SEARCH · PubMed Health

Results for “FIBROIN”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 433 records · Page 24Linked to original sources

PubMed1995

Attachment and growth of cultured fibroblast cells on silk protein matrices.

The attachment and growth of L-929 cells on films made of Bombyx mori silk proteins--fibroin and sericin and their mixtures--was studied by a cell culture method. Both cell attachment and growth were dependent on a minimum of around 90% sericin in the mixture. The results from electron micrography as well as from the DSC measurements supported the notion that the mixture of the two proteins fibroin and sericin has a phase-separated structure in the solid state. The observed minimum of sericin in the cell attachment and growth is thought to be a result of this phase-separated structure. Films of pure component proteins (i.e., 100% fibroin or sericin) exhibited as high a cell attachment and growth as collagen, a widely used mammalian cell culture substrate. However, a morphological study of the attached cells revealed that the cells attached to silk fibroin were extended and had a spindle shape, just like the cells attached to collagen, while the cells attached to the silk sericin had a different shape. It is concluded, therefore, that the attachment condition on silk fibroin is ideal for the viability, growth and function of the cells.

Amino Acids↗

PubMed1986

The cylindrical or tubiliform glands of Nephila clavipes.

The cylindrical or tubiliform glands of the spider Nephila clavipes have been studied and compared to the large ampullates on which we have previously reported. The three pairs of cylindrical or tubiliform glands secrete the fibroin for the organism's egg case. Their solubilized luminar contents migrate as a homogeneous band in Sodium dodecyl sulfate polyacrylamide gel electrophoresis and turn out to be a larger protein than that produced by the large ampullates. The excised cylindrical glands remain metabolically active for several hours in a simple culture medium, where fibroin synthesis can be monitored through the incorporation of 14C alanine. The glands' response to a fibroin production stimulus does not reach the magnitude displayed by the large ampullates, but this is to be expected since their products supply different functions in this organism. This fibroin also seems to be elongated discontinuously. Translational pauses have been detected in the secretory epithelium of cylindrical and large ampullate glands of Nephila as well as in the silk glands of Bombyx mori. Since these glands produce the fibroin for the females egg case, they should prove to be an interesting model system.

Alanine↗

PubMed2001

Syntheses of glycine and L-serine by their interconversion in the posterior silkgland of the silkworm, Bombyx mori.

It was observed by solution-state 13C NMR spectroscopy that a great portion of the 13C of [1-13C]L-serine fed to the 5th instar larvae of the silkworm, Bombyx mori was incorporated into C1 of glycine in silk fibroin. [1-13C]Glycine was detected along with [1-13C]serine in fibroin of the posterior silkgland cultured in a medium containing [1-13C]serine. This formation of [1-13C]glycine was inhibited by addition of aminopterin to the culture medium. These findings suggest that an active conversion from serine to glycine, which needs tetrahydrofolate, occurs in the posterior silkgland for fibroin synthesis. Moreover, the solid-state 13C CP/MAS spectrum of the fibroin prepared from cocoons spun by larvae fed with [13C]formate revealed that serine C3 was labelled specifically with 13C, suggesting that the reverse conversion from glycine to serine took place in the silkworm. The posterior silkgland has the ability to synthesize not only fibroin but also its major materials, glycine and serine.

Animals↗

PubMed2006

Characterization and expression of a cDNA encoding a tubuliform silk protein of the golden web spider Nephila antipodiana.

Spider silks are renowned for their excellent mechanical properties. Although several spider fibroin genes, mainly from dragline and capture silks, have been identified, there are still many members in the spider fibroin gene family remain uncharacterized. In this study, a novel silk cDNA clone from the golden web spider Nephila antipodiana was isolated. It is serine rich and contains two almost identical fragments with one varied gap region and one conserved spider fibroin-like C-terminal domain. Both in situ hybridization and immunoblot analyses have shown that it is specifically expressed in the tubuliform gland. Thus, it likely encodes the silk fibroin from the tubuliform gland, which supplies the main component of the inner egg case. Unlike other silk proteins, the protein encoded by the novel cDNA in water solution exhibits the characteristic of an alpha-helical protein, which implies the distinct property of the egg case silk, though the fiber of tubuliform silk is mainly composed of beta-sheet structure. Its sequence information facilitates elucidation of the evolutionary history of the araneoid fibroin genes.

Amino Acid Sequence↗

PubMed2006

Electrospun silk-BMP-2 scaffolds for bone tissue engineering.

Silk fibroin fiber scaffolds containing bone morphogenetic protein 2 (BMP-2) and/or nanoparticles of hydroxyapatite (nHAP) prepared via electrospinning were used for in vitro bone formation from human bone marrow-derived mesenchymal stem cells (hMSCs). BMP-2 survived the aqueous-based electrospinnig process in bioactive form. hMSCs were cultured for up to 31 days under static conditions in osteogenic media on the scaffolds (silk/PEO/BMP-2, silk/PEO/nHAP, silk/PEO/nHAP/BMP-2) and controls (silk/PEO, silk/PEO extracted). Electrospun silk fibroin-based scaffolds supported hMSC growth and differentiation toward osteogenic outcomes. The scaffolds with the co-processed BMP-2 supported higher calcium deposition and enhanced transcript levels of bone-specific markers than in the controls, indicating that these nanofibrous electrospun silk scaffolds were an efficient delivery system for BMP-2. X-ray diffraction (XRD) analysis revealed that the apatite formed on the silk fibroin/BMP-2 scaffolds had higher crystallinity than on the silk fibroin scaffold controls. In addition, nHAP particles were incorporated into the electrospun fibrous scaffolds during processing and improved bone formation. The coexistence of BMP-2 and nHAP in the electrospun silk fibroin fibers resulted in the highest calcium deposition and upregulation of BMP-2 transcript levels when compared with the other systems. The results suggest that electrospun silk-fibroin-based scaffolds are potential candidates for bone tissue engineering. Furthermore, the mild aqueous process required to spin the fibers offers an important option for delivery of labile cytokines and other components into the system.

Adult↗

PubMed2000

Conformational transitions in model silk peptides.

Protein structural transitions and beta-sheet formation are a common problem both in vivo and in vitro and are of critical relevance in disparate areas such as protein processing and beta-amyloid and prion behavior. Silks provide a "databank" of well-characterized polymorphic sequences, acting as a window onto structural transitions. Peptides with conformationally polymorphic silk-like sequences, expected to exhibit an intractable beta-sheet form, were characterized using Fourier transform infrared spectroscopy, circular dichroism, and electron diffraction. Polymorphs resembling the silk I, silk II (beta-sheet), and silk III (threefold polyglycine II-like helix) crystal structures were identified for the peptide fibroin C (GAGAGS repetitive sequence). Two peptides based on silk amorphous sequences, fibroin A (GAGAGY) and fibroin V (GDVGGAGATGGS), crystallized as silk I under most conditions. Methanol treatment of fibroin A resulted in a gradual transition from silk I to silk II, with an intermediate state involving a high proportion of beta-turns. Attenuated total reflectance Fourier transform infrared spectroscopy has been used to observe conformational changes as the peptides adsorb from solution onto a hydrophobic surface. Fibroin C has a beta-strand structure in solution but adopts a silk I-like structure upon adsorption, which when dried on the ZnSe crystal contains silk III crystallites.

Amino Acid Sequence↗

PubMed2002

Comparative architecture of silks, fibrous proteins and their encoding genes in insects and spiders.

The known silk fibroins and fibrous glues are thought to be encoded by members of the same gene family. All silk fibroins sequenced to date contain regions of long-range order (crystalline regions) and/or short-range order (non-crystalline regions). All of the sequenced fibroin silks (Flag or silk from flagelliform gland in spiders; Fhc or heavy chain fibroin silks produced by Lepidoptera larvae) are made up of hierarchically organized, repetitive arrays of amino acids. Fhc fibroin genes are characterized by a similar molecular genetic architecture of two exons and one intron, but the organization and size of these units differs. The Flag, Ser (sericin gene) and BR (Balbiani ring genes; both fibrous proteins) genes are made up of multiple exons and introns. Sequences coding for crystalline and non-crystalline protein domains are integrated in the repetitive regions of Fhc and MA exons, but not in the protein glues Ser1 and BR-1. Genetic 'hot-spots' promote recombination errors in Fhc, MA, and Flag. Codon bias, structural constraint, point mutations, and shortened coding arrays may be alternative means of stabilizing precursor mRNA transcripts. Differential regulation of gene expression and selective splicing of the mRNA transcript may allow rapid adaptation of silk functional properties to different physical environments.

Alternative Splicing↗

PubMed2005

Modular evolution of egg case silk genes across orb-weaving spider superfamilies.

Spider silk proteins (fibroins) are renowned for their extraordinary mechanical properties and biomimetic potential. Despite extensive evolutionary, ecological, and industrial interest in these fibroins, only a fraction of the known silk types have been characterized at the molecular level. Here we report cDNA and genomic sequences of the fibroin TuSp1, which appears to be the major component of tubuliform gland silk, a fiber exclusively synthesized by female spiders for egg case construction. We obtained TuSp1 sequences from 12 spider species that represent the extremes of phylogenetic diversity within the Orbicularia (orb-weaver superfamilies, Araneoidea and Deinopoidea) and finer scale sampling within genera. TuSp1 encodes tandem arrays of an approximately 200-aa-long repeat unit and individual repeats are readily aligned, even among species that diverged >125 million years ago. Analyses of these repeats across species reveal the strong influence of concerted evolution, resulting in intragenic homogenization. However, deinopoid TuSp1 repeats also contain insertions of coding, minisatellite-like sequences, an apparent result of replication slippage and nonreciprocal recombination. Phylogenetic analyses of 37 spider fibroin sequences support the monophyly of TuSp1 within the spider fibroin gene family, consistent with a single origin of this ortholog group. The diversity of taxa and silks examined here confirms that repetitive architecture is a general feature of this gene family. Moreover, we show that TuSp1 provides a clear example of modular evolution across a range of phylogenetic levels.

Amino Acid Sequence↗

PubMed1997

Translational pauses during the synthesis of proteins and mRNA structure.

Translational pauses are observed during a spider fibroin synthesis (1,2). The spider major ampullate (dragline) silk of the spider Nephila clavipes is composed of multiple proteins. The amino acid sequences of the partial cDNA clones for the two major dragline silk fibroin components (Spidroin 1 and 2) exhibit repetitive motifs (3,4). Our detailed inspection of the nucleotide sequences of the repetitive motifs revealed highly selective site-specific codon usage patterns within a motif, suggesting that the secondary structure of the spider fibroin mRNA is optimized by the nucleotide sequence of the fibroin gene. The results, combined with our preceding results on silk fibroin from Bombyx mori (5) suggest that translational pauses of spider silk are interpreted in terms of the mRNA secondary structure.

Amino Acid Sequence↗

PubMed2006

Cartilage tissue engineering with silk scaffolds and human articular chondrocytes.

Adult cartilage tissue has poor capability of self-repair, especially in case of severe cartilage damage due to trauma or age-related degeneration. Autologous cell-based tissue engineering using three-dimensional (3-D) porous scaffolds has provided an option for the repair of full thickness defects in adult cartilage tissue. Mesenchymal stem cells (MSCs) and chondrocytes are the two major cell sources for cartilage tissue engineering. Silk fibroin as a naturally occurring degradable fibrous protein with unique mechanical properties, excellent biocompatibility and process-ability has demonstrated strong potential for skeletal tissue engineering. The present study combined adult human chondrocytes (hCHs) with aqueous-derived porous silk fibroin scaffolds for in vitro cartilage tissue engineering. The results were compared with a previous study using the same scaffolds but using MSCs to generate the cartilage tissue outcomes. Culture-expanded hCHs attached to, proliferated and re-differentiated in the scaffolds in a serum-free, chemically defined medium containing TGF-beta1, based on cell morphology, levels of cartilage-related gene transcripts, and the presence of a cartilage-specific ECM. Cell density was critical for the redifferentiation of culture-expanded hCHs in the 3-D aqueous-derived silk fibroin scaffolds. The level of cartilage-related transcripts (AGC, Col-II, Sox 9 and Col-II/Col-I ratio) and the deposition of cartilage-specific ECM were significantly upregulated in constructs initiated with higher seeding density. The hCH-based constructs were significantly different than those formed from MSC-based constructs with respect to cell morphology, zonal structure and initial seeding density needed to successfully generate engineered cartilage-like tissue. These results suggest fundamental differences between stem cell-based (MSC) and primary cell-based (hCH) tissue engineering, as well as the importance of suitable scaffold features, in the optimization of cartilage-related outcomes in vitro. The present work diversifies cell sources in combination with silk fibroin-based tissue engineering applications. Together with our previous studies, the present results show great promise for engineered 3-D silk fibroin scaffolds in autologous cell-based skeletal tissue engineering.

Animals↗

PubMedDate not supplied

Structural analysis of silk with 13C NMR chemical shift contour plots.

The polymorphic structures of silk fibroins in the solid state were examined on the basis of a quantitative relationship between the 13C chemical shift and local structure in proteins. To determine this relationship, 13C chemical shift contour plots for C alpha and C beta carbons of Ala and Ser residues, and the C alpha chemical shift plot for Gly residues were prepared using atomic co-ordinates from the Protein Data Bank and 13C NMR chemical shift data in aqueous solution reported for 40 proteins. The 13C CP/MAS NMR chemical shifts of Ala, Ser and Gly residues of Bombyx mori silk fibroin in silk I and silk II forms were used along with 13C CP/MAS NMR chemical shifts of Ala residues of Samia cynthia ricini silk fibroin in beta-sheet and alpha-helix forms for the structure analyses of silk fibroins. The allowed regions in the 13C chemical shift contour plots for C alpha and C beta carbons of Ala and Ser residues for the structures in silk fibroins, i.e. Silk II, Silk I and alpha-helix, were determined using their 13C isotropic NMR chemical shifts in the solid state. There are two area of the phi,psi map which satisfy the observed Silk I chemical shift data for both the C alpha and C beta carbons of Ala and Ser residues in the 13C chemical shift contour plots.

Alanine↗

PubMed1985

Efficiency of in vitro transcription of Dictyostelium discoideum actin gene is affected by the nucleotide sequence of the transcription initiation region.

The actin gene of Dictyostelium discoideum is transcribed faithfully but with very low efficiency in a cell-free system containing HeLa cell extract [Takiya, S., Tabata, T., Iwabuchi, M., Hirose, S., & Suzuki, Y. (1984) J. Biochem. (Tokyo) 95, 1367-1377]. Using the same in vitro system, we determined that the promoter activity of the actin 5 gene is 100-200 times weaker than that of the silkworm fibroin gene. To clarify the cause of the low transcription efficiency, various chimeric genes were constructed from the actin and fibroin genes, and their transcription efficiencies were examined in vitro. Both the TATA box and the transcription initiation site of the two natural genes functioned in the transcription of the chimeric genes, the efficiency of which was especially affected by the transcription initiation region. In chimeric genes having the upstream sequence of the actin gene and a downstream sequence including the transcription initiation site of the fibroin gene, the transcription efficiency was higher than one-third of that of the natural fibroin gene. In chimeric genes having the actin transcription initiation region and an upstream sequence of the fibroin gene, the transcription efficiency was as low as that of the natural actin gene. We concluded that the transcription initiation site is a part of the promoter and an essential region for directing faithful and efficient initiation of gene transcription.

Actins↗

PubMed1977

On the introduction of disulfide crosslinks into fibrous proteins and bovine serum albumin.

Hydroxyl groups in serine side chains of collagen, silk fibroin, and bovine serum albumin (BSA) were converted to SH by tosylation. In collagen film, 50% of the serine OH groups could be thiolated at most. In fibroin, only 13% because of its compact beta-pleated sheet structure and low susceptibility to swelling. The SH groups introduced are near enough together to form -S-S- bonds by oxidation. The residual SH content after oxidation was 0.1% in collagen and 0.03 to 0.25% in fibroin. Disulfide crosslinking increased the shrinkage temperature of collagen and fibroin and decreased the amount of shrinkage. BSA was crosslinked to dimers (MBSA) according to gel permeation chromatography and sedimentation analysis by the analytical centrifuge. Because these crosslinked proteins can be metabolized by the usual processes, in contrast to those crosslinked by artificial, nonphysiological bridges, they may be used for biological or medical purposes.

Amino Acids↗

PubMed1992

Essential role of duplications of short motif sequences in the genomic evolution of Bombyx mori.

The Bombyx fibroin gene has a discrete mosaic structure of various repetitive sequences, which may have evolved through various repeating arrangements. Detailed sequence analysis of the fibroin gene containing coding and noncoding regions revealed that the whole sequence could be arranged as an array of short repetitive sequences. A portion of the intron of the fibroin gene is one of interspersed repetitive elements. We cloned a 1.5-kb DNA fragment of the Bombyx genome that contains interspersed elements homologous to the intron sequence. Sequence comparison between the intron and the 1.5-kb fragment shows that partial duplication has frequently occurred in evolutionary progress, and the resultant repetitive blocks of short motif sequences are abundant in the genome. These facts suggest that tandem duplication of the short motif sequence is an important rearrangement in genomic evolution of the fibroin gene.

Animals↗

PubMed2000

Discontinuous translation and mRNA structure of the coding region.

An analysis of the published data of the electrophoresis of the products of silk fibroin exhibiting discontinuities or pauses in the translational process showed that about 60 times of pauses occur during the translation of the mRNA from silk fibroin. In addition, we estimated that silk fibroin gene as a whole (15 kb) is composed of about 60 alternate, crystalline-noncrystalline, arrays of nucleotide sequence elements each connected by the boundary sequence, on the basis of the nucleotide sequence of the partial cDNA clone of the heavy chain gene of Bombyx mori available in the literature. The coincidence of the total number of pauses with that of the alternate arrays suggests that one translational pausing site exists in each of the unit (alternate array + boundary) nucleotide sequence in the silk fibroin mRNA template.

Animals↗

PubMed2002

13C CP/MAS NMR study on structural heterogeneity in Bombyx mori silk fiber and their generation by stretching.

It is important to resolve the structure of Bombyx mori silk fibroin before spinning (silk I) and after spinning (silk II), and the mechanism of the structural transition during fiber formation in developing new silk-like fiber. The silk I structure has been recently resolved by (13)C solid-state NMR as a "repeated beta-turn type II structure." Here, we used (13)C solid-state NMR to clarify the heterogeneous structure of the natural fiber from Bombyx mori silk fibroin in the silk II form. Interestingly, the (13)C CP/MAS NMR revealed a broad and asymmetric peak for the Ala Cbeta carbon. The relative proportions of the various heterogeneous components were determined from their relative peak intensities after line shape deconvolution. Namely, for 56% crystalline fraction (mainly repeated Ala-Gly-Ser-Gly-Ala-Gly sequences), 18% distorted beta-turn, 13% beta-sheet (parallel Ala residues), and 25% beta-sheet (alternating Ala residues). The remaining fraction of 44% amorphous Tyr-rich region, 22% in both distorted beta-turn and distorted beta-sheet. Such a heterogeneous structure including distorted beta-turn can be observed for the peptides (AG)(n) (n > 9 ). The structural change from silk I to silk II occurs exclusively for the sequence (Ala-Gly-Ser-Gly-Ala-Gly)(n) in B. mori silk fibroin. The generation of the heterogeneous structure can be studied by change in the Ala Cbeta peak of (13)C CP/MAS NMR spectra of the silk fibroin samples with different stretching ratios.

Alanine↗

PubMed1977

[Studies on protein biosynthesis in the silk gland of Bombyx mori L. silkworm].

The incorporation of (14C) lysine (to characterize the biosynthesis of cellular proteins) and (14C) glycine (for silk fibroin) in free and membrane-bound polyribosomes was studied in fibroin portion of the silk gland of Bombyx mori silkworm in the V instar. It was shown that although the membrane-bound polyribosomes are found in posterior silk gland from the beginning of the V instar, the fibroin biosynthesis in the membranebound polyribosomes takes place predominantly in the second part of the V instar. On the other hand the cellular proteins are synthesized mostly in the free polyribosomes in the first half of the V instar. In the second half of the V instar in the sucrose gradient zone corresponding to free polyribosomes, monoribosomes unable to synthesize protein for the absence of mRNA are present. Nevertheless, these ribosomes isolated from the fibroin part of the silk gland in the end of the V instar do synthesize polyphenylalanine in the presence of poly (U), and aminoacyl-t-RNA-synthetases and tRNA's obtained from the posterior silk gland.

Amino Acyl-tRNA Synthetases↗

PubMed1996

13C NMR of Nephila clavipes major ampullate silk gland.

The major ampullate glands of the spider Nephila clavipes contain approximately 0.2 microliter each of a highly concentrated (approximately 50%) solution of silk fibroin. Therefore, the reservoir of silk in these glands presents an ideal opportunity to observe prefolded conformations of a protein in its native state. To this end, the structure and conformation of major ampullate gland silk fibroin within the glands of the spider N. clavipes were examined by 13C NMR spectroscopy. These results were compared to those from silk protein first drawn from the spinneret and then denatured. The 13C NMR chemical shifts, along with infrared and circular dichroism data, suggest that the silk fibroin in the glands exists in dynamically averaged helical conformations. Furthermore, there is no evidence of proline residues in U-(13)C-D-glucose-labeled silk. This transient prefolded "molten fibril" state may correspond to the silk I form found in Bombyx mori silk. There is no evidence of the final beta-sheet structure in the ampullate gland silk fibroin before final silk processing. However, the conformation of silk in the glands appears to be in a highly metastable state, as plasticization with water produces the beta-sheet structure. Therefore, the ducts connecting the ampullate glands to the spinnerets play a larger role in silk processing than previously thought.

Alanine↗