Nine assorted papers on mulberry, silkworm physiology, molecular biology and seri medicine.
1. Micromorphological Characterization of Ten Mulberry Cultivars (Morus spp)
Magda Biasiolo, Maria Teresa Da Canal, and Noemi Tornadore
Economic Botany Volume 58, Issue 4 (December 2004) pp. 639–646
The micromorphological features of the vegetative and reproductive structures of ten mulberry cultivars grown at the Specialized Sericultural Section of the Agricultural Zoology Experimental Institute of Padua, northeastern Italy, were examined by SEM in order to determine the charactetistics that were the most valuable taxonomically. The observed specimens (leaves, flowers, seeds, and pollen grains) showed micromorphological differences regarding leaf hairiness, quantity of waxes, quality of epidermis cuticle and tepal hairiness. The effects of differing environments in altering the floral sex ratios of this basically monoecious group of plants were also investigated. However, no significant differences between the micromorphology of the seeds and the pollen grains of these selected cultivars were detected. The authors are hopeful that the information gained in this study may prove useful in the future creation of an exhaustive and final catalogue “descriptor” of cultivated varieties belonging to the genus Morus L.
2. Developmental Profile of Annexin IX and its Possible Role in Programmed Cell Death of the Bombyx mori Anterior Silk Gland
Yu Kaneko, Keiko Takaki, Masafumi Iwami, and Sho Sakurai
Zoological Science Volume 23, Issue 6 (June 2006) pp. 533–542
During pupal metamorphosis, the anterior silk gland (ASG) of the silkworm, Bombyx mori, undergoes programmed cell death (PCD), which is triggered by 20-hydroxyecdysone (20E). Annexin IX (ANX IX) has been identified as a 20E-inducible gene in dying ASGs, and we show here that its expression is down-regulated in tissues destined to die but not in tissues that survive pupal metamorphosis. ANX IX expression was high in the ASGs during the feeding period, when the ecdysteroid titer was low, and decreased in response to the rising ecdysteroid titer that triggered pupal metamorphosis. Before gut purge, in vitro exposure of the ASGs to 20E levels corresponding to the ecdysteroid concentration present at the time of gut purge caused a decrease in ANX IX messenger RNA levels. Expression profiles of EcR and USP, and the 20E concentration-responses of these genes, indicate the importance of the relative abundance of EcR-A and EcR-B1 isoforms in ANX IX regulation. These results suggest an involvement of ANX IX in the determination of PCD timing by delaying or suppressing the response to the increase in hemolymph ecdysteroid concentration during the prepupal period.
3. Release of Ecdysteroid-Phosphates from Egg Yolk Granules and Their Dephosphorylation during Early Embryonic Development in Silkworm, Bombyx mori
Ryouichi Yamada, Yumi Yamahama, and Haruyuki Sonobe
Zoological Science Volume 22, Issue 2 (February 2005) pp. 187–198
Newly laid eggs of many insect species store maternal ecdysteroids as physiologically inactive phosphoric esters. In the silkworm Bombyx mori, we previously reported the presence of a specific enzyme, called ecdysteroid-phosphate phosphatase (EPPase), which catalyzes the dephosphorylation of ecdysteroid-phosphates to increase the amount of free ecdysteroids during early embryonic development. In this study, we demonstrated that (1) EPPase is found in the cytosol of yolk cells, (2) ecdysteroid-phosphates are localized in yolk granules, being bound to the yolk protein vitellin (Vn), and (3) Vn-bound ecdysteroid-phosphates are scarcely hydrolyzed by EPPase, although free ecdysteroid-phosphates are completely hydrolyzed by EPPase. Thus, we investigated the mechanism by which ecdysteroid-phosphates dissociate from the Vn-ecdysteroid-phosphate complex, and indicated that the acidification of yolk granules causes the dissociation of ecdysteroid-phosphates from the Vn-ecdysteroid-phosphate complex and thereby ecdysteroid-phosphates are released from yolk granules into the cytosol. Indeed, the presence of vacuolar-type proton-translocating ATPase in the membrane fraction of yolk granules was also verified by Western blot analysis. Our experiments revealed that Vn functions as a reservoir of maternal ovarian ecdysteroid-phosphates as well as a nutritional source during embryonic development. This is the first report showing the biochemical mechanism by which maternal Vn-bound ecdysteroid-phosphates function during early embryonic development.
4. Establishment of a Sandwich ELISA System to Detect Diapause Hormone, and Developmental Profile of Hormone Levels in Egg and Subesophageal Ganglion of the Silkworm, Bombyx mori
Norio Kitagawa, Kunihiro Shiomi, Kunio Imai, Teruyuki Niimi, Toshinobu Yaginuma, and Okitsugu Yamashita
Zoological Science Volume 22, Issue 2 (February 2005) pp. 213–221
In the silkworm Bombyx mori, diapause hormone (DH) is produced in the female subesophageal ganglion (SG) and induces embryonic diapause by targeting developing ovaries. DH is processed from a precursor protein consisting of DH, pheromone biosynthesis activating neuropeptide (PBAN) and three other neuropeptides (SGNPs). Because these five neuropeptides share a common sequence, FXPRLamide, at the C-terminus, a direct and specific assay for DH itself is required in order to understand the profile of concentration changes. In this study, we produced a mouse monoclonal antibody (anti-DH[N] mAb) against the N-terminal region of DH and developed a sandwich enzyme-linked immunosorbent assay using the anti-DH[N] mAb and a rabbit polyclonal antibody against the C-terminus of DH. This procedure enabled us to specifically quantify the DH molecule at femtomolar levels (equivalent to 1/10 of SG). We then plotted DH levels in eggs and SGs during embryonic and post-embryonic development. DH was present in late-stage embryos that had been destined for the production of both diapause and nondiapause eggs. DH levels in SG gradually increased in both types during larval development and peaked at the early pupal stage. At the middle pupal stage, DH levels in SG and SG-brain complex decreased markedly in the diapause-egg producing type, thus indicating active release of DH into the hemolymph. From 5th instar larva to adult, no sexual differences in DH levels were observed in SGs or SG-brain complexes from diapause and nondiapause egg-producing types.
5. Functional analysis of four Gloverin-like genes in the silkworm, Bombyx mori. Kawaoka S, Katsuma S, Daimon T, Isono R, Omuro N, Mita K, Shimada T.
Arch Insect Biochem Physiol. 2007 Dec 12; [Epub ahead of print]
To identify genes involved in the innate immunity of the silkworm Bombyx mori, we constructed a cDNA library from the fat body of Escherichia coli-challenged B. mori larvae. Based on the expressed sequence tag (EST) data and whole genome shotgun sequence analysis, we found four Gloverin-like genes, BmGlov1-4, in the Bombyx genome. Northern blot and RT-PCR analysis showed that BmGlov1-4 were induced in the larval fat body after an immune challenge by the injection of E. coli; however, less induction was observed after the injection of a yeast Candida albicans. In silico sequence analysis revealed the presence of a motif homologous to NF-kappaB binding site in the upstream region of each BmGlov gene. Moreover, we expressed recombinant BmGlov1-4 proteins using the baculovirus expression system, and found that all the recombinant BmGlov1-4 significantly inhibited the growth of E. coli. Arch. Insect Biochem. Physiol. 2007. (c) 2007 Wiley-Liss, Inc.
6. Solubilization of the Ecdysone Binding Protein from Anterior Silk Gland Cell Membranes of the Silkworm, Bombyx mori.
M Elmogy, M Iwami, and S Sakurai
Zoolog Sci. 2007; 24: 971
We previously provided preliminary evidence for the presence of a putative membrane ecdysone receptor (mEcR) anchored in the plasma membranes of anterior silk glands (ASGs) in Bombyx mori. This receptor may act in concert with the conventional EcR in 20E-dependent programmed cell death of these glands. We report here, for the first time, the solubilization of mEcR from ASG membranes using the zwitterionic detergent CHAPS in the presence of NaCl. Our results show by ligand binding assay that mEcR solubilized this way is functionally active and retains 75% of its native binding activity. We also defined experimental conditions that yielded protein/detergent complexes with partial binding activity, which makes it possible to purify the membrane-bound ecdysone binding protein.
7. A silkworm baculovirus model for assessing the therapeutic effects of antiviral compounds: characterization and application to the isolation of antivirals from traditional medicines.
Y Orihara, H Hamamoto, H Kasuga, T Shimada, Y Kawaguchi, and K Sekimizu
J Gen Virol. 2008; 89: 188
Ganciclovir, foscarnet, vidarabine and ribavirin, which are used to treat viral infections in humans, inhibited the proliferation of a baculovirus (Bombyx mori nucleopolyhedrovirus) in BmN4 cells, a cultured silkworm cell line. These antiviral agents inhibited the proliferation of baculovirus in silkworm body fluid and had therapeutic effects. Using the silkworm infection model, the antiviral activity of Kampo medicines was screened and it was found that cinnamon bark, a component of the traditional Japanese medicine Mao-to, had a therapeutic effect. Based on the therapeutic activity, the antiviral substance was purified. Nuclear magnetic resonance analysis of the purified fraction revealed that the antiviral activity was due to cinnzeylanine, which has previously been isolated from Cinnamomum zeylanicum. Cinnzeylanine inhibits the proliferation of herpes simplex virus type 1 in Vero cells. These results suggest that the silkworm-baculovirus infection model is useful for screening antiviral agents that are effective for treating humans infected with DNA viruses.
8. Antidiabetic Properties of 2,5-Dihydroxy-4,3'-Di(beta-D-Glucopyranosyloxy)-trans-Stilbene from Mulberry (Morus bombycis Koidzumi) Root in Streptozotocin-Induced Diabetic Rats.
Heo SI, Jin YS, Jung MJ, Wang MH.
J Med Food. 2007 Dec;10(4):602-7.
We investigated the antidiabetic properties of 2,5-dihydroxy-4,3-di(beta-D-glucopyranosyloxy)-trans-stilbene (DGTS) isolated from Morus bombycis Koidzumi in streptozotocin (STZ)-induced diabetic rats. The DGTS prevented the increase in aspartate aminotransferase, alanine aminotransferase, and blood urea nitrogen levels in serum of diabetic rats. At doses of 200-800 mg/kg, DGTS improved hyperglycemia in the rats, and the hypoglycemic effect of DGTS was comparable to that of tolbutamide. The histological observations showed that DGTS prevented atrophy of pancreatic beta-cells and vascular degenerative changes in the islets. DGTS reversed STZ-induced diabetes and had antioxidant activity in assays of FeCl(2)/ascorbic acid-induced lipid peroxidation in the rats. Levels of cytochrome P450 2E1 mRNA, as measured by reverse transcription-polymerase chain reaction, were lower in the livers of the DGTS-treated rats than those of the control group. These results suggest that DGTS might be beneficial in the treatment of type 1 diabetes
9. Structural Disorder in Silk Proteins Reveals the Emergence of Elastomericity. Dicko C, Porter D, Bond J, Kenney JM, Vollrath F.
Biomacromolecules. 2007 Dec 14; [Epub ahead of print]
Spider silks combine basic amino acids into strong and versatile fibers where the quality of the elastomer is attributed to the interaction of highly adapted protein motifs with a complex spinning process. The evaluation, however, of the interaction has remained elusive. Here, we present a novel analysis to study silk formation by examining the secondary structures of silk proteins in solution. Using the seven different silks of Nephila edulis as a benchmark system, we define a structural disorder parameter (the folding index, gamma). We found that gamma is highly correlated with the ratio of glycine present. Testing the correlation between glycine content and the folding index (gamma) against a selected range of silks, we find quantitatively that, in order to achieve specialization with changes in mechanical performance, the spider's silks require higher structural flexibility at the expense of reduced stability and consequently an increased conversion-energy cost. Taken together, our biophysical and evolutionary findings reveal that silk elastomericity evolved in tandem with specializations in the process of silk spinning.
1. Micromorphological Characterization of Ten Mulberry Cultivars (Morus spp)
Magda Biasiolo, Maria Teresa Da Canal, and Noemi Tornadore
Economic Botany Volume 58, Issue 4 (December 2004) pp. 639–646
The micromorphological features of the vegetative and reproductive structures of ten mulberry cultivars grown at the Specialized Sericultural Section of the Agricultural Zoology Experimental Institute of Padua, northeastern Italy, were examined by SEM in order to determine the charactetistics that were the most valuable taxonomically. The observed specimens (leaves, flowers, seeds, and pollen grains) showed micromorphological differences regarding leaf hairiness, quantity of waxes, quality of epidermis cuticle and tepal hairiness. The effects of differing environments in altering the floral sex ratios of this basically monoecious group of plants were also investigated. However, no significant differences between the micromorphology of the seeds and the pollen grains of these selected cultivars were detected. The authors are hopeful that the information gained in this study may prove useful in the future creation of an exhaustive and final catalogue “descriptor” of cultivated varieties belonging to the genus Morus L.
2. Developmental Profile of Annexin IX and its Possible Role in Programmed Cell Death of the Bombyx mori Anterior Silk Gland
Yu Kaneko, Keiko Takaki, Masafumi Iwami, and Sho Sakurai
Zoological Science Volume 23, Issue 6 (June 2006) pp. 533–542
During pupal metamorphosis, the anterior silk gland (ASG) of the silkworm, Bombyx mori, undergoes programmed cell death (PCD), which is triggered by 20-hydroxyecdysone (20E). Annexin IX (ANX IX) has been identified as a 20E-inducible gene in dying ASGs, and we show here that its expression is down-regulated in tissues destined to die but not in tissues that survive pupal metamorphosis. ANX IX expression was high in the ASGs during the feeding period, when the ecdysteroid titer was low, and decreased in response to the rising ecdysteroid titer that triggered pupal metamorphosis. Before gut purge, in vitro exposure of the ASGs to 20E levels corresponding to the ecdysteroid concentration present at the time of gut purge caused a decrease in ANX IX messenger RNA levels. Expression profiles of EcR and USP, and the 20E concentration-responses of these genes, indicate the importance of the relative abundance of EcR-A and EcR-B1 isoforms in ANX IX regulation. These results suggest an involvement of ANX IX in the determination of PCD timing by delaying or suppressing the response to the increase in hemolymph ecdysteroid concentration during the prepupal period.
3. Release of Ecdysteroid-Phosphates from Egg Yolk Granules and Their Dephosphorylation during Early Embryonic Development in Silkworm, Bombyx mori
Ryouichi Yamada, Yumi Yamahama, and Haruyuki Sonobe
Zoological Science Volume 22, Issue 2 (February 2005) pp. 187–198
Newly laid eggs of many insect species store maternal ecdysteroids as physiologically inactive phosphoric esters. In the silkworm Bombyx mori, we previously reported the presence of a specific enzyme, called ecdysteroid-phosphate phosphatase (EPPase), which catalyzes the dephosphorylation of ecdysteroid-phosphates to increase the amount of free ecdysteroids during early embryonic development. In this study, we demonstrated that (1) EPPase is found in the cytosol of yolk cells, (2) ecdysteroid-phosphates are localized in yolk granules, being bound to the yolk protein vitellin (Vn), and (3) Vn-bound ecdysteroid-phosphates are scarcely hydrolyzed by EPPase, although free ecdysteroid-phosphates are completely hydrolyzed by EPPase. Thus, we investigated the mechanism by which ecdysteroid-phosphates dissociate from the Vn-ecdysteroid-phosphate complex, and indicated that the acidification of yolk granules causes the dissociation of ecdysteroid-phosphates from the Vn-ecdysteroid-phosphate complex and thereby ecdysteroid-phosphates are released from yolk granules into the cytosol. Indeed, the presence of vacuolar-type proton-translocating ATPase in the membrane fraction of yolk granules was also verified by Western blot analysis. Our experiments revealed that Vn functions as a reservoir of maternal ovarian ecdysteroid-phosphates as well as a nutritional source during embryonic development. This is the first report showing the biochemical mechanism by which maternal Vn-bound ecdysteroid-phosphates function during early embryonic development.
4. Establishment of a Sandwich ELISA System to Detect Diapause Hormone, and Developmental Profile of Hormone Levels in Egg and Subesophageal Ganglion of the Silkworm, Bombyx mori
Norio Kitagawa, Kunihiro Shiomi, Kunio Imai, Teruyuki Niimi, Toshinobu Yaginuma, and Okitsugu Yamashita
Zoological Science Volume 22, Issue 2 (February 2005) pp. 213–221
In the silkworm Bombyx mori, diapause hormone (DH) is produced in the female subesophageal ganglion (SG) and induces embryonic diapause by targeting developing ovaries. DH is processed from a precursor protein consisting of DH, pheromone biosynthesis activating neuropeptide (PBAN) and three other neuropeptides (SGNPs). Because these five neuropeptides share a common sequence, FXPRLamide, at the C-terminus, a direct and specific assay for DH itself is required in order to understand the profile of concentration changes. In this study, we produced a mouse monoclonal antibody (anti-DH[N] mAb) against the N-terminal region of DH and developed a sandwich enzyme-linked immunosorbent assay using the anti-DH[N] mAb and a rabbit polyclonal antibody against the C-terminus of DH. This procedure enabled us to specifically quantify the DH molecule at femtomolar levels (equivalent to 1/10 of SG). We then plotted DH levels in eggs and SGs during embryonic and post-embryonic development. DH was present in late-stage embryos that had been destined for the production of both diapause and nondiapause eggs. DH levels in SG gradually increased in both types during larval development and peaked at the early pupal stage. At the middle pupal stage, DH levels in SG and SG-brain complex decreased markedly in the diapause-egg producing type, thus indicating active release of DH into the hemolymph. From 5th instar larva to adult, no sexual differences in DH levels were observed in SGs or SG-brain complexes from diapause and nondiapause egg-producing types.
5. Functional analysis of four Gloverin-like genes in the silkworm, Bombyx mori. Kawaoka S, Katsuma S, Daimon T, Isono R, Omuro N, Mita K, Shimada T.
Arch Insect Biochem Physiol. 2007 Dec 12; [Epub ahead of print]
To identify genes involved in the innate immunity of the silkworm Bombyx mori, we constructed a cDNA library from the fat body of Escherichia coli-challenged B. mori larvae. Based on the expressed sequence tag (EST) data and whole genome shotgun sequence analysis, we found four Gloverin-like genes, BmGlov1-4, in the Bombyx genome. Northern blot and RT-PCR analysis showed that BmGlov1-4 were induced in the larval fat body after an immune challenge by the injection of E. coli; however, less induction was observed after the injection of a yeast Candida albicans. In silico sequence analysis revealed the presence of a motif homologous to NF-kappaB binding site in the upstream region of each BmGlov gene. Moreover, we expressed recombinant BmGlov1-4 proteins using the baculovirus expression system, and found that all the recombinant BmGlov1-4 significantly inhibited the growth of E. coli. Arch. Insect Biochem. Physiol. 2007. (c) 2007 Wiley-Liss, Inc.
6. Solubilization of the Ecdysone Binding Protein from Anterior Silk Gland Cell Membranes of the Silkworm, Bombyx mori.
M Elmogy, M Iwami, and S Sakurai
Zoolog Sci. 2007; 24: 971
We previously provided preliminary evidence for the presence of a putative membrane ecdysone receptor (mEcR) anchored in the plasma membranes of anterior silk glands (ASGs) in Bombyx mori. This receptor may act in concert with the conventional EcR in 20E-dependent programmed cell death of these glands. We report here, for the first time, the solubilization of mEcR from ASG membranes using the zwitterionic detergent CHAPS in the presence of NaCl. Our results show by ligand binding assay that mEcR solubilized this way is functionally active and retains 75% of its native binding activity. We also defined experimental conditions that yielded protein/detergent complexes with partial binding activity, which makes it possible to purify the membrane-bound ecdysone binding protein.
7. A silkworm baculovirus model for assessing the therapeutic effects of antiviral compounds: characterization and application to the isolation of antivirals from traditional medicines.
Y Orihara, H Hamamoto, H Kasuga, T Shimada, Y Kawaguchi, and K Sekimizu
J Gen Virol. 2008; 89: 188
Ganciclovir, foscarnet, vidarabine and ribavirin, which are used to treat viral infections in humans, inhibited the proliferation of a baculovirus (Bombyx mori nucleopolyhedrovirus) in BmN4 cells, a cultured silkworm cell line. These antiviral agents inhibited the proliferation of baculovirus in silkworm body fluid and had therapeutic effects. Using the silkworm infection model, the antiviral activity of Kampo medicines was screened and it was found that cinnamon bark, a component of the traditional Japanese medicine Mao-to, had a therapeutic effect. Based on the therapeutic activity, the antiviral substance was purified. Nuclear magnetic resonance analysis of the purified fraction revealed that the antiviral activity was due to cinnzeylanine, which has previously been isolated from Cinnamomum zeylanicum. Cinnzeylanine inhibits the proliferation of herpes simplex virus type 1 in Vero cells. These results suggest that the silkworm-baculovirus infection model is useful for screening antiviral agents that are effective for treating humans infected with DNA viruses.
8. Antidiabetic Properties of 2,5-Dihydroxy-4,3'-Di(beta-D-Glucopyranosyloxy)-trans-Stilbene from Mulberry (Morus bombycis Koidzumi) Root in Streptozotocin-Induced Diabetic Rats.
Heo SI, Jin YS, Jung MJ, Wang MH.
J Med Food. 2007 Dec;10(4):602-7.
We investigated the antidiabetic properties of 2,5-dihydroxy-4,3-di(beta-D-glucopyranosyloxy)-trans-stilbene (DGTS) isolated from Morus bombycis Koidzumi in streptozotocin (STZ)-induced diabetic rats. The DGTS prevented the increase in aspartate aminotransferase, alanine aminotransferase, and blood urea nitrogen levels in serum of diabetic rats. At doses of 200-800 mg/kg, DGTS improved hyperglycemia in the rats, and the hypoglycemic effect of DGTS was comparable to that of tolbutamide. The histological observations showed that DGTS prevented atrophy of pancreatic beta-cells and vascular degenerative changes in the islets. DGTS reversed STZ-induced diabetes and had antioxidant activity in assays of FeCl(2)/ascorbic acid-induced lipid peroxidation in the rats. Levels of cytochrome P450 2E1 mRNA, as measured by reverse transcription-polymerase chain reaction, were lower in the livers of the DGTS-treated rats than those of the control group. These results suggest that DGTS might be beneficial in the treatment of type 1 diabetes
9. Structural Disorder in Silk Proteins Reveals the Emergence of Elastomericity. Dicko C, Porter D, Bond J, Kenney JM, Vollrath F.
Biomacromolecules. 2007 Dec 14; [Epub ahead of print]
Spider silks combine basic amino acids into strong and versatile fibers where the quality of the elastomer is attributed to the interaction of highly adapted protein motifs with a complex spinning process. The evaluation, however, of the interaction has remained elusive. Here, we present a novel analysis to study silk formation by examining the secondary structures of silk proteins in solution. Using the seven different silks of Nephila edulis as a benchmark system, we define a structural disorder parameter (the folding index, gamma). We found that gamma is highly correlated with the ratio of glycine present. Testing the correlation between glycine content and the folding index (gamma) against a selected range of silks, we find quantitatively that, in order to achieve specialization with changes in mechanical performance, the spider's silks require higher structural flexibility at the expense of reduced stability and consequently an increased conversion-energy cost. Taken together, our biophysical and evolutionary findings reveal that silk elastomericity evolved in tandem with specializations in the process of silk spinning.
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