoscience
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• v.4 no.2
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• pp.37-40
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• 2015

To examine the photosensitized biomolecules damaging activity, dimethoxyP(V)tetrakis(2-naphthyl)porphyrin (NP) and dimethoxyP(V)tetraphenylporphyrin (PP) were synthesized. The naphthyl moiety of NP hardly deactivated the photoexcited P(V)porphyrin ring in ethanol. In aqueous solution, the naphthyl moiety showed the quenching effect on the photoexcited porphyrin ring, possibly through electron transfer and self-quenching by a molecular association. Binding interaction between human serum albumin (HSA), a water soluble protein, and these porphyrins could be confirmed by the absorption spectral change. The apparent association constant of NP was larger than that of PP. It is explained by that more hydrophobic NP can easily bind into the hydrophobic pockets of HSA. The photoexcited PP effectively induced damage of the tryptophan residue of HSA, through electron transfer-mediated oxidation and singlet oxygen generation. NP also induced HSA damage during photo-irradiation and the contributions of the electron transfer and singlet oxygen mechanisms were speculated. The electron transfer-mediated mechanism to the photosensitized protein damage should be advantageous for photodynamic therapy in hypoxic condition. The quantum yield of the HSA photodamage by PP was significantly larger than that of NP. The quenching effect of the naphthyl moiety is considered to suppress the photosensitized protein damage. In conclusion, the naphthalene substitution to the P(V)porphyrins can enhance the binding interaction with hydrophobic biomacromolecules such as protein, however, this substitution may reduce the photodynamic effect of P(V)porphyrin ring in aqueous media.

• Development and Reproduction
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• v.10 no.3
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• pp.177-184
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• 2006

Retinol-binding protein(RBP) plays an important role in the specific transport of retinol to target cells through the blood stream in higher vertebrates. In order to clarify the effects of 4-nonylphenol(4-NP) on RBP mRNA expression in the rockfish, Sebastes schlegeli which is common in coastal waters of Korea and commercially important species, the cDNA library was constructed from the liver, and a partial fragment of the RBP gene was cloned. The deduced amino acid sequence from the RBP mRNA showed a high homology to the amino acid sequence from Sparus aurata(80%), Oncorhynchus mykiss(72%) or Anguilla anguilla(78%). Effects of 4-NP on RBP and vitellogenin(VTG) mRNA expression level in rockfish were examined by the northern blot analysis. In female and male rockfish injected with 4-NP(10 mg/kg BW, lower dose), there was no changes in the levels of VTG mRNA expression in the liver. The RBP mRNA levels, however, decreased at 48 hours after the injection in male. In the rockfish injected with 4-NP(25 mg/kg BW, higher dose), the level of VTG mRNA expression increased after 24 hours, regardless of sex. The level of RBP mRNA expression decreased at 48 hours after the injection in both sexes. These data indicate that estrogenic mimics such as 4-NP exhibit a contrasting effect on RBP and VTG gene expression in rockfish.