The Bombyx mori homolog of the Broad-Complex gene (BmBR-C) is transcribed from two different promoters separated by 101kbp. In our previous report where we used semi-quantitative RT-PCR to amplify the total RNA prepared from tissues at different stages, the distal (Pdist) and the proximal (Pprox) promoters appeared to respond differentially to ecdysone signals. To explore the expression control mechanism of BmBR-C in response to ecdysone, transfection assays using Bombyx BM-N cells were performed with luciferase reporter genes under the control of various Pdist or Pprox fragments. In these experiments, the promoter regions located between 3 and 5kbp upstream from the proximal transcription start site (TSS), and between 2 and 5kbp upstream from the distal TSS, appeared to function in the down-regulation of Pprox and the up-regulation of Pdist, respectively, in response to 20-hydroxyecdysone (20E). In electrophoretic mobility shift assays (EMSAs), proteins bound to the probe containing the candidate ecdysone-responsive element (cEcRE), located 3 kbp upstream from the distal TSS, were detected predominantly in the nuclear extract prepared from 20E-treated cells; however oligonucleotides containing the typical EcRE (that of the hsp27 or fbp1 genes of Drosophila) could not compete with probe for binding to these proteins. Furthermore, the level of responsiveness of Pdist to 20E declined, but did not disappear, after deletion of the cEcRE. These results suggest that the functional ecdysone receptor (EcR) may bind to elements other than the cEcRE, or that transcription from Pdist is activated by an ecdysone-responsive factor, such as βFTZ-F1, rather than a functional EcR.

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To analyze the spinning behavior of flossy cocoon mutants in the silkworm, Bombyx mori, the spinning characteristics of two mutant larvae: WO7+, which produced spherical-shaped flossy cocoons, and WO7q constructed peanut-shaped tight cocoons were recorded on videotapes from two different angles and analyzed for spinning behavior by the three dimensional computer graphics software, 3DASBS. WO7+ silkworms constructed an appropriate shape of cocoon very rapidly. On the other hand, WO7q silkworms produced cocoon very slowly and needed more time to form a full cocoon shape than WO7+. Both silkworms showed different spinning rates at different spinning stages, although the speed of the WO7q silkworm was comparatively slower in the later spinning stage. The silkworm which constructed a flossy cocoon spun primarily in a C-letter shape changed its direction frequently. On the other hand, the larva of peanut-shaped cocoons often assumed S-letter shapes and displayed a desire to change at a considerably lower frequency. Since the silkworms w07+ and w07q are segregated in the strain w07 which has been maintained for more than 25 years using the same sib mating, different characteristics shown in this experiment should be considered for genetical reasons.

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A microarray-based gene expression analysis was carried out to identify Trypanosoma-inducible genes in the silkworm Bombyx mori. A Bombyx cysteine proteinase inhibitor (BCPI) gene was upregulated following Trypanosoma brucei brucei GUTat3.1 injection. The BCPI was shown to encode 105 amino acids and putative signal peptide sequences were found. The expression profiles of parasite-inducible transcripts were confirmed by real-time quantitative polymerase chain reaction. BCPI is expressed specifically in the fat body and hemocytes following T. b. brucei challenge. The expression level of BCPI increased significantly 3 h after the parasite injection. A homology search showed that BCPI contains a region homologous to the propeptide sequence of cysteine proteinase from protozoan parasite species including T. b. brucei and T. congolense. BCPI possesses the conserved motif (Gly-X-Asn-X-Phe-X-Asp) and these residues might be capable of inhibiting the corresponding enzymes. Therefore, BCPI may play an important role in defense mechanisms against invading protozoan parasites. These results suggest that BCPI would be a good candidate for use in development of peptidyl inhibitors against cysteine proteinases in trypanosomal species.

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The spatial and temporal characteristics of cell divisions in the dorsal epidermis of the 5th segment of the silkworm, Bombyx mori, were investigated from the 3rd instar to the wandering stage. Whole-mount immunohistochemistry using an antibody specific for M phase nuclei was used to examine the mitotic characteristics, and changes in cell size and ploidy level were also examined. During the 3rd and 4th instar stages, mitoses occurred during mid-feeding stage, but not in the early and late feeding stages and molting stage. By contrast, in the final 5th instar mitoses occurred during the period from the late feeding stage to the wandering stage. The spatial characteristics of mitotic cell division were also stage specific. Mitoses occurred in the whole segment in the 3rd instar, whereas in the 4th and 5th instars mitotic figures were observed primarily in the anterior half of the segment, with few in the posterior half. Cell size and ploidy level appeared to correlate with cell division, resulting in a clear boundary along the transverse middle line of the larval segment. These results suggest that there are three types of mitotic classes in the epidermis during larval development in the silkworm, namely early instar-specific, penultimate instar-specific and final instar-specific.

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The knobbed mutant of the silkworm, Bombyx mori, is a dermal mutant that is characterized as having paired knobs at specific regions of larval segments. In order to clarify the details of cell proliferation which induce knob formation, we tried to detect mitotic cells of larval epidermis by whole mount immunohistochemistry using an antibody specific for M-phase nuclei. The epidermal mitotic activities were compared between the knobbed and the surrounding unknobbed regions during the 4th larval instar. A comparison was also performed in the corresponding epidermal regions between the mutant and normal strains. Mitosis in epidermis of the unknobbed region was mainly observed to occur at 48h (mid feeding stage) after ecdysis. Conversely, numerous mitotic figures were observed in the knob region at 24 and 48h after ecdysis. Clear mitotic figures were also observed at the stage just preceding the molting period (72h). Z-axis observations of propidium iodide stained epidermis using a confocal laser-scanning microscope indicated that the cell nuclei of knob regions were considerably longer and more slender than those of the unknobbed epidermis during the early and middle 4th instar stages. These results suggest that mitosis occurs at the knob regions for most of the intermolt/feeding stage, and that the prolonged cell proliferation observed in the epidermal cells results in an increase of cell number, causing conspicuous outgrowths of integument which takes the form of swollen knobs.

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Accumulating evidence suggests that amyloid β-peptide (1-42) plays an important role in the etiology of Alzheimer’s disease. In a previous study, we reported that mulberry leaf extract inhibits the amyloid β-peptide (1-42) fibril formation and protects hippocampal neurons against amyloid β-peptide (1-42)-induced cell death. In the present study, we compared the amyloid β (1-42) fibril-destabilizing activities of leaf extracts from 258 varieties of the mulberry. Our results showed that for proper utilization, it is necessary to screen mulberry leaves for high Aβ42 fibril-destabilizing activity.

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