To evaluate the N-glycosylation property of an insect cell line, NISES-AnPe-428 (AnPe), which was derived from embryos of the Chinese oak silkworm, Antheraea pernyi, and used in a newly developed baculovirus expression vector (BEV) system, N-glycans of purified prothoracicotropic hormone (PTTH) produced by AnPe cells infected with a recombinant A. pernyi nucleopolyhedrovirus (AnpeNPV), were digested with glycoamidase A (from sweet almond), reductively aminated with 2-aminopyridine, and identified by two-dimensional high performance liquid chromatography (2D-HPLC) mapping technique. For comparison, N-glycans of PTTH produced by Spodoptera frugiperda cells (Sf9) infected with a recombinant Autographa californica NPV (AcNPV) were also analyzed similarly. Consequently, similarities and differences in the N-glycan structure composition between AnPe and Sf9 were revealed. N-glycans from AnPe contained 41.8% high mannose type, 32.8% paucimannosidic type, 12.5% monoantennary type, and 1.3% biantennary, complex type with terminal N-acetylglucosamine (GlcNAc) residues, while those from Sf9 contained 46.5% high mannose type, 29.8% paucimannosidic type, 7.7% monoantennary type, and no detectable biantennary complex type. In addition, one of the monoantennary type N-glycans with a terminal GlcNAc residue identified in AnPe was not detected in Sf9. The proportion of N-glycans with an α1, 6-linked fucose residue at the innermost core GlcNAc residue was 41.4% in AnPe and 23.4% in Sf9, respectively. In both cell lines, mammalian-like complex N-glycans with terminal galactose and/or sialic acid residues were not identified. These results indicated that a reaction catalyzed by GlcNAc transferase II (GnTII), a key enzyme to form biantennary, complex N-glycans, occurs more efficiently in AnPe than in Sf9, suggesting that the proportion of complex N-glycans in AnPe cells may be largely enhanced by inhibiting the N-acetylglucosaminidase activity and/or supplying sufficient amounts of enzymes and substrates required for the sugar chain extension in the Golgi.

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The Baculovirus immediate-early gene ie1 product IE1 is a viral regulatory protein of nucleopolyhedrovirus (NPV) and acts as an essential transactivator in viral replication. In this study, IE1-transactivation of promoters in mammalian cells was examined using the Bombyx mori (Bm) NPV ie1 (Bie1), BmNPV p35 (Bp35), Drosophila melanogaster heat-shock protein 70 (Dhsp), Drosophila melanogaster elongation factor 1 alpha (Delf) and SV40 early (SV40e) promoters. It was demonstrated that BmNPV IE1 could transactivate the Bie1 promoter in BHK cells approximately 2-fold as well as in insect cells. The deletion mutant analysis revealed that only the core promoter region (from the TATA-box to CAGT initiation motif) of Bie1 was required for hrs-independent transactivation by IE1 in BHK cells. Furthermore, IE1-transactivation of core promoters both in BHK cells and in Vero cells was demonstrated for the Dhsp, Delf and SV40e promoters. These results suggest that baculovirus IE1 maintains the potential for transactivation of the core promoter in mammalian cells although flanking sequences may prevent it.

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The purpose of this study was to determine whether the regeneration of hemopoietic organ occurs in the silkworm, Bombyx mori, after locally targeted irradiation with heavy ion beams. Hemopoietic organs of 4th and 5th instar larvae were selectively irradiated with carbon ions (¹²C⁵⁺, 220 MeV, 18.3 MeV/u, 100, 250, 500 Gy), and morphological changes of these organs in the 5th instar were studied using light and electron microscopy. Carbon ions were considered to pass through the entire hemopoietic organ because the beam depth (range in water = 1.2 mm) was more than enough to reach the organ. Light microscopic observations suggested a possibility that the irradiated hemopoietic organs regenerate at a high frequency. The organs seemed to be able to regenerate even after irradiated at 500 Gy when a JH analog, methoprene, was applied to prolong the larval life.
Electron microscopic observations showed that the irradiation of carbon ions (100 Gy) caused serious damage to the hemopoietic organs in the early 5th instar. They lost acellular sheaths surrounding the organs and islets, and characteristic features of cell disruption and melt necrosis were observed. However, the cells of the hemopoietic organs appeared to be proliferating again in the late 5th instar: the organs were again surrounded by acellular sheaths, and were composed of spherical islets packed with many cells (both immature and mature hemocytes). These results indicate that the hemopoietic organs regenerate after irradiation with heavy ions at a high dose of more than 100 Gy.

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Hemocyte density in the hemolymph changes drastically during larval development of the silkworm, Bombyx mori. Hemocyte density increases abruptly at the wandering stage and subsequently decreases during the spinning and prepupal stages. This pattern is also observed in larvae whose hemopoietic organs (HPOs) had been selectively subjected to heavy-ion irradiation, and in larvae whose entire HPOs had been extirpated surgically at the pre-molting stage during the 4th instar. These results and the relatively small number of hemocytes present in HPOs during the final 5th instar indicate that the HPOs do not play an important role in increasing the number of hemocytes in circulation, at least during the wandering stage. Application of juvenile hormone analog (JHA) called methoprene, during the early 5th instar, inhibited the sharp increase of the hemocyte density, while injecting 5th instar day 2 larvae with a low dose of 20-OH ecdysone brought about a slight but significant increase in hemocyte density. Tracking bromodeoxyuridine (BrdU) incorporated hemocytes reveals that the percentage of labeled hemocytes was the highest (26%) during the mid-instar (day 3) in the control larvae, but highest during the wandering stage in both HPOs-extirpated larvae (40%) and heavy-ion-irradiated larvae (37%). These results suggest that juvenile hormone and ecdysone are involved in the change of hemocyte density, and that mitosis of circulating hemocytes plays an essential role for elevation of hemocyte density during the wandering stage.

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The formation of protein yolk spheres during oogenesis in the egg character mutation, vit, of Bombyx mori, was studied by light microscopy and transmission electron microscopy of sections of the resin-embedded follicles. The vit ovary exhibited the normal polytrophic meroistic form, in which the follicle has an oocyte, nurse cells and follicular epithelial cells. Also the accumulations of lipid droplets, glycogen granules and small yolk spheres took place normally in the vit oocytes during oogenesis. However, abnormality of the mutant appeared because large protein yolk spheres did not form. Instead, many small spheres were accumulated, which were not observed in the normal. Since the vit egg has been reported to have the egg specific protein but lack the two major yolk proteins, vitellin and the 30 kDa proteins, we infer that the vit mutation affects the incorporation of the precursors for these proteins from hemolymph to oocyte.

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Ultrastructure of sensilla on the maxilla of the silkworm, Bombyx mori L. was observed by light microscopy and by both scanning and transmission electron microscopy. Structural differences among different silkworm strains, if any, were also examined. Cuticular specializations reveal presence of eight sensilla on the maxillary galea and eight basiconic sensilla on the apical tip of the maxillary palpus. The two galeal styloconic sensilla are innervated by five neurons with one modified as a structure like a tubular body. Cuticular specializations of conical pegs of the galeal styloconic sensillum and the number of basiconic sensilla on the apical tip of the maxillary palpus vary among different strains and individuals. These structural differences are attributed to strain differences and not to feeding habits of strains.

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When mulberry leaves were irradiated with UV for various durations, the prolonged UV exposure significantly increased the level of proline as compared to the control plants. Also, the lipid peroxidation increased in the UV stressed plants. Moreover, the level of proline in treated leaf tissues increased within 5 h of incubation and reached a maximum level in 25 h. Conversely, mulberry moracins began accumulating after 20 h of UV stress. The transition in the lipid peroxidation began to decrease when moracins began to accumulate, suggesting that moracins have a protective role in UV stressed plants. The results obtained in this study imply that both proline and mulberry moracins are free radical quenchers. However, moracins were ∼1000 times more effective than proline in scavenging activity, suggesting that proline may be a primary protectant, and moracins, which accumulate at a later stage under UV-stress, act as a secondary protectant.

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