• Journal of Microbiology and Biotechnology
• /
• v.25 no.11
• /
• pp.1960-1965
• /
• 2015

Rabbit hemorrhagic disease virus (RHDV) is highly contagious and often causes fatal disease that affects both wild and domestic rabbits of the species Oryctolagus cuniculus. A highly pathogenic RHDV variant (RHDVa) has been circulation in the Korean rabbit population since 2007 and has a devastating effect on the rabbit industry in Korea. A highly pathogenic RHDVa was isolated from naturally infected rabbits, and the gene encoding the VP60 protein was cloned into a baculovirus transfer vector and expressed in insect cells. The hemagglutination titer of the Sf-9 cell lysate infected with recombinant VP60 baculovirus was 131,072 units/50 μl and of the supernatant 4,096 units/50 μl. Guinea pigs immunized twice intramuscularly with a trial inactivated RHDVa vaccine containing recombinant VP60 contained 2,152 hemagglutination inhibition (HI) geometric mean titers. The 8-week-old white rabbits inoculated with one vaccine dose were challenged with a lethal RHDVa 21 days later and showed 100% survival rates. The recombinant VP60 protein expressed in a baculovirus system induced high HI titers in guinea pigs and rendered complete protection, which led to the development of a novel inactivated RHDVa vaccine.

• Journal of Microbiology and Biotechnology
• /
• v.9 no.2
• /
• pp.206-212
• /
• 1999

The gene orf7(oxi III) was expressed using an E. coli system in anticipation that it would encode dTDP-glucose 4,6-dehydratase which is involved in the biosynthesis of the olivose moiety of chlorothricin produced from Streptomyces antibioticus Tu99. The solubility of the expressed protein increased up to 20% under optimal induction conditions. The expressed protein was purified from the E. coli BL 21(DE3) cell lysate by a 28.5-fold purification in two chromatography steps with a 38% recovery to near homogeneity. The molecular weight and N-terminal amino acid sequence of the purified protein correlated with the predicted mass and sequence deduced from the orf7 gene. The purified protein was a homodimer with a subunit relative molecular weight of 38,000 Dalton. The expressed protein was found to exhibit dTDP-glucose 4,6-dehydratase activity and be highly specific for dTDP-glucose as a substrate. The values of K'm and V'max for dTDP-glucose were 28 $\mu$M and 295 nmol $min^{-1} (mg protein)^{-1}$, respectively. dTTP and dTDP were strong inhibitors of this enzyme.$NAD^+$, the coenzyme for dTDP-glucose 4,6-dehydratase, was tightly bound to the expressed protein.

• Journal of Microbiology and Biotechnology
• /
• v.12 no.1
• /
• pp.65-70
• /
• 2002

A TGF-${\beta}1$/GFP monomeric fusion protein was cloned from pPK9A and pGFP-Cl plasmid by PCR amplification. The fusion protein was expressed in a $Bac-To-Bac^{TM}$ baculovirus expression system. A 45 kDa fusion protein was purified using an Ni-NTA column with 300 mM imidazol from a cell lysate infected with recombinant viruses for 72 h post-infection. The fusion protein cross-reacted with the commercial $TGF-{\beta}1$ polyclonal Ab as well as Ab raised against a precursor, monomeric $TGF-{\beta}1$, and GFP. The binding activity of the fusion protein with a $TGF-{\beta}1$ receptor was examined. Fluorescence was observed in Mv1Lu cells, yet not in insect cells treated with the fusion protein. No fluorescence was detected in Mv1Lu cells incubated with the fusion protein treated with Ab prior to the binding reaction, or with GFP alone, thereby indicating that the binding of the fusion protein was specific to $TGF-{\beta}1$ with a receptor.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.3 no.2
• /
• pp.82-86
• /
• 1998

The maltose binding protein (MBP) fusion protein system is a versatile tool to express and isolate recombinant proteins in E. coli. In this system, MBP fusion proteins are efficiently isolated from whole cell lysate using amylose conjugated agarose beads and then eluted by competition with free maltose. Since MBP is a rather large molecule (∼42 kDa), for further experiments, the MBP part is usually proteolytically cleaved from the fusion protein and subsequently removed by ion-exchange chromatography or rebinding to amylose columns after washing out excess and MBP-bound maltose. In the present study, we have developed an improved method for the removal of cleaved MBP, which is advantageous over conventional methods. In this method, factor Xa cleaved MBP fusion proteins were incubated with Sepharose beads conjugated with MBP specific monoclonal antibodies and then precipitated buy centrifugation, resulting in highly purified proteins in the supernatant.

• BMB Reports
• /
• v.40 no.3
• /
• pp.302-314
• /
• 2007

N type calcium channels (CaV2.2) play a key role in the gating of transmitter release at presynaptic nerve terminals. These channels are generally regarded as parts of a multimolecular complex that can modulate their open probability and ensure their location near the vesicle docking and fusion sites. However, the proteins that comprise this component remain poorly characterized. We have carried out the first open screen of presynaptic CaV2.2 complex members by an antibody-mediated capture of the channel from purified rat brain synaptosome lysate followed by mass spectroscopy. 589 unique peptides resulted in a high confidence match of 104 total proteins and 40 synaptosome proteome proteins. This screen identified several known CaV2.2 interacting proteins including syntaxin 1, VAMP, protein phosphatase 2A, $G_{o\alpha}$, G$\beta$ and spectrin and also a number of novel proteins, including clathrin, adaptin, dynamin, dynein, NSF and actin. The unexpected proteins were classified within a number of functional classes that include exocytosis, endocytosis, cytoplasmic matrix, modulators, chaperones, and cell-signaling molecules and this list was contrasted to previous reports that catalogue the synaptosome proteome. The failure to detect any postsynaptic density proteins suggests that the channel itself does not exhibit stable trans-synaptic attachments. Our results suggest that the channel is anchored to a cytoplasmic matrix related to the previously described particle web.

• Toxicological Research
• /
• v.18 no.2
• /
• pp.183-190
• /
• 2002

The mechanism by which catechin-mediated cytotoxicity against tumor cells remains to be elusive. To elucidate the mechanical mights of anti-tumor effects, (-)epigallocatechin-gallate (EGCG) of catechin was applied to human prostate cancer DU 145 cells. Cell viability was measured by crystal violet staining. Cell lysates were wed to measure the catalytic activity of caspases by using fluorogenic peptide: Ac-DEVD-AMC for caspase-3 protease, Z-IETD-AFC for caspase-8 protease, Ac-LEHD-AFC for caspase-9 protease as substrates. The equal amounts of protein from cell lysate was separated on SDS-PAGE and analyzed by western blotting with anti-Fas antibody, anti-FasL antibody, anti-BCL2 antibody and anti-Bax antibody. (-)EGCG induced the death of DUl45 cells, which was revealed as apoptosis shown by DNA fragmentation. (-)EGCG induced the activation of caspase family cysteine proteases including caspase-3, -8 and -9 proteases in DU145 cells. Also, (-)EGCG increased the expression of Fas and Fas ligand (FasL) protein in DU145 colls. The expression level of BCL2 was decreased in (-)EGCG treated DU145 cells, whereas Bax protein was increased in a time-dependent manner. We suggest that (-)EGCG-induced apoptosis of DU145 cells is mediated by signaling pathway involving caspase family cysteine protease, mitochondrial BCL2-family protein and Fas/FasL.

• Journal of Preventive Medicine and Public Health
• /
• v.47 no.2
• /
• pp.124-128
• /
• 2014

Objectives: To evaluate the monthly variation in the airborne $(1{\rightarrow}3)-{\beta}$-D-glucan level throughout one year and its relationship with climatic factors (temperature, relative humidity, wind speed, hours of daylight, cloud cover, and pollen counts). Methods: A total of 106 samples were collected using a two-stage cyclone sampler at five outdoor sampling locations (on top of 5 university buildings). The kinetic limulus amebocyte lysate assay was used to obtain $(1{\rightarrow}3)-{\beta}$-D-glucan levels. Results: Airborne $(1{\rightarrow}3)-{\beta}$-D-glucan levels were significantly higher in the spring, particularly in April, and temperature was significantly related to $(1{\rightarrow}3)-{\beta}$-D-glucan levels (r=0.339, p<0.05). Conclusions: $(1{\rightarrow}3)-{\beta}$-D-glucan levels may be highest in the spring, and outdoor temperature may influence $(1{\rightarrow}3)-{\beta}$-D-glucan levels.

• The Korean Journal of Food And Nutrition
• /
• v.32 no.6
• /
• pp.738-744
• /
• 2019

Recently, the prevalence of hyperlipidemia has been increasing, and consequently, the need to identify safe and effective treatments to control this chronic disease has also increased. The beneficial effects of probiotics have been revealed by several studies over the past few years, including their effects on hypertriglyceridemia. However, the mechanisms of action of probiotics are still unclear. The anti-obesity effects of Lactobacillus plantarum Q180 on lipid accumulation have already been demonstrated using an in vitro HepG2 cell model, and therefore, we investigated its efficacy and mechanism of action. Lipid accumulation was induced in HepG2 cells by palmitic acid treatment and then the cells were incubated with L. plantarum Q180 lysate or supernatant to investigate changes in lipid accumulation and expression of lipid metabolism-related genes. The results showed that the L. plantarum Q180-treated group exhibited significantly lower levels of lipid accumulation and mRNA expression of lipid synthesis- and adipogenesis-related genes than the palmitic acid-treated group did. These results indicate that L. plantarum Q180 may contribute to alleviating hypertriglyceridemia by inhibiting lipid synthesis.

• Korean Journal of Veterinary Research
• /
• v.38 no.3
• /
• pp.559-564
• /
• 1998

The polymerase chain reaction(PCR) assay was used for rapid diagnosis from blood and organ samples experimentally infected with Listeria monocytogenes. This method used a pair of primers based on a unique region in the 16S rRNA sequence of L monocytogenes. Procedure A was based on dilution of the blood sample followed by lysis of bacterial cell and direct analysis of the lysate with PCR. In artificially infected blood samples with L monocytogenes, it was possible to detect fewer than 40 cells per ml of blood. However, L monocytogenes was detected low rates on infected organs by the direct PCR. In procedure B, enrichment cultivation was used to increase numbers of bacteria before lysis and PCR. L monocytogenes was detected from 23 samples of 24 liver and spleen, respectively, and 18 samples of 24 blood were found to be positive by PCR on a subset of 72 organ samples, whereas L monocytogenes were detected on 63 organ samples in classical culture technique. It was required to analyze including enrichment steps were 6h and 18h on the procedure A and B, respectively.

• Journal of Nutrition and Health
• /
• v.21 no.2
• /
• pp.113-121
• /
• 1988

The purpose of this study was to investigate the effects of dietary zinc on immune response in mice. Weanling male mice was placed individually in stainless steel cages and fed a zinc dificient diet and control diet. All mice were given deionized water ad libitum. The introduction of extraneous zinc was minimized in all cage by washing feed jars and water bottles sequentially with 4mM EDTA and conc-nitiric acid followed by deionized water. After 4 and 5 weeks of the diets, mice were immunized with lx 106 Naegleria fowleri intraperitoneally. Mice were weighed once a week. The results from this study are summarized as followed ; 1) Mice fed the zinc dificient diet showed growth retardation. After 3 weeks of diets, mean body weight of zinc deficient mice was 21.4g and that of control was 25.0g. This difference is singnificant statistically (p<0.01). The more time passed, the more remarkable difference was found. 2) The weigth of organs were measured on liver, kidney, spleen, thymus, heart, lung, brain. Difference in weight were observed only in liver and spleen. 3) Proliferative response of spleen cells of zinc deficient mice to con A was lower than that of control mice after one week on immunization(p<0.005). 4) Stimulation index was lower in zinc deficient mice to phytohemagglutinin after two weeks on immunization (p<0.05). 5) Blastogenesis of speen cells of zinc deficient mice to Naegleria fowleric lysate was lower after 10 days on immunization (p<0.05). 6) Immunoglobulin G antribody titers of zinc deficient mice sera by ELISA was lowered to control mice after 5 weeks on immunization (p<0.005).