Toshiharu Furusawa

Limited degradation of vitellin and egg-specific protein in Bombyx eggs during embryogenesis

Abstract The metabolic fate of yolk proteins was followed by examining changes in subunit structure and immunochemical properties during embryogenesis in Bombyx mori . SDS-polyacrylamide gel electrophoresis of extracts from newly laid eggs showed eight major polypeptides that corresponded to the subunits of three yolk proteins, vitellin (Vtn), egg-specific protein (ESP) and the 30 kDa proteins. During embryogenesis, the peptides corresponding to Vtn and ESP decreased, and four new peptides (96, 55, 46 and 36 kDa) appeared at specific stages, but they disappeared suddenly before larval hatching. Vtn and ESP peptides stained with dyes specific for lipids (Sudan black), for sugars (Schiffs PAS reagent) and for phosphate (methyl green), but the newly appearing peptides stained only with Schiffs PAS reagent. Using a specific antiserum bound-protein A affinity column, two of the new peptides (55 and 36 kDa) were shown to be derived from ESP, and the other two peptides (96 and 46 kDa) from Vtn, suggesting that limited proteolysis of ESP and Vtn occurs during embryogenesis. In contrast, the 30 kDa proteins remained almost unchanged throughout the egg stage.

Double-stranded ribonuclease activity in the digestive juice and midgut of the silkworm, Bombyx mori

Abstract 1. 1. Nuclease activity capable of degrading Bombyx CPV double-stranded RNA (CPV-dsRNA) was detected in the midgut homogenate and digestive juice of the silkworm, Bombyx mori by using 3.5% polyacrylamide gel electrophoresis (PAGE). 2. 2. The optimal pH range for the reaction was pH 7–10, under which yeast single-stranded RNA was not digested. The breakdown products in the reaction at pH 7 showed a pattern of three distinct bands besides CPV-dsRNA fragments in the PAGE, which indicates specific cleavage of CPV-dsRNA. 3. 3. The products at pH 9 were heterodispersed, giving a continuous smear indicative of random degradation. 4. 4. The activity was elevated by the addition of Mg 2+ , Mn 2+ , Ca 2+ , Na + , NH 4 + or K + to the reaction mixture, but inhibited by EDTA. 5. 5. The 0–40% ammonium sulfate saturated fraction of the 1200 g supernatant of midgut homogenate and digestive juice contained nuclease activity which caused degradation of double-stranded, poly(I) • poly(C) and CPV-dsRNA at pH 9.0.

De novo gene expression and antisense inhibition in cultured cells of BmTRN-1, cloned from the midgut of the silkworm, Bombyx mori, which is homologous with mammalian TIA-1/R

A cDNA encoding a 388 amino acid TIA-1-type RNA-binding protein (BmTRN-1) was isolated from midgut cDNAs of the silkworm, Bombyx mori, via homologous cloning, in order to characterize its function. The deduced amino acid sequence, most likely encoded by a single copy gene, has significant homology with human TIA-1 and TIAR known as apoptotic regulators and recently reported to function as important factors for either splicing or translation. RT-PCR analysis showed that the BmTRN-1 gene was vigorously transcribed in the midgut at the gut purge stage, indicating a possible relation to the tissue-decomposing process in larval-pupal metamorphosis. We also show that inhibition of the expression of BmTRN-1 by a transfected oligonucleotide encoding the antisense sequence caused a remarkable rise in protein expression from artificially constructed cDNAs encoded by plasmid vectors in Bombyx cells, depending on the constructed ORF sequences of the introduced cDNAs. Furthermore, it was shown that the transcripts from the cDNAs introduced into the cells increased under the antisense-inhibition of BmTRN-1 when the protein levels of these cDNAs also rose, demonstrating that BmTRN-1 could act as a regulator especially of the mechanism eliminating transcripts with possible targets for BmTRN-1 recognition in the authentic post-transcription process.

Expression of freeze-responsive proteins, Fr10 and Li16, from freeze-tolerant frogs enhances freezing survival of BmN insect cells

To date, two novel freeze‐responsive proteins, Fr10 and Li16, have been discovered in the wood frog, Rana sylvatica, and likely support freezing survival. Although previous studies have established tissue distribution of each protein, there have been no studies that explore their functional consequences in intolerant cells. To assess the ability of Fr10 and Li16 to confer freeze tolerance, we transfected each protein into a freeze‐intolerant silkworm cell line (BmN). Selected controls were the transfection of an unrelated protein (CAT) and a no‐transfection sample. Li16 and Fr10 showed 1.8 ± 0.1‐ and 1.7 ± 0.2‐fold, respectively, greater survival after freezing at –6°C for 1 h than did transfection controls. To investigate how these novel proteins protect cells from freezing damage, protein structures were predicted from primary amino acid sequences. Analysis of the structures indicated that Fr10 is a secreted protein and may be a new type IV antifreeze protein, whereas Li16 may have intracellular membrane associated functions. This study shows that freezing protection can be provided to intolerant cells by the overexpression of transfected Li16 and Fr10 frog proteins. Results from this study will provide new insights into adapting intolerant cells for medical organ cryoprotection using a natural vertebrate model of tolerance.—Biggar, K. K., Kotani, E., Furusawa, T., Storey, K. B., Expression of freeze‐responsive proteins, Fr10 and Li16, from freeze‐tolerant frogs enhances freezing survival of BmN insect cells. FASEB J. 27, 3376–3383 (2013). www.fasebj.org

Stage-dependent limited degradation of vitellin and its localization in the Japanese oak silkworm, Anthereae yamamai, during embryonic development

Abstract 1. 1. Analysis of the content and pre-larval protein in Antheraea yamamai egg by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting showed stage-dependent degradation of vitellin; on day 8–9 when pre-larva of A. yamamai appeared to enter diapause, a heavy subunit (180 kDa) of vitellin was cleaved into five polypeptides in the 130 and 180 kDa region of the gel. 2. 2. The degraded vitellin polypeptides were detected in the contents of the alimentary canal, and then maintained during diapause. After diapause termination, these polypeptides of vitellin were degraded on day 4 from the beginning of resumed embryonic development. 3. 3. Concomitantly with the vitellin degradation at the two limited stages, a 34 kDa protease band appeared at pH 9 in gel by casein SDS-PAGE. 4. 4. In the hemolymph of pre-larva, two subunits (180 and 40 kDa) of vitellin were conserved and also protease activity could not be detected throughout embryonic development. 5. 5. Therefore, there appear to be differences in the degradation and utilization of vitellin between hemolymph and digestive juice of A yamamai pre-larvae.