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Development stage-specific expression of fibroin in the silk worm Bombyx mori is regulated translationally.

The contents of fibroin H RNA as a function of development have been quantitated in the posterior silk glands of Bombyx mori larvae on different days of 4th and 5th instars. The fibroin RNA levels increased during the feeding stages of larvae and the RNA got completely degraded during the interim moult. The patterns of accumulation of fibroin RNA were similar in both the instars. Although there was considerable increase in the fibroin RNA content during the 5th larval instar, the relative abundance of fibroin RNA in the total RNA was fairly constant during the 4th and 5th instars. The increased content of fibroin RNA in 5th instar was the consequence of an overall increase in transcription accompanying the development progress, rather than specific increase only in fibroin transcription. The contents of fibroin protein in the 4th and 5th instars of development have also been quantitated making use of a sensitive radioimmune assay with a purified, antifibroin antibody. There were substantial differences between 4th and 5th instars in the absolute fibroin contents as well as the relative proportion of fibroin in the total proteins. These results implied that although the fibroin gene was transcribed at the same efficiency during the 4th and 5th instars, the translational efficiency was much lower during the 4th instar. The extent of polyadenylation of fibroin RNA was similar in both instars. However, there was a two-fold increase in the polysome association of fibroin RNA in the 5th instar. Over and above this, there was substantial increase during the 5th instar in the contents of those tRNAs. (e.g. Gly, Ala and Ser) which are abundantly represented in fibroin and therefore directly related to the expression of fibroin. The increased polysome association of fibroin mRNA and the adequate supply of cognate tRNAs in the 5th instar, together contributes to the translational regulation of fibroin in a developmental stage-specific manner. Based on these observations, we propose that translational regulation plays a major role in the development stage-specific synthesis of fibroin in Bombyx mori.

Animals↗
PubMed2006

Structure and properties of regenerated Antheraea pernyi silk fibroin in aqueous solution.

Antheraea pernyi silk fibroin fibers were dissolved by aqueous lithium thiocyanate to obtain regenerated A. pernyi silk fibroin solution. By means of circular dichroism, (13)C NMR and Raman spectroscopy, the molecular conformation of regenerated A. pernyi silk fibroin in aqueous solution was investigated. The relationship of environmental factors and sol-gel transformation behavior of regenerated A. pernyi silk fibroin was also studied. The molecular conformations of regenerated A. pernyi silk fibroin mainly were alpha-helix and random coil in solution. There also existed a little beta-sheet conformation. It was obviously different with Bombyx mori silk fibroin, whose molecular conformation in solution was only random coil but no alpha-helix existence. With the increase of temperature and solution concentration and with the decrease of solution pH value, the gelation velocity of regenerated A. pernyi silk fibroin solution increased. Especially, it showed that A. pernyi silk fibroin was more sensitive to temperature than B. mori silk fibroin during the sol-gel transformation. The velocity increased obviously when the temperature was above 30 degrees C. During the sol-gel transformation, the molecular conformation of regenerated A. pernyi silk fibroin changed from random coil to beta-sheet structure. The results of these studies provided important insight into the preparation of new biomaterials by silk fibroin protein.

Animals↗
PubMed2001

Differential expression of the fibroin gene in developmental stages of silkworm, Antheraea mylitta (Saturniidae).

Fibroin gene expression during the larval developmental stages of the Saturniid silkworm, Antheraea mylitta, was analyzed. Northern blot analysis of larval silk gland total RNA using the fibroin gene as a probe showed that fibroin is expressed in the intermoult stages and repressed during the moulting stages. Abundance of fibroin transcripts gradually increased from the third to fifth intermoult stage, reaching a peak in the fifth intermoult. Transcripts declined during the early spinning stage. Western blot analysis of fibroin protein production with anti-fibroin antibody confirmed the differential fibroin expression, in accordance with fibroin mRNA synthesis. Dot blot hybridization of genomic DNA isolated from each larval developmental stage with the labelled fibroin gene showed that at the genomic level, the relative concentration of the fibroin gene was constant throughout the developmental stages. Our data confirm that fibroin gene expression in A. mylitta, like in B. mori, is transcriptionally controlled and shows differential temporal variations.

Animals↗
PubMed2005

[Role of titanium mesh coated with silk fibroin in rabbit tracheal reconstruction].

OBJECTIVE: To Compare the curative effects between titanium mesh coated with or without silk fibroin and in the treatment of larger trachea circumferential defects. To explore the merits of titanium mesh coated with silk fibroin in promoting mucous healing and decreasing related complications. METHODS: Twenty-five New Zealand white rabbits were chosen , divided randomly as follows: group one: titanium mesh coated with silk fibroin (n = 12), group two: pure titanium mesh without silk fibroin (n = 12); Titanium mesh coated with silk fibroin and those titanium without silk fibroin were embedded in the anterior of 7-12th tracheal rings respectively, in 2 weeks we removed the anterior and lateral wall of 7-12th tracheal rings about 8 mm x 6mm (more than half ring) and used the two different materials to reconstruct tracheal defects. All these rabbits were euthanized in 4, 8 and 12 weeks and tracheal CT scan, tracheal endoscopy, histopathological examination and scanning electron microscopic examinations were performed. All complications such as death, local inflammation, tracheal stenosis were documented. Group three, leave one normal rabbit as controlled study , removed the anterior and lateral wall of 7-12th tracheal rings, and histopathological examination, scanning electron microscopy were performed as controlled study. RESULTS: The CT scan, tracheal endoscopy of those with silk fibroin showed there was no apparent tracheal stenosis. Histopathological examination showed there was no obvious granulation tissue and scar. The mucous membrane and cilia grow normally. The silk fibroin was not entirely absorbed and the remains showed good biocompatibility. The scanning electron microscopy showed that the cilia grew better and more orderly than that of group two, and in 12 weeks, the cilia became normal. In group two, there were 5 rabbits died in succession from suffocation in 4, 5, 7, 17, 60 days after surgery. All rabbits had local or general subcutaneous emphysema. The CT scan, tracheal endoscopy and pathological examination showed that there were apparent granulation tissue, scar hypertrophy, and tracheal stenosis. The mortality of group two (41.7%) for suffocation compare to that of group one (0), the incidence rate of subcutaneous emphysema in group two (100%) to that of group one (25.0%) and the average cross section stenosis area in group two (18.15 +/- 7.85) mm2 to that of group one (3.15 +/- 1.58) mm2, there are significant statistically differences in these postoperative complications (P <0.05) and stenosis degree (P <0.05) between the two groups. In addition, there was one rabbit died of diarrhea in each group except group three. CONCLUSIONS: Silk fibroin and titanium mesh coated with silk fibroin showed good biocompatibility. Titanium mesh coated with silk fibroin used in tracheal reconstruction of rabbits can promote agglutination of mucous membrane and decrease postoperative complications, such as subcutaneous emphysema, tracheostenosis. It may be a prospective biomaterial which can be used in clinical circumferential trachea defect reconstruction.

Animals↗
PubMed2006

Preparation of 3-D regenerated fibroin scaffolds with freeze drying method and freeze drying/foaming technique.

Although three-dimensional fibroin scaffolds have been prepared with freeze drying method, the porosity and pore sizes still can not satisfy the requirement of tissue engineering. In this article, fibroin porous scaffold with high porosity and > 100 microm diameter interconnected pores was firstly prepared with freeze drying method through adjusting fibroin concentration. The morphology of different scaffolds lyophilized from different fibroin concentration was observed by SEM. A novel freeze drying improved method, freeze drying/foaming technique, was also devised to prepare fibroin scaffolds at different fibroin concentrations. Using the said method, the porosity and pore size of fibroin scaffolds prepared from 12% concentration were 85.8 +/- 4% and 109 +/- 20 microm respectively with yield strength up to 450 +/- 6 KPa while the porosity and pore size of fibroin scaffolds prepared from 8% concentration were 96.9 +/- 3.6% and 120 +/- 30 microm respectively with yield strength up to 30 +/- 1 KPa. The freeze drying/foaming technique produced scaffolds with a useful combination of high yield strength, interconnected pores, and pore sizes greater than 100 microm in diameter. Through adjusting fibroin concentration and thawing time, the porosity, pore sizes and mechanical properties could be controlled to satisfy the different requirements of tissue engineering. The results suggested that fibroin scaffolds prepared with the above methods could be formed for utility in biomaterial application.

Animals↗