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PubMed2005

Charge-selective recognition at fibroin-modified electrodes for analytical application.

A novel fibroin-modified electrode with charge recognition is reported. The characteristics of silk fibroin membranes have been exploited for analytical applications. The membrane, with an isoelectric point of pH 4.5, was applied to graphite and carbon-fiber electrodes. The modified electrode was negatively charged in solutions of pH > 4.5, and so rejected anions and attracted cations. In solutions of pH < 4.5 the electrode was positively charged, and so rejected cations and attracted anions. The pH-responsive charge recognition of the modified electrode was investigated for some neurocompounds. A fibroin carbon-fiber electrode was used for in-vivo determination of the concentration of the cationic neurotransmitter dopamine (DA).

Animals↗

PubMed1986

Conformational study of 13C-enriched fibroin in the solid state, using the cross polarization nuclear magnetic resonance method.

Silk fibroin with the alanyl carboxyl carbon enriched with 13C was obtained by giving a diet containing 13C-enriched alanine to the larvae of Bombyx mori and Antheraea pernyi at the fifth instar. Sericin-free fibroin fibers were prepared from cocoons, and gut was made from the liquid silk in the gland. The final 13C content was about 13%. Cross polarization/magic angle sample spinning spectra at 25 MHz and 75 MHz were measured for each sample at different orientations. Spectra were simulated using the principal values and orientations of the shielding tensor in the alanine crystal. The results indicate that the beta-structure of the fibroin may be a little more flattened than the typical pleated sheet beta-structure.

Animals↗

PubMed1988

Specific codon usage pattern and its implications on the secondary structure of silk fibroin mRNA.

We have identified two distinctive regions of the repetitive unit nucleotide sequence of fibroin mRNA of Bombyx mori. The codon usage for the major amino acids, glycine, alanine and serine is distinctly different in these two regions, indicating that it is determined by the fibroin mRNA or gene structure but not by the tRNA population. Comparative computer analyses of nucleotide substitutions in the unit sequence suggest that selection has operated on the codon usage to optimize the secondary structure characteristic of the fibroin mRNA.

Amino Acid Sequence↗

PubMed1991

Stimulation of fibroin synthesis elicits ultrastructural modifications in spider silk secretory cells.

Electron microscopic studies of unstimulated and stimulated spider fibroin glands show that the fibroin synthesis stimulus evokes visible changes in both the endoplasmic reticulum and Golgi apparatus of their secretory epithelium. Gradual increase in distension of the reticulum accompanies the increase of evoked fibroin synthetic activity. The flattened translucent Golgi vesicles, seen in inactive cells, display a gradual increase in size and number, also with time. The stimulation also elicits a gradual transition in the gland's luminal membrane, during which the microvilli on the lining gradually disappear acquiring an electron dense appearance. Correlations of the observed transitions to the gland's increase in rate of elicited synthetic activity are discussed. The parallelisms between the ultrastructural modifications observed in the spider secretory cells with those described in the silkworm glands during their progression through the fifth instar have been stressed.

Animals↗

PubMed1975

The size of fibroin messenger RNA and its polyadenylic acid content.

The molecular weight of fibroin messenger RNA has been measured by its contour length in the electron microscope and its electrophoretic mobility under conditions of complete denaturation. These methods give values of 5.6 times 10-6 and 6.0 times 10-6 daltons, respectively. The average molecular weight of 5.8 times 10-6 (1.6 times 10-4 base residues) would be expected to code for a protein of 414,000 daltons, a value about 12 percent larger than the most recently published size for fibroin. When the mRNA is prepared carefully to avoid shear breakage, it is as homogeneous in size as ribosomal RNA. Between 43 percent and 81 percent of the mRNA molecules bind to oligo(dT)-cellulose and are presumed to contain a poly(A) sequence. This sequence, isolated after ribonuclease digestion, comprises about 0.6 percent of the molecule, or an estimated 100 nucleotides. The poly(A) has been localized at the end of the molecule by hybridizing with a small poly (dT:dA) piece and subsequent visualization by electron microscopy. This observation, along with the ability of reverse transcriptase to transcribe fibroin mRNA with an oligo(dT) primer, demonstrates that the poly(A) is at the 3' end of the mRNA.

Adenine Nucleotides↗

PubMed1992

Structure of the Bombyx mori fibroin light-chain-encoding gene: upstream sequence elements common to the light and heavy chain.

Two overlapping genomic clones containing the fibroin light-chain (Fib-L)-encoding gene (Fib-L) were obtained from the cosmid library of the silkworm, Bombyx mori J-139, by hybridization with the Fib-L cDNA clone. Sequencing of the 14.6-kb region revealed that Fib-L was 13,472 bp long containing seven exons, and that the gene contained a large first intron which occupied about 60% of the gene. Comparison of restriction patterns of the J-139 Fib-L with those of eight other B. mori breeds producing normal-level fibroin demonstrated that considerable restriction-fragment length polymorphisms were present in regions containing the first intron and the 3'-flanking sequence. However, sizes of the Fib-L mRNA and the Fib-L polypeptide were very similar among the nine breeds tested, suggesting that the exon sequences and the splice signals were all well conserved. 5'-Flanking regions of Fib-L and the fibroin heavy-chain (Fib-H)-encoding gene (Fib-H) compared in this study contained three 18-30-bp sequences of high similarity and many 8-10-bp common elements, six of which coincided with the binding sites of homeodomain proteins. Gel retardation assays with the nuclear extracts of the posterior and middle silk glands suggested that protein factors present in the posterior silk-gland nuclei could bind to a set of those common upstream elements.

Amino Acid Sequence↗

PubMed2005

Silk fibroin as a novel coating material for controlled release of theophylline.

The aim of this study was to explore potential use of the silk fibroin (SF) as an aqueous coating material for theophylline tablets. We have examined the film forming and coating properties of heat-treated fibroin, SF solution having different amounts of polyethylene glycol (PEG) and 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) cross-linked SF. Heat-treated SF material possessed a brittle structure, which resulted in poor film forming and coating properties. The optimum PEG amount in SF solution was determined as 17% (by weight) for an acceptable film forming and zero order release profile. EDC cross-linked SF has shown a very good film forming and coating property with a potential for sustaining the drug release from coated theophylline tablets. Dissolution data for coated theophylline tablets were analyzed using Ritger and Peppas equation to describe the mechanism of drug release. Drug release from the EDC coated tablets followed zero-order kinetics. Release rate constants were found to be 0.26, 0.19, 0.16%min(-1) for single-coated, double coated, and triple coated tablets, respectively. These results clearly demonstrated that silk fibroin has high utility as a novel aqueous coating material for controlled release products.

Animals↗

PubMed2005

A fibroin secretion-deficient silkworm mutant, Nd-sD, provides an efficient system for producing recombinant proteins.

The silkworm Nd-s(D) mutant is silk fibroin-secretion deficient. In the mutant, a disulfide linkage between the heavy (H) and light (L) chains, which is essential for the intracellular transport and secretion of fibroin, is not formed because of a partial deletion of the L-chain gene. To utilize the inactivity of the mutant L-chain, we investigated the possibility of using the Nd-s(D) mutant for the efficient production of recombinant proteins in the silkworm. A germ line transformation of the mutant with a normal L-chain-GFP fusion gene was performed. In the transgenic mutant, normal development of the posterior silk gland (PSG) was restored and it formed a normal cocoon. The biochemical analysis showed that the transgenic silkworms expressed the introduced gene in PSG cells, produced a large amount of the recombinant protein, secreted it into the PSG lumen, and used it to construct the cocoon. The molar ratio of silk proteins, H-chain:L-chain-GFP:fibrohexamerin, in the lumen and cocoon in the transgenic silkworm was 6:6:1, and the final product of the fusion gene formed about 10% of the cocoon silk. This indicates that the transgenic mutant silkworm possesses the capacity to produce and secrete the recombinant proteins in a molar ratio equal to that of the fibroin H-chain, contributing around half molecules of the total PSG silk proteins.

Animals↗

PubMed2006

Preparation and characterization of novel nanocomposite films formed from silk fibroin and nano-TiO2.

This paper describes the synthesis and characterization of new regenerated silk fibroin (SF)/nano-TiO(2) composite films. The preparation method, based on the sol-gel technique using butyl titanate as oxide precursor, could avoid reagglomeration of the prepared nanoparticles. Samples were characterized mainly by X-ray diffraction (XRD), ultra-violet (UV) spectroscopy, atomic force microscopy (AFM), Fourier transform infrared (FT-IR) spectroscopy, and thermogravimetric analysis (TGA). The UV and AFM results indicated that TiO(2) nanoparticles could be well dispersed inside the SF film, and the size of TiO(2) was about 80nm. The XRD and FT-IR analysis implied that the formation of nano-TiO(2) particles may induce the conformational transition of silk fibroin to a typical Silk II structure partly with the increasing of crystallinity in the composite films. Compared to the pure SF films, the mechanical and thermal properties of composite films were improved, and the solubility in water was decreased due to the conformational transition of silk fibroin to Silk II structure.

Fibroins↗

PubMed2002

Acid hydrolysis of silk fibroins and determination of the enrichment of isotopically labeled amino acids using precolumn derivatization and high-performance liquid chromatography-electrospray ionization-mass spectrometry.

Silk fibroins from moth larvae and spiders are composed of highly repetitive Ala- and Gly-rich blocks that determine their structure, properties, and function. To investigate the metabolic integration of isotopically labeled amino acids in the excreted silk, the enrichment of ingested tracers was determined after acid hydrolysis of the fibroins. Thus, spiders and moth larvae were fed with stable isotope tracers such as [1-13C]Ala or [1-13C]Gly and silked. After hydrolysis of the silk proteins, the corresponding amino acids were derivatized with Nalpha-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (Marfey's reagent) and separated by liquid chromatography. The isotopical enrichment of the amino acids was determined by online electrospray mass spectrometry and calculated by newly developed software. Depending on the feeding protocol, enrichments of up to 58% in Gly and 31% in Ala were found in the investigated silks. The highly enriched silk fibroins are suitable for further structural investigation such as solid-state nuclear magnetic resonance.

Acetic Acid↗

PubMed1990

Fibroin gene promoter contains a cluster of homeodomain binding sites that interact with three silk gland factors.

By nuclease protection and mobility shift assays, we have shown that the 5' flanking sequence of the Bombyx mori fibroin gene, which is known to be required for a maximal level of transcription in vitro, contains five regions (A to E) that bind protein factors from the posterior silk gland extract. A silk gland-specific factor (SGF-1) and a ubiquitous factor (FBF-A1) were found to interact with the proximal A region, while the related B region can only bind SGF-1. The three distal regions (C, D and E) bind one posterior silk gland-specific factor (SGF-2) and two ubiquitous factors (SGF-3 and -4). SGF-1 might play an important role in the expression of silk protein genes because it also binds to a similar site in the sericin-1 gene and potential SGF-1 binding sites can be found in two other silk protein genes of Bombyx mori and the fibroin gene of Antheraea yamamai. The three distal regions for SGF-2, -3 and -4 contain ten base-pairs of A + T-rich repeats that resemble the consensus binding site (TCAATTAAAT) of a number of homeodomain proteins. The TAAT motif in the core of these regions is shown to be important for the binding of these three proteins and, as described elsewhere, two Drosophila homeodomain proteins, ZEN (zerknüllt) and EVE (even-skipped). Interestingly, SGF-3 appears to be a Bombyx octamer binding protein and may also be involved in the regulation of the sericin-1 gene. The possibility that the fibroin gene and other silk protein genes may be a group of target genes for some members of the homeobox gene family is discussed.

Animals↗

PubMed2002

Poly(D,L-lactic acid) surfaces modified by silk fibroin: effects on the culture of osteoblast in vitro.

The objective of this study was to modify the surface of poly(D,L-lactic acid) (PDLLA) with different molecular weight of silk fibroins, and assess the effects of the modified surfaces on the functions of rat osteoblasts cultured in vitro. The properties of the modified PDLLA surface and the control one were investigated by contact angle and electron spectroscopy for chemical analysis (ESCA). The former indicated the variation of hydrophilicity and the latter suggested that the modified PDLLA film using silk fibroin is enriched with nitrogen atoms. The biocompatibility of the PDLLA film may be altered and in turn affects the seeded cell functions. Therefore, attachment and proliferation of osteoblasts seeded on the modified PDLLA films and the control one were examined. Cell morphologies on these films were studied by scanning electron microscopy (SEM) and cell viability was evaluated by MTT assay. In addition, differentiated cell function was assessed by measuring the alkaline phosphatase (ALP) activity. These results suggest that the silk fibroin-modified PDLLA surface can improve the interaction between osteoblasts and the PDLLA films.

Alkaline Phosphatase↗

PubMed2002

Influence of different surface modification treatments on poly(D,L-lactic acid) with silk fibroin and their effects on the culture of osteoblast in vitro.

The objective of this study was to investigate the efficiency of two treatments for poly(D,L-lactic acid) (PDLLA) surface modification using silk fibroin. one chemical treatment and one physical treatment: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (WSC) and entrapment. The properties of control films, WSC-modified and entrapment-treated PDLLA films were investigated by water contact angle measurement and electron spectroscopy for chemical analysis (ESCA). The water-contact angle measurement indicated the change of hydrophilicity and the ESCA analysis suggested that the modified PDLLA film using silk fibroin became enriched with nitrogen atoms. The biocompatibility of PDLLA film might be altered, which in turn would affect the functions of cells that were seeded on it. Therefore, attachment and proliferation of osteoblasts that were seeded on modified PDLLA films and control films were examined. Cell viability was evaluated by the MTT assay and differentiated cell function was assessed by measuring alkaline phosphatase activity. These results suggested that silk fibroin was used to modify PDLLA surface via WSC and that entrapment could improve the interactions between osteoblasts and PDLLA films. The entrapment treatment was more effective thin WSC treatment to accomplish the goal of surface modification.

Alkaline Phosphatase↗

PubMed2004

Identification of fibroin-derived peptides enhancing the proliferation of cultured human skin fibroblasts.

We previously reported that the fibroin of the silkworm Bombyx mori enhanced the proliferation of cultured human skin fibroblasts. In this work, the fibroin was digested by chymotrypsin, and the resulting peptide fragments were fractionated and assayed for their biological activity. Two peptides that promoted fibroblast growth were isolated and identified to be VITTDSDGNE and NINDFDED. Both sequences are found in the N-terminal region of the fibroin polypeptide and are thought to be the active principle of fibroblast growth-promoting activity.

Amino Acid Sequence↗

PubMed1998

Use of Bombyx mori silk fibroin as a substratum for cultivation of animal cells.

The growth of animal cells on silk fibroin-coated plates was examined. The anchorage-dependent cells showed almost the same growth on both fibroin- and collagen-coated plates, and it was 30-50% higher than that on polystyrene plates coated with hydrophilic groups. On the other hand, the growth of the anchorage-independent hybridomas on the three different plates did not show a significant difference. The cells grown on the respective plates produced their products with the same efficiency despite the difference in the chemical properties of the plates. In this paper, it is demonstrated that silk fibroin can be used as the substratum for the culture of animal cells in place of collagen.

Animals↗

PubMed2001

Purification and characterization of fibroin from the tropical Saturniid silkworm, Antheraea mylitta.

The fibroin protein isolated from the posterior silkgland of the tropical Saturniid silkworm Antheraea mylitta, was solubilized in lithium dodecyl sulfate and purified by gel filtration. The major fraction from gel filtration was analyzed by SDS-PAGE under non-reducing and reducing conditions. One major protein band of ca 395 kDa was obtained under non-reducing conditions and a doublet band of approximately 197 kDa under reducing conditions. The appearance of a single spot in two-dimensional electrophoresis confirmed the purity of the protein indicating that it may be a homodimeric protein of two similar sized polypeptides. Amino acid composition analysis showed that, like other Saturniid fibroins, it is rich in glycine, alanine and serine amino acids. N-terminal amino acid sequence shows significant homology with other Antheraea species. The enzymatic deglycosylation analysis indicates that the fibroin protein is glycosylated and the oligosaccharides are O-linked to the protein backbone by N-acetylgalactoseamine moiety which conforms to a Core 1 mucin-type glycosylation pattern.

Amino Acid Sequence↗

PubMed1999

Hydrophobic interaction of P25, containing Asn-linked oligosaccharide chains, with the H-L complex of silk fibroin produced by Bombyx mori.

Fibroin light (L-) chain and P25 are low molecular weight protein components of silk fibroin which are secreted from the posterior silk gland cells of the silkworm, Bombyx mori. The primary structure of L-chain was determined previously by cDNA cloning and peptide analysis, but that of P25 has only been deduced from its genomic sequence. Our previous studies with specific antibodies against L-chain and P25 have shown that L-chain and H-chain are linked by disulfide bond(s) but P25 is not covalently linked to H-chain. Here, we present evidence that P25 associates with the H-L complex primarily by hydrophobic interactions and that P25 is a glycoprotein containing Asn-linked oligosaccharide chains. From the analysis of three fibroin-secretion-deficient 'naked pupa' mutant breeds [Nd(2), Nd-s and Nd-sD], it is suggested that P25 interacts with H-chain in the absence of H-L linkage but its content of oligosaccharide is reduced when the H-L linkage is not formed. From these results, models are presented implying that the H-L complex and P25 are associated to form a higher-order complex of specific conformation during the processes of intracellular transport and secretion, and that the Asn-linked glycosylation of P25 is partially altered under such conditions.

Animals↗

PubMed2004

Effects of pH and calcium ions on the conformational transitions in silk fibroin using 2D Raman correlation spectroscopy and 13C solid-state NMR.

Silk fibroin exists in a number of different states, such as silk I and silk II, with different properties largely defined by differences in secondary structure composition. Numerous attempts have been made to control the transitions from silk I to silk II in vitro to produce high-performance materials. Of all the factors influencing the structural compositions, pH and some metal ions play important roles. This paper focuses on the influence of pH and Ca(2+) ions on the conformational transition from silk I to silk II in regenerated (redissolved) Bombyx mori fibroin. One- and two-dimensional correlation Raman spectroscopy was used to describe qualitatively the transitions in secondary structure in silk I, silk II, and their intermediates as pH and Ca(2+) ion concentration were changed, while (13)C cross polarization magic angle spinning (CP/MAS) solid-state NMR was used to quantify these changes. We showed that conditions (low pH, pH 5.2; a defined range of Ca(2+) ion concentrations; gradual water removal) that mimic natural silk spinning promote the formations of beta-sheet and distorted beta-sheet characteristic of silk II or silk II-related intermediate. In contrast, higher pH (pH 6.9-8.0) and higher Ca(2+) ion concentrations maintain "random coil" conformations typical of silk I or silk I-related intermediate. These results help to explain why the natural silk spinning process is attended by a reduction in pH from 6.9 to 4.8 and a change in the Ca(2+) ion concentration in the gland lumen as fibroin passes from the posterior division through the secretory pathway to the anterior division.

Animals↗