• Biotechnology and Bioprocess Engineering:BBE
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• 제3권2호
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• pp.87-90
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• 1998

Hemolymph is oxidized and darkens visibly during the collection from silkworm due to the activity of tyrosinase in it. Toxic quinones are produced by the oxidation and consequently inhibit the cell growth. Heat treatment can be used to prevent the oxidation ; however, the oxidation may occur during the collection of hemolymph before it is heat-treated. It makes the hemolymph collection difficult especially on a large-scale preparation. Hemolymphs collected from 257 different strains of silkworms were examined to select the slowly oxidized hemolymphs. Hemolymphs collected from mutant strains such as Lemone, TBO, Cre, Y4, and wEb showed relatively slow color changes. Oxidation rates of the hemolymphs were measured by the absorbance change using a spectrophotometer. The hemolymph of wEb showed the slowest oxidation. The absorbance of this mutant hemolymph reached the saturation value at 20$^{\circ}C$ in 450 min, whereas the total oxidation time of the wild-type (Baekokjam) hemolymph at the same temperature was 120 min. We tested if this mutant hemolymph is useful as a medium supplement for insect cell culture. Cell growth rate and final cell concentration in the medium supplemented with the wEb hemolymph were almost same as those in the medium supplemented with the wild-type hemolymph. Hemolymph is collected on a small scale by clipping the abdominal leg; however, this method is not appropriate fro large scale preparation. Centrifugation after chopping the silkworm hemolymph by a blending mixer is a more appropriate procedure for large scale collection. Slowly oxidized wEb hemolymph resulted in higher cell concentration than the wild-type hemolymph when hemolymph was collected by the large scale preparation method.

• Journal of Microbiology and Biotechnology
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• 제17권5호
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• pp.739-744
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• 2007

Insect cell lines and the control of infection for obtaining the maximum amount of polyhedrin-Cry1Ac-polyhedrin fusion protein from Bactrus in monolayer and suspension culture systems were tested. Growth rates of the Trichoplusia ni(High-Five) cell line in both culture systems were better than the other insect cell lines, Spodoptera frugiferda(Sf-9, Sf-21), Trichoplusia ni(Tn5), and Spodoptera exigua(Se301). The expression of the fusion protein in a monolayer culture showed that Se301 cells were 2.3-4.8 times more productive on a per cell basis than the other cell lines. However, in suspension culture, only High-Five cells were productive. High-Five cells infected with Bactrus at a multiplicity of infection(MOI) of 5 and a cell density of $3.0{\times}10^5$ cells per ml were more productive than the other infection condition in a suspension culture suitable for a large-scale production of baculovirus. In conclusion, for the large-scale production of Bactrus in vitro, High-Five cells showing good growth and high productivity are suitable.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권3호
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• pp.275-279
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• 2005

Insect cell transformants, stably expressing human $GFP_{uv}-{\beta}1$, 3-N-acetylglucosaminyltransferase 2 $({\beta}3GnT2)$ as the green fluorescent protein $(GFP_{uv})-fused$ protein, were efficiently isolated on Western blot by the quantification of the densitometric intensity of the fusion protein. From almost 150 transformants containing the fusion gene linked to three different types of signal sequence, two transformants, Tn-pXme4a and -pX28a, were successfully selected, showing 8.3 and 8.6 mU/mL ${\beta}3GnT$ activity, respectively. This method requires a screening time almost one-half that required in the isolation of stably transformed cells with high expression levels, and at the same time allows the handling a large number of transformants.

• 한국미생물·생명공학회지
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• 제50권1호
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• pp.157-163
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• 2022

Alcoholic liver disease (ALD), which encompasses alcoholic steatosis, alcoholic hepatitis, and alcoholic cirrhosis, is a major cause of morbidity and mortality worldwide. Although the economic and health impacts of ALD are clear, few advances have been made in its prevention or treatment. We recently demonstrated that the insect-derived antimicrobial peptide CopA3 exerts anti-apoptotic and anti-inflammatory activities in various cell systems, including neuronal cells and colonic epithelial cells. Here, we tested whether CopA3 inhibits ethanol-induced liver injury in mice. Mice were intraperitoneally injected with ethanol only or ethanol plus CopA3 for 24 h and then liver injury and inflammatory responses were measured. Ethanol enhanced the production of proinflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, interferon (IFN)-γ, and IL-10. It also induced hepatocyte apoptosis and ballooning degeneration in hepatocytes. Notably, all these effects were eliminated or significantly reduced by CopA3 treatment. Collectively, our findings demonstrate that CopA3 ameliorates ethanol-induced liver cell damage and inflammation, suggesting the therapeutic potential of CopA3 for treating ethanol-induced liver injury.

• BMB Reports
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• 제39권3호
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• pp.263-269
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• 2006

In the genome of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus, open reading frame 39 (Ha39) is the only gene predicted to encode an RNA recognition protein. Computer analysis revealed that Ha39 homologues were found in 15 NPVs, but not in GVs. Its transcripts were detected from 3 through 72 hours post infection (h p.i.) using RT-PCR and Northern blot analysis. The protein was detected in infected-cell lysates from 6 h p.i. Western blot assay of ODV and BV preparations revealed that Ha39 encodes a structural protein associated with BVs. Additionally, immunofluorescence microscopy demonstrated that the protein was present within cytoplasm in virus-infected cells, but not in the nuclear region.

• International Journal of Industrial Entomology and Biomaterials
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• 제4권1호
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• pp.57-62
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• 2002

Cationic liposomes complexed with DNA have been extensively utilized for the delivery of reporter or therapeutic genes both in culture and in vivo. We investigated and determined the optimum conditions of a cationic liposome, composed of dimethyldioctadecy-lammonium bromide (DDAB) and dioleoyl phosphati-dylethanolamine UOPE), mediated a reporter plasmid expressing luciferase into insect cell lines (Sf-21 and Bm-N) and silkworm larvae. Together the data demonstrated that Bombyx mori nuclear polyhedrosis virus (BmNPV) genomic DNA (128 kb) was successfully transfected into Bm-5 cells using this liposome. These results suggest that DDAB/DOPE liposome will be useful as delivery agents for gene transfer to insect cells both in vitro and in vivo.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2002년도 생물공학의 동향 (X)
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• pp.62-65
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• 2002

We have previously shown that the addition of silkworm hemolymph to a culture medium increases the longevity of insect and mammalian cells by inhibiting apoptosis. This indicates that the component which inhibits apoptosis is contained in the silkworm hemolymph, The apoptosis-inhibiting component was isolated from silkwonn hemolymph and characterized in our previous study. A database search using the N-terminal amino acid sequence of this component as a template resulted in a 95% homology with a low molecular weight lipoprotein, the so called ’30K protein' of unknown function. In this study, the 30K protein gene was expressed in mammalian and insect cells to confirm the apoptosis-inhibiting effect. The overexpression of 30K protein in mammalian cell inhibited the staurosporin-induced apoptosis by the prevention of the activation of caspase 3. Using an Autographa californicanuclear polyhedrosis virus (AcNPV) system, the 30K protein was overexpressed also in insect cells. The expression of the 30K protein increased the longevity of baculovirus-infected insect cells by inhibiting apoptosis. These results suggest that the 30K protein is a novel anti-apoptotic protein.

• 대한의생명과학회지
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• 제8권4호
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• pp.211-215
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• 2002

The baculovirus/Sf9 cell expression can be employed as a powerful system for producing large amounts of the human erythrocyte glucose transporter, GLUT1 heterologously In order to exploit the system further, it is necessary to develop a convenient method for demonstrating that the transporter expressed in insect cells is biologically active. To achieve this, we have expressed the human CLUT1 in insect cells and photolabelled the expressed protein with [$^3$H] cytochalasin B, a potent inhibitor of the human erythrocyte glucose transporter. Subsequently, the labelled proteins were analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Membranes labelled with [$^3$H] cytochalasln B in the presence of L-Glucose yielded a single sharp peak of labelling of apparent $M_r$ 45,000 on SDS/polyacrylamide gels. The mobility of this peak corresponded exactly to that of the band detected by anti-glucose transporter antibodies on Western blots of membranes prepared from insect cells infected with recombinant virus. In addition, the sharpness of the radioactive peak provides further evidence for the conclusion that the expressed protein is much less heavily and heterogeneously glycosylated than its erythrocyte counterpart. No peak of labelling was seen with the membranes prepared from non-infected Sf9 cells. Furthermore, the incorporation of label into this peak was completely inhibited by the presence of 500 mM-D-Glucose during tile photolabelling procedure, showing the stereoselectivity of the labelling. These evidences clearly show that human glucose transporter expressed in insect cells exhibits native-like biological activity, and that photolabelling with [$^3$H] cytochalasin B can be a convenient means for analysing the biological activity of the transport protein expressed in insect cells.

• 한국잠사곤충학회지
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• 제49권2호
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• pp.47-50
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• 2007

누에 체액은 공기 중의 산소와 접하게 되면 도파퀴논(dopaquinone)이라는 세포 성장을 저해하는 물질을 생성하게 된다. 체액의 산화를 방지하기 위해서는 일반적으로 열처리에 의해서 효소를 불활성화 시키는 방법이 이용되고 있으나 대량 채혈 시에는 열처리 이전에 이미 산화가 진행되는 문제점이 있다. 이러한 문제점을 해결하기 위하여 누에 중 체액 산화가 더디게 진행되는 누에 계통을 선발하게 되었다. 누에체액을 채취하여 경과시간별로 산화정도(변색)를 육안으로 확인하여 TBO, $Y_4$, 백안$E^b$과잉지 3품종을 선발하고 spectrophotometer를 이용하여 산화반응 속도를 측정하였다. 백옥잠 체액과 각각의 산화지연 누에 체액의 산화속도를 400 nm에서 측정한 결과 백옥잠은 120분인데 비하여 체액산화 지연계통인 $Y_4$는 330분, TBO는 360분 그리고 백안$E^b$과잉지는 450분에 산화가 완료되었다. 산화지연 누에계통의 체액의 곤충세포 증식에 미치는 영향을 조사한 결과 대규모 채혈한 체액산화지연 계통의 체액은 야성형 계통(백옥잠)에서 소규모 채취한 체액에는 미치지 못하였으나 대규모 채취한 체액보다는 우수하였다.

• The Plant Pathology Journal
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• 제32권4호
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• pp.340-346
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• 2016

To understand how Bursaphelenchus xylophilus kills pine trees, the differences between the effects of B. xylophilus and B. mucronatus on pine trees are usually compared. In this study, the migration and attacking ability of a non-pathogenic B. mucronatus in Pinus thunbergii were investigated. The distribution of B. mucronatus and the number of dead epithelial cells resulting from inoculation were compared with those of the pathogenic B. xylophilus. Although B. mucronatus is non-pathogenic in pines, its distribution pattern in P. thunbergii was the same as that of B. xylophilus. We therefore concluded that the non-pathogenicity of B. mucronatus could not be attributed to its migration ability. The sparse and sporadic attacking pattern of B. mucronatus was also the same as that of B. xylophilus. However, the number and area of the dead epithelial cells in pine cuttings inoculated with B. mucronatus were smaller than in those cuttings inoculated with B. xylophilus, meaning that the attacking ability of B. mucronatus is weaker than that of B. xylophilus. Therefore, we concluded that the weaker attacking ability of B. mucronatus might be the factor responsible for the non-pathogenicity.