누에 핵다각체병 바이러스의 Ecdysteroid UDP-glucosyltransferase 유전자가 누에의 발육에 미치는 영향

강경돈,이은정,Shizuo George Kamita,성수일 한국잠사학회 1998 한국잠사곤충학회지 Vol.40 No.2

The baculovirus egt gene encodes an ecdysteroid UDP-glucosyltransfernse(EGT) which catalyzes the transfer of glurose from UDP-glucose to the insect molting hormone ecdysteroid resulting in a functionally inactive ecdysteroid. In baculovirus-infected insect larvae, EGT has been shown block molting and pupation. In this study, we compared the development of 4th and 5th instar silkworm, Bombyx mori, larvae injected with either wild-type Bombyx mori nucleopolyhedrovirus (BmNPV) or a mutant BmNPV(BmEGTZ) in which the egt gene was disrupted by the insertion of a lacZ gene cassette. Larvae injected with BmEGTZ died roughly 12 h more rapidly compared to identical larvae infected with BmNPV. In addition, BmEGTZ-infected larvae prematurely stopped feeding and gain less weight compared to BmNPV-infected larvae. In order to investigate why BmEGTZ-infected larvae died more rapidly than BmNPV-infected larvae, the array of hemolymph proteins in BmEGTZ- or BmNPV-infected larvae were analyzed by SDS-PAGE. The hemolymph of BmEGTZ-infected larvae showed virus-specific proteins, including polyhedrin, about 12 h earlier than the hemolymph of BmNPV-infected larvae

Comparative Analysis of α-glucosidase Activity in Bombyx mori and Antheraea yamamai

강경돈,Shizuo George Kamita,Koichi Suzuki,성수일 한국잠사학회 2010 International Journal of Industrial Entomology Vol.21 No.2

α-Glucosidase (EC 3.2.1.20) is a glycosidase that hydrolyzes disaccharides, oligosaccharides, and polysaccharides resulting in the release of α-D-glucose. In this study, α-glucosidase activity in the hemolymph and midgut of the mulberry silkworm Bombyx mori and Japanese oak silkmoth Antheraea yamamai was measured using maltose,sucrose, trehalose, and p-nitrophenyl α-D-glucopyranoside as substrates. In general, hemolymph α-glucosidase activity was higher in B. mori than in A. yamamai. In contrast, midgut α-glucosidase activity was higher in A. yamamai than in B. mori for all of the substrates tested. α-Glucosidase activity in the midgut of both B. mori and A. yamamai showed similar responses to changes in pH and temperature for all of the substrates tested. Native (7.5%) PAGE of hemolymph and midgut proteins from B. mori and A. yamamai followed by staining with 4-methylumbelliferyl α-D-glucoside (MUG) indicated that the α-glucosidases of these related lepidopterans are functionally similar but structurally different. In comparison to α-glucosidase activity from A. yamamai, α-glucosidase activity from B. mori was generally less sensitive to the α-glucosidase inhibitors, 1-deoxynojirimycin (DNJ), acarbose, and voglibose when the activity was determined using maltose,sucrose, and trehalose.

Comparative Analysis of $\alpha$-glucosidase Activity in Bombyx mori and Antheraea yamamai

Kang, Kyung-Don,Kamita, Shizuo George,Suzuki, Koichi,Seong, Su-Il Korean Society of Sericultural Science 2010 International Journal of Industrial Entomology Vol.21 No.2

[ $\alpha$ ]Glucosidase (EC 3.2.1.20) is a glycosidase that hydrolyzes disaccharides, oligosaccharides, and polysaccharides resulting in the release of α-D-glucose. In this study, $\alpha$-glucosidase activity in the hemolymph and midgut of the mulberry silkworm Bombyx mori and Japanese oak silkmoth Antheraea yamamai was measured using maltose, sucrose, trehalose, and p-nitrophenyl $\alpha$-D-glucopyranoside as substrates. In general, hemolymph $\alpha$-glucosidase activity was higher in B. mori than in A. yamamai. In contrast, midgut $\alpha$-glucosidase activity was higher in A. yamamai than in B. mori for all of the substrates tested. $\alpha$-Glucosidase activity in the midgut of both B. mori and A. yamamai showed similar responses to changes in pH and temperature for all of the substrates tested. Native (7.5%) PAGE of hemolymph and midgut proteins from B. mori and A. yamamai followed by staining with 4-methylumbelliferyl $\alpha$-D-glucoside (MUG) indicated that the $\alpha$-glucosidases of these related lepidopterans are functionally similar but structurally different. In comparison to $\alpha$-glucosidase activity from A. yamamai, $\alpha$-glucosidase activity from B. mori was generally less sensitive to the $\alpha$-glucosidase inhibitors, 1-deoxynojirimycin (DNJ), acarbose, and voglibose when the activity was determined using maltose, sucrose, and trehalose.

Ecdysteroid Stimulates Virus Transmission in Larvae Infected with Bombyx mori Nucleopolyhedrovirus

Kang, Kyung-Don,Lee, Eun-Jung,Kamita, Shizuo George,Maeda, Susumu,Seong, Su-Il Korean Society for Biochemistry and Molecular Biol 2000 Journal of biochemistry and molecular biology Vol.33 No.1

Most baculoviruses have an ecdysteroid UDP-glucosyltransferase (egt) gene, whose product inactivates ecdysteroid within the infected host. Bomhyx mori larvae infected with BmEGTZ, a mutant B. mori nucleopolyhedrovirus (BmNPV) in which the egt gene has been inactivated, die more rapidly compared to larvae infected with wild-type BmNPV. In this study, the profile of hemolymph proteins, and progression of virus infection in BmEGTZ- and BmNPV-infected B. mori larvae, was analyzed by SDS-PAGE and histochemically. These analyses showed that virus-encoded and virus-induced proteins were expressed quicker in BmEGTZ-infected larvae than in BmNPV-infected larvae. This suggests that the decrease in time to death, following BmEGTZ infection, results from the stimulation of virus-specific protein expression. In order to examine the effect of ecdysteroid on virus transmission, the profile of hemolymph proteins, and progression of virus infection, were analyzed following an ecdysteroid injection of BmEGTZ- or BmNPV-infected larvae. In the BmNPV-infected larvae, ecdysteroid treatment had no apparent effect on hemolymph protein expression. This suggests that the injected ecdysteroid was inactivated by the BmNPV-expressed ecdysteroid UDP-glucosyltransferase. An Ecdysteroid injection into BmEGTZ-infected larvae increased the speed of virus-specific protein expression and virus transmission. These results suggest that ecdysteroid stimulates protein expression, which in tum results in the stimulation of virus transmission.

Ecdysteroid Stimulates Virus Transmission in Larvae Infected with Bombyx mori Nucleopolyhedrovirus

Lee, Eun Jung,Maeda, Susumu,Kang, Kyung Don,Kamita, Shizuo George,Seong, Su Il 생화학분자생물학회 2001 BMB Reports Vol.33 No.1

Most baculoviruses have an ecdysteroid UDP-glucosyltransferase (egt) gene, whose product inactivates ecdysteroid within the infected host. Bombyx mori larvae infected with BmEGTZ, a mutant B. mori nucleopolyhedrovirus (BmNPV) in which the egt gene has been inactivated, die more rapidly compared to larvae infected with wild-type BmNPV In this study, the profile of hemolymph proteins, and progression of virus infection in BmEGTZ- and BmNPV-infected B. mori larvae, was analyzed by SDS-PAGE and histochemically These analyses showed that virus-encoded and virus-induced proteins were expressed quicker in BmEGTZ-infected larvae than in BmNPV infected larvae. This suggests that the decrease in time to death, following BmEGTZ infection, results from the stimulation of virus-specific protein expression. In order to examine the effect of ecdysteroid on virus transmission, the profile of hemolymph proteins, and progression of virus infection, were analyzed following an ecdysteroid injection of BmEGTZ- or BmNPV infected larvae. In the BmNPV infected larvae, ecdysteroid treatment had no apparent effect on hemolymph protein expression. This suggests that the injected ecdysteroid was inactivated by the BmNPV expressed ecdysteroid UDP-glucosyltransferase. An Ecdysteroid injection into BmEGTZ-infected larvae increased the speed of virus-specific protein expression and virus transmission. These results suggest that ecdysteroid stimulates protein expression, which in turn results in the stimulation of virus transmission.

Effect of 1-deoxynojirimycin on the Replication of Baculoviruses, Bombyx Mori Nucleopolyhedrovirus and Autographa Californica Multiple Nucleopolyhedrovirus

Kang, Kyung-Don,Park, Joo-Sung,Cho, Yong-Seok,Park, Young-Shik,Lee, Jae-Yeon,Hwang, Kyo-Yeol,Yuk, Won-Jeong,Kamita, Shizuo George,Suzuki, Koichi,Seong, Su-Il Korean Society of Sericultural Science 2011 International Journal of Industrial Entomology Vol.23 No.1

1-Deoxynojirimycin (DNJ) is an alkaloid that is found at relatively high concentrations in mulberry leaf and tissues of the silkworm, $Bombyx$ $mori$. DNJ is a well known inhibitor of ${\alpha}$-glucosidase, an enzyme that is involved in the early stages of the $N$-linked glycoprotein synthesis pathway. ${\alpha}$-Glucosidase activity in the cell extract from $B.$ $mori$-derived Bm5 cells showed approximately 40-fold less sensitivity to DNJ than ${\alpha}$-glucosidase activity in the cell extract from $Spodoptera$ $frugiperda$-derived Sf9 cells. The replication of $B.$ $mori$ nucleopolyhedrovirus (BmNPV) was not inhibited when it was propagated in BmN cells that were grown in medium containing up to 10 mM DNJ. In contrast, the replication of $Autographa$ $californica$ multiple NPV (AcMNPV) was reduced by 67% when it was propagated in Sf9 cells that were grown in medium containing 10 mM DNJ. The viability of Bm5 and Sf9 cells that were grown in medium containing up to 10 mM DNJ was not affected. Our results suggested that the reduced replication of AcMNPV was the result of the higher sensitivity of ${\alpha}$-glucosidase activity in Sf9 cells to DNJ.

1-Deoxynojirimycin 생산 균주 Bucillus subtilis MORI 3K-85의 단백질 분석

조용석(Yong Seok Cho),강경돈(Kyung-Don Kang),박영식(Young Shik Park),이재연(Jae Yeon Lee),김현수(Hyun Su Kim),육원정(Won Jeong Yuk),Shizuo George Kamita,황교열(Kyo Yeol Hwang),성수일(Su-Il Seong) 한국생물공학회 2011 KSBB Journal Vol.26 No.6

In our previous study, we isolated and characterized a 1-deoxynojirimycin (DNJ)-producing bacterium, Bacillus subtilis MORI, from chungkookjang, a Korean traditional food. B. subtilis MORI was subjected to γ-irradiation and the resulting bacteria were screened for increased DNJ production. A mutant was identified that produced 7.6 times more DNJ and named B. subtilis MORI 3K-85. In this study, the protein profiles of both strains were compared by one-dimensional and two-dimensional gel electrophoresis (1-DE and 2-DE, respectively) under both native and denaturing conditions. The 1-DE native-PAGE and 1-DE SDS-PAGE analyses identified 5 and 7 bands, respectively, that were found at higher concentrations in B. subtilisMORI 3K-85 than in B. subtilis MORI. Similarly, 2-DE analyses identified 20 protein spots which were found at higher concentrations in B. subtilis MORI 3K-85. The peptide mass profiles of these 20 proteins were analyzed by MALDI-TOF and compared with peptide sequences of B. subtilis and B. amyloliquefaciens in the MASCOT database. This screening suggested that three dehydrogenases, an aldolase, a synthetase, an isomerase, a reductase, and a peroxidase are elevated in B. subtilis MORI 3K-85. Based on this data, one or more of the elevated 8 enzymes might be related to the DNJ biosynthetic pathway.

Research Articles : Effect of 1-deoxynojirimycin on the Replication of Baculoviruses, Bombyx Mori Nucleopolyhedrovirus and Autographa Californica Multiple Nucleopolyhedrovirus

( Kyung Don Kang ),( Joo Sung Park ),( Yong Seok Cho ),( Young Shik Park ),( Jae Yeon Lee ),( Kyo Yeol Hwang ),( Won Jeong Yuk ),( Shizuo George Kamita ),( Koichi Suzuki ),( Su Il Seong ) 한국잠사학회 2011 International Journal of Industrial Entomology Vol.23 No.1

1-Deoxynojirimycin (DNJ) is an alkaloid that is found at relatively high concentrations in mulberry leaf and tissues of the silkworm, Bombyx mori. DNJ is a well known inhibitor of α-glucosidase, an enzyme that is involved in the early stages of the N-linked glycoprotein synthesis pathway. α-Glucosidase activity in the cell extract from B. mori-derived Bm5 cells showed approximately 40-fold less sensitivity to DNJ than α- glucosidase activity in the cell extract from Spodoptera frugiperda-derived Sf9 cells. The replication of B. mori nucleopolyhedrovirus (BmNPV) was not inhibited when it was propagated in BmN cells that were grown in medium containing up to 10 mM DNJ. In contrast, the replication of Autographa californica multiple NPV (AcMNPV) was reduced by 67% when it was propagated in Sf9 cells that were grown in medium containing 10 mM DNJ. The viability of Bm5 and Sf9 cells that were grown in medium containing up to 10 mM DNJ was not affected. Our results suggested that the reduced replication of AcMNPV was the result of the higher sensitivity of α-glucosidase activity in Sf9 cells to DNJ.