Contributions to Silkworm Biochemistry V. — Time of Biosynthesis of Fibroin and its Location in the Silk ofBombyx MoriL. (European Race)
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BmSuc1, a novel animal-type β-fructofuranosidase (β-FFase, EC 3.2.1.26) encoding gene, was cloned and identified for the first time in the silkworm, Bombyx mori. BmSuc1 was specifically and highly expressed in the midgut and silk gland of Bombyx mori. Until now, the function of BmSuc1 in the silk gland was unclear. In this study, it was found that the expression changes of BmSuc1 in the fifth instar silk gland were consistent with the growth rate of the silk gland. Next, with the aid of the CRISPR/Cas9 system, the BmSuc1 locus was genetically mutated, and homozygous mutant silkworm strains with truncated β-FFase (BmSUC1) proteins were established. BmSuc1 mutant larvae exhibited stunted growth and decreased body weight. Interestingly, the molecular weight of part of Sericin1 (Ser1) in the silk gland of the mutant silkworms was reduced. The knockout of BmSuc1 reduced the sericin content in the silkworm cocoon shell, and the mechanical properties of the mutant line silk fibers were also negatively affected. These results reveal that BmSUC1 is involved in the synthesis of Ser1 protein in silk glands and helps to maintain the homeostasis of silk protein content in silk fibers and the mechanical properties of silk fibers, laying a foundation for the study of BmSUC1 regulation of silk protein synthesis in silk glands.
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Silk is a functional protein biomaterial produced by a variety of insects like flies, silkworms, scorpions, spiders, and mites. Silk synthesized by silkworms is extensively studied for its applications in tissue engineering and wound healing. Silk is undoubtedly a natural biocompatible material with humans and has its role in medical treatments from ancient times. The silk worm protein comprises two types of proteins namely fibroin and sericin. Silk fibroin makes up approximately 70% of cocoon weight and has wide applications in textiles and in all biomedical applications owing to its biocompatible, nontoxic, biodegradable, less immunogenic, and noncarcinogenic nature. It possesses outstanding toughness and mechanical strength, while silk sericin possesses high defensive ability against ultraviolet light and oxidation. Silk fibroin has been known to induce wound healing by increasing cell proliferation and growth and migrating various types of cells which are involved in different stages of wound healing process. With several silk varieties like silk worm fibroin, silk sericin, recombinant silk materials, and native spider silk have been investigated for its wound healing applications over the last several decades. With an objective of harnessing the silk regenerative properties, plentiful strategies have been studied and applied to develop bioartificial skin grafts and bioactive wound dressings in recent times. This review gives a detailed insight into the structure, general properties, fibroin structure-properties relationship, and biomedical applications of silk fibroin.
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The mRNA that codes for silk fibroin in the silkworm Bombyx mori can be isolated in greater than 90% purity as judged by partial sequence analysis (Suzuki and Brown, 1972). Molecular hybridization experiments using highly purified fibroin mRNA have established that there are one to three genes per haploid complement of DNA in all tissues investigated, including the posterior silk gland where fibroin is synthesized (Suzuki et al., 1972).
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Abstract This study focuses on the conformational characterization of differently processed Bombyx mori silk fibroin samples by Raman spectroscopy. The Raman spectra of silk fibroin film and liquid silk are discussed in comparison with those of the crystalline fractions of Bombyx mori silk fibroin (Cp, chymotryptic precipitate) with Silk I (Silk I‐Cp) and Silk II (Silk II‐Cp) structures. The complete 1800–200 cm −1 Raman spectrum of Silk I‐Cp is reported for the first time. The amide I and amide III modes were found to be scarcely suitable for the spectroscopic characterization of silk fibroin in the Silk I form in the presence of a random coil conformation. Raman marker bands for the Silk I form were identified in other spectral ranges at about 1415, 950, 930, 865, 260 and 230 cm −1 . On the basis of the above findings, the comparison of the Raman spectra of film, liquid silk and Silk I‐Cp in the range 1000–800 cm −1 clearly indicates that in addition to random coil, both film and liquid silk contain local domains of Silk I structure; their amount is higher in liquid silk, as indicated by the relative intensity of the bands at about 950, 930 and 865 cm −1 and by the I 1415 / I 1455 intensity ratio. The assignments of the bands at about 1275 and 1107 cm −1 are also discussed. These bands were previously assigned to the presence of α‐helical conformation in Bombyx mori silk but, from the results reported, they should rather be attributed to the Silk I form. Copyright © 2001 John Wiley & Sons, Ltd.
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Recently, much interest has been paid to the separation of silk produced by Bombyx mori from silk produced by other species and tracing the beginnings of silk cultivation from wild silk exploitation. In this paper, significant differences between silks from Bombyx mori and other species were found by microscopy and spectroscopy, such as morphology, secondary structure, and amino acid composition. For further accurate identification, a diagnostic antibody was designed by comparing the peptide sequences of silks produced by Bombyx mori and other species. The results of the noncompetitive indirect enzyme-linked immunosorbent assay (ELISA) indicated that the antibody that showed good sensitivity and high specificity can definitely discern silk produced by Bombyx mori from silk produced by wild species. Thus, the antibody-based immunoassay has the potential to be a powerful tool for tracing the beginnings of silk cultivation. In addition, combining the sensitive, specific, and convenient ELISA technology with other conventional methods can provide more in-depth and accurate information for species identification.
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The study aimed to investigate and compare some characteristics of Bombyx mori and Eri (Philosamia ricini) silks in different forms; with and without sericin. The protein contents were measured and find out the composition of the silk fibroin and sericin proteins by Lowry method. The secondary structure and thermal behavior of all kind of silk were determined by FT-IR and TA instrument, respectively. The B. mori composed of more amount of sericin content than that of Eri silk. FT-IR spectra indicated that the Eri silk was similar profile of silk with and without sericin, whereas B. mori silk showed dramatically differed. With sericin, B. mori composed of higher ratio random coil and alpha-helix structures than beta-structure. With thermogravimetric analysis, both B. mori and Eri silk fibers without sericin showed higher stability than that silk fiber with sericin. This is due to the crystalline region of hydrophobic amino acid composed in the fibroin core protein. The differential scanning calorimetry thermogram of B. mori was differed from Eri silk fiber. It is a promising that characteristics of the silk were influenced by both silk components and silk varieties.
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Event Abstract Back to Event Silk protein as a cell additive You-Young Jo1, Haeyong Kweon1 and Kwang-Gill Lee1 1 National Academy of Agricultureal Science, Department of Agricultural BIology, Korea Introduction: A substance for the cell proliferation, additives such as cell cytokine is a very expensive. It is known that silk protein supports effectively cell proliferation[1]-[4]. Although there are many varieties of Bombyx mori silkworm, the effect of silkworm varieties on cell proliferation has not been considered in detail. To develop the cell additives using silk proteins, we studied that effects on cell proliferation and characteristics of silk obtained from Korea various silkworm varieties. Materials and Methods: Silk sericin was prepared under high temperature high pressure condition. Silk fibroin was prepared using CaCl2 : H2O : ethanol (1:8:2) with different dissolution time(1, 3, 5 hour). The characteristics of each silk protein were analyzed by performing Tensile strength, amino acid composition, porosity, XRD and gel electrophoresis. We investigated the attachment, proliferation, morphology of the cells and the expression levels of genes related to cell growth. Results: There are differences in silk cocoon from different silkworm varieties. The porosity of cocoons was 73~85%. The crystal structure of each cocoon was different by XRD analysis[5]. Also amino acid composition was different. But the molecular weight of silk proteins was same. The proliferation was accelerated in the presence all of silk sericin and silk fibroin. Especially, this was the best Baekokjam silk sericin in cell proliferation. The Baekokjam silk serin was upregulated the EGF gene expression up to 5 times. Conclusion: We expect that silk proteins could be a preferable culture medium supplement for stimulating the proliferation of cell. Then, these results suggest silk as a new material for medium supplement. This study was carried out with the support of "Research Program for Agricultural Science & Technology Development (Project No. PJ010006)", National Academy of Agricultural Science, Rural Development Administration, Republic of KoreaReferences:[1] Aramwit P, Kanokpanont S, De-Eknamkul W, Kamei K, Srichana T (2009) The effect of sericin with variable amino-acid content from different silk strains on the production of collagen and nitric oxide. Journal of Biomaterials Science-Polymer Edition 20 1295~1306.[2] Bisceglie V (1933) Uber die antineoplasticsche immunitat: heterologe Einplflanzung von tumoren in Huhner-embryonen. Ztschr. Krebsforsch 40 122~140.[3] Cha HM, Kim SM, Choi YS (2015) Serum-free media supplement from silkworm gland for the expansion of mesenchymal stem cells. Tissue Engineering and Regenerative Medicine 12, 53~59.[4] Chen F, David P, Vollrath F (2012) Morphology and structure of silkworm cocoons. Materials Science and Engineering C 32, 772~778.[5] Um IC, Kwon HY, Lee KG, Park YH (2003) The role of formic acid in solution stability and crystallization of silk protein polymer. International Journal of Biological Macromolecules 33, 203~213. Keywords: Cell Proliferation, Gene Expression, Biocompatibility Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Cellular migration and biomaterials Citation: Jo Y, Kweon H and Lee K (2016). Silk protein as a cell additive. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.00401 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers You-Young Jo Haeyong Kweon Kwang-Gill Lee Google You-Young Jo Haeyong Kweon Kwang-Gill Lee Google Scholar You-Young Jo Haeyong Kweon Kwang-Gill Lee PubMed You-Young Jo Haeyong Kweon Kwang-Gill Lee Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.
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