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 127 records · Page 7Linked to original sources

PubMed1981

Promoter sequence of fibroin gene assigned by in vitro transcription system.

We have shown that the silk fibroin gene from Bombyx mori is faithfully transcribed in an in vitro transcription system of the HeLa cell extract prepared by the method of Manley et al. [Manley, J. L., Fire, A., Cano, A., Sharp, P. A. & Gefter, M. L. (1980) Proc. Natl. Acad. Sci. USA 77, 3855-3859]. Using this system and a series of deletion mutants of fibroin gene, we have assigned the promoter sequence of fibroin gene. The 5' boundary of the promoter is around nucleotide position -29, indicating that most of the T-A-T-A-A-A-A sequence (-30 to -24) is essential for the promoter function, where the transcription initiation point of fibroin gene is assigned as nucleotide position +1 [Tsuda, M., Ohshima, Y. & Suzuki, Y. (1979) Proc. Natl. Acad. Sci. USA 76, 4872-4876]. The 3' boundary is around nucleotide position +6. However, to support the efficient, faithful transcription, some additional (more than 26 but less than 41) nucleotides of nonspecific origin are required at the 5' side of -29. Functions ascribed to the promoter region are discussed.

Animals↗

PubMed2000

Silk fibroin of Bombyx mori is secreted, assembling a high molecular mass elementary unit consisting of H-chain, L-chain, and P25, with a 6:6:1 molar ratio.

Silk fibroin produced by the silkworm Bombyx mori consists of a heavy chain, a light chain, and a glycoprotein, P25. The heavy and light chains are linked by a disulfide bond, and P25 associates with disulfide-linked heavy and light chains by noncovalent interactions. Quantitative enzyme-linked immunosorbent assay revealed that molar ratios of the heavy chain, light chain, and P25 were 6:6:1, both in cocoons and in fibroin secreted into the lumen of posterior silk gland. Trace amounts of fibroin produced by three "naked pupa" mutants of B. mori lacked the light chain, but the molar ratio of heavy chain and P25 was also 6:1. Gel filtration chromatography and sedimentation equilibrium analysis demonstrated that a large protein complex of approximately 2.3 MDa, designated an elementary unit of fibroin having 6:6:1 molar ratios of the heavy chain, light chain, and P25, existed in posterior silk gland cells. Inaccessibility of biotinylated concanavalin A to the native elementary unit and partial dissociation of the elementary unit after incubation with excess N-glycosidase F or endoglycosidase H suggest that a single molecule of P25 is located internally and plays an important role in maintaining integrity of the complex.

Amino Acid Sequence↗

PubMed2003

Correlation between fibroin amino acid sequence and physical silk properties.

The fiber properties of lepidopteran silk depend on the amino acid repeats that interact during H-fibroin polymerization. The aim of our research was to relate repeat composition to insect biology and fiber strength. Representative regions of the H-fibroin genes were sequenced and analyzed in three pyralid species: wax moth (Galleria mellonella), European flour moth (Ephestia kuehniella), and Indian meal moth (Plodia interpunctella). The amino acid repeats are species-specific, evidently a diversification of an ancestral region of 43 residues, and include three types of regularly dispersed motifs: modifications of GSSAASAA sequence, stretches of tripeptides GXZ where X and Z represent bulky residues, and sequences similar to PVIVIEE. No concatenations of GX dipeptide or alanine, which are typical for Bombyx silkworms and Antheraea silk moths, respectively, were found. Despite different repeat structure, the silks of G. mellonella and E. kuehniella exhibit similar tensile strength as the Bombyx and Antheraea silks. We suggest that in these latter two species, variations in the repeat length obstruct repeat alignment, but sufficiently long stretches of iterated residues get superposed to interact. In the pyralid H-fibroins, interactions of the widely separated and diverse motifs depend on the precision of repeat matching; silk is strong in G. mellonella and E. kuehniella, with 2-3 types of long homogeneous repeats, and nearly 10 times weaker in P. interpunctella, with seven types of shorter erratic repeats. The high proportion of large amino acids in the H-fibroin of pyralids has probably evolved in connection with the spinning habit of caterpillars that live in protective silk tubes and spin continuously, enlarging the tubes on one end and partly devouring the other one. The silk serves as a depot of energetically rich and essential amino acids that may be scarce in the diet.

Amino Acid Sequence↗

PubMed2005

Egg case protein-1. A new class of silk proteins with fibroin-like properties from the spider Latrodectus hesperus.

Spiders produce multiple types of silk that exhibit diverse mechanical properties and biological functions. Most molecular studies of spider silk have focused on fibroins from dragline silk and capture silk, two important silk types involved in the survival of the spider. In our studies we have focused on the characterization of egg case silk, a third silk fiber produced by the black widow spider, Latrodectus hesperus. Analysis of the physical structure of egg case silk using scanning electron microscopy demonstrates the presence of small and large diameter fibers. By using the strong protein denaturant 8 M guanidine hydrochloride to solubilize the fibers, we demonstrated by SDS-PAGE and protein silver staining that an abundant component of egg case silk is a 100-kDa protein doublet. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called ecp-1, which encodes for one of the protein components of the 100-kDa species. BLAST searches of the NCBInr protein data base using the primary sequence of ECP-1 revealed similarity to fibroins from spiders and silkworms, which mapped to two distinct regions within the ECP-1. These regions contained the conserved repetitive fibroin motifs poly(Ala) and poly(Gly-Ala), but surprisingly, no larger ensemble repeats could be identified within the primary sequence of ECP-1. Consistent with silk gland-restricted patterns of expression for fibroins, ECP-1 was demonstrated to be predominantly produced in the tubuliform gland, with lower levels detected in the major and minor ampullate glands. ECP-1 monomeric units were also shown to assemble into higher aggregate structures through the formation of disulfide bonds via a unique cysteine-rich N-terminal region. Collectively, our findings provide new insight into the components of egg case silk and identify a new class of silk proteins with distinctive molecular features relative to traditional members of the spider silk gene family.

Alanine↗

PubMed1984

In monkey COS cells only the TATA box and the cap site region are required for faithful and efficient initiation of the fibroin gene transcription.

The DNA sequences necessary for faithful and efficient initiation of transcription of Bombyx mori fibroin gene have been studied in vivo using monkey COS cells and a SV40 origin vector. Transcriptional analysis of 5' deletion genes and of exact substitution genes including a series of single base substitution mutants indicates that the TATA box and the cap region are required for faithful and efficient initiation of the fibroin gene transcription in vivo, whereas far upstream sequences and the CAAT-like sequences are not required. In addition, single base substitution genes of the cap site region have clearly shown that there exists a strict sequence requirement of the cap region of the fibroin gene. These results define the basic promoter of the fibroin gene to the region spanning the TATA box and the cap site region.

Animals↗

PubMed1989

Factors involved in preferential transcription of the fibroin gene.

Transcription factors for class II genes from a tissue source, the posterior silk glands of Bombyx mori, were fractionated by stepwise elution on a phosphocellulose column into five fractions: A (0.04 M KCl flow through), A' (0.1 M KCl eluate), B (0.3 M KCl eluate), C (0.5 M KCl eluate) and D (1 M KCl eluate). The minimal requirement for reconstruction of accurate initiation of transcription of the fibroin gene, as well as of the adenovirus 2 major late promoter, was the combination of fractions A, B and D, suggesting that transcription factors from B. mori can recognize general signals of the promoters for class II genes and that basic transcription factors are conserved even in distantly diverged species of eukaryotes. To detect activities stimulating the transcription governed by the promoter of fibroin gene, each fraction was tested for its function by supplementing a basal amount of HeLa cell extract. When circular templates were used, stimulatory activities specific for the fibroin gene were detected in fraction D. This preferential transcription is composed of at least three activities; the first and the second dependent on the upstream elements and the third dependent on the sequence downstream from the TATA box. However, when linear templates were used the preference for the fibroin gene was apparently lost and transcription activation by fraction D became general.

Animals↗

PubMed1986

Participation of the upstream region of the fibroin gene in the formation of transcription complex in vitro.

The addition of exogenous histones has an inhibitory effect on fibroin gene transcription in posterior silk gland extracts. The histones probably disturb a process in complex formation, because when transcription complexes were constructed by preincubation of the templates with the extracts, the inhibitory effect of histones was greatly reduced. Transcription of a fibroin gene construct, pFb5' delta-238, having the upstream region beyond the TATA box was relatively less inhibited than that of pFb5' delta-44 lacking the upstream region. This tendency toward differential inhibition was observed in the silk gland extracts but not in a HeLa cell extract and persisted even after complex formation in the silk gland extracts, suggesting a specific interaction of the upstream region with some factors in the extracts. The complexes formed on pFb5' delta-44 are probably more susceptible to the inhibitory effect of histones. On the basis of these results we propose a participation of the upstream region of the fibroin gene in the formation of stable transcription complexes at the promoter through an interaction with specific factors in the silk gland. Since the transcription-enhancing effect via the upstream region is augmented at a high histone/DNA ratio, it may mimic the in vivo situation in which the fibroin gene can be transcribed in the posterior silk gland even in the presence of excess suppressive materials.

Animals↗

PubMed2000

Sulfated fibroin, a novel sulfated peptide derived from silk, inhibits human immunodeficiency virus replication in vitro.

We prepared two kinds of sulfated silk fibroins, SclFib30 and SclFib31, which contain different amounts of sulfate. These sulfated silk fibroins have anti-HIV-1 activity in vitro, apparently due to interference with the adsorption of virus particles to CD4+ cells, and completely blocked virus binding to the cells at a concentration of 100 microg/ml. Sulfated fibroins also abolished cell-to-cell infection-induced syncytium formation upon cocultivation of MOLT-4 and MOLT-4/HIV-IIIB cells, suggesting that they would interfere with gp120 and prevent the formation of gp120/CD4 complex. Silk is used in biomaterials such as surgical sutures and is believed to be a safe material for humans. In accordance with low anticoagulant activity and high anti-HIV-1 activity against both X4 HIV-1 and R5 HIV-1 strains, sulfated silk fibroins have potential as antiviral material such for a vaginal anti-HIV formulation.

Animals↗

PubMed1980

Secretory mechanism of fibroin, a silk protein, in the posterior silk gland cells of Bombyx mori.

There are two microtubule-microfilament systems in the posterior silk gland cells of Bombyx mori. One is a radial microtubule system; the other is a circular microtubule-microfilament system. These two systems are presumably concerned with the intracellular transport of secretory granules of fibroin and the secretion of fibroin into the lumen, respectively. Conventional and scanning electron microscopic observations of the two microtubule-microfilament systems in the posterior silk gland cells are reported. Scanning electron micrographs showed that a number of parallel linear cytoplasmic processes ran circularly on the luminal surface of the posterior silk gland cells. These processes were assumed to correspond to the circular microtubule-microfilament systems. The effects of cytochalasin (B or D), a secretion stimulating agent of fibroin, on the intracellular recording of membrane potential from the posterior silk gland cells are also reported. Exposure to cytochalasin resulted in depolarization of the membrane potential of the gland cells. Possible functional roles of the two microtubule-microfilament systems in the secretory mechanism of fibroin are discussed with reference to the effects of antimitotic reagents and cytochalasin on these two systems.

Animals↗

PubMed2006

[Progress of silk fibroin in the cell scaffold of tissue engineering].

Recent researches about the application of silk fibroin in cell culture suggested that silk fibroin displayed high rate of cell attachment and growth in vitro culture of most kinds of cells, equivalent to collagen. So silk fibroin can be used for cell scaffold material of tissue engineering, and can be applied to several fields such as tissue engineering of skin, cartilage and blood vessel. The related researches and the prospect of the application of silk fibroin in cell scaffold of tissue engineering are reviewed in this paper.

Animals↗

PubMed1979

The nucleotide sequence adjacent to poly(A) in silk fibroin messenger RNA.

The 3'-terminal nucleotide sequence of fibroin mRNA has been determined. A cDNA transcript primed by oligo(dT) hybridized to the poly(A) segment of fibroin mRNA was used to determine the sequence immediately adjacent to poly(A). A specific primer complementary to this sequence then allowed synthesis of 5'-terminally homogeneous transcripts amenable to analysis by rapid sequencing methods. The 3' terminus of fibroin mRNA contains an unusually long oligo(U) stretch, as well as the sequence A2UA3 recently found in 3' non-coding regions of other eukaryotic mRNAs. It lacks the oligonucleotides characteristic of the repetitive coding region of fibroin mRNA.

Animals↗

PubMed1982

Efficient and strand-selective in vitro transcription initiation by purified RNA polymerase II from a unique site of the fibroin gene.

Using purified RNA polymerase II and the cloned fibroin gene of supercoiled form, we found a novel in vitro transcription initiation site. An analysis by S1 nuclease mapping showed that the transcripts synthesized by RNA polymerase II purified from three different sources gave primarily a common protection band, the 5' terminus of which was located at a specific site about the nucleotide position +25 of the fibroin gene. Such a transcript was not detected in the in vivo RNA; however, this site-specific transcription revealed a coding-strand selectivity and a high efficiency accounting for nearly 90% of total in vitro RNA synthesis. An analysis using a series of 5' deletion mutants of the fibroin gene indicates that this transcription initiation depends on a unique DNA sequence about the nucleotide position +25, which includes an inverted repeat and a sequence homologous to that around the in vivo initiation site of the fibroin gene.

Animals↗

PubMed1996

Entrapment of both glucose oxidase and peroxidase in regenerated silk fibroin membraneCharacterization of the membrane structure and its application to an amperometric glucose sensor employing methylene green as an electron transfer mediato.

Two enzmyes, glucose oxidase and peroxidase, were for the first time simultaneously immobilized in regenerated silk fibroin membrane. The structure and morphology of the regenerated silk fibroin membrane containing both glucose oxidase and peroxidase were investigated with IR spectra and SEM. The bienzymes do not change the structures of the regenerated silk fibroin in the membrane, which has an islands-sea structure. For the first time, an amperometric methylene green mediating sensor for glucose based on co-immobilization of both glucose oxidase and peroxidase in regenerated silk fibroin was constructed. Cyclic voltammetry and amperometry were used to test the suitability of methylene green shuttling electrons between peroxidase and the glassy carbon electrode. The bienzyme-based system offers fast response and high sensitivity of the sensor to glucose. The effects of pH, temperature, and the concentration of the mediator on the response current were evaluated, and the dependence of the Michaelis-Menten constant K(m)(app) on the concentration of the mediator was investigated.

Journal Article↗

PubMed2001

Structure of Bombyx mori silk fibroin before spinning in solid state studied with wide angle x-ray scattering and (13)C cross-polarization/magic angle spinning NMR.

The structure of a crystalline form of Bombyx mori silk fibroin, commonly found before the spinning process (known as silk I), has been proposed as a repeated beta-turn type II-like structure by combining data obtained from solid-state two dimensional spin-diffusion nuclear magnetic resonance and rotational-echo double-resonance (T. Asakura et al., J Mol Biol, in press). In this paper, the WAXS pattern of alanine-glycine alternating copolypeptide, (Ala-Gly)(15) with silk I form which was used for a silk I model of B. mori silk fibroin was observed. The pattern calculated with the silk I model proposed by us is well reproduced the observed one, indicating the validity of the proposed silk I model. In addition, two peptides of the other repeated sequences which contain Tyr or Val residues in the silk fibroin,23 were synthesized; (Ala-Gly-Tyr-Gly-Ala-Gly)(5) and (X-Gly)(15) where X is Tyr for the 7th, 15th and 23th residues, and Val for the 11th residue and Ala for other residues. There are no sharp peaks in the WAXS patterns, and therefore both samples are in the non-crystalline state. This is in agreement with the (13)C CP/MAS NMR result, where the conformation is mainly random coil.

Amino Acid Sequence↗

PubMed2004

Structure and structural changes of the silk fibroin from Samia cynthia ricini using nuclear magnetic resonance spectroscopy.

The structure of silk fibroin from a wild silkworm, S. c. ricini, the amino acid sequence of which consists of repeated poly-Ala and Gly-rich regions, was examined by using solution and solid-state NMR methods. The structural transition of the silk fibroin in aqueous solution was monitored by using 13C solution NMR spectroscopy as a function of temperature. The fast exchange with respect to the chemical shift between the helix and coil conformations was observed in the poly-Ala region and the slow conformational change from alpha-helix to random coil was observed for the Gly residue adjacent to the N-terminal Ala residue of the poly-Ala region. The torsion angles of several Ala and Gly residues in the model peptide, GGAGGGYGGDGG(A)12GGA-GDGYGAG, were determined by the conformation-dependent 13C chemical shifts, rotational echo double resonance (REDOR) and 2D spin-diffusion NMR methods. The solid-state NMR analysis leads to the precise silk structure before spinning, where the poly-Ala sequence takes a typical alpha-helix pattern with a tightly winded helical structure at both terminal regions of the poly-Ala sequence. This is expected to stabilize the alpha-helical structure of the poly-Ala region in S. c. ricini silk fibroin from the silkworm.

Amino Acids↗

PubMed1996

An amperometric new methylene blue N-mediating sensor for hydrogen peroxide based on regenerated silk fibroin as an immobilization matrix for peroxidase.

A simple and effective procedure was described for the immobilization of peroxidase in regenerated silk fibroin membrane prepared from waste silk. The membranes of regenerated silk fibroin with or without peroxidase, before or after the ethanol treatment, were characterized by ir spectra. An amperometric H202 sensor, based on the immobilized peroxidase in regenerated silk fibroin membrane, in the use of new methylene blue N as an electron transfer mediator, was fabricated. The characteristics of the sensor with respect to linearity, response time, effect of pH and temperature, stability, and reproducibility were investigated. Dependences of Michaelis-Menten constant KMapp on the concentration of the mediator, and the applied potential were also studied and the results were presented. The sensor was highly sensitive to H2O2 with a detection limit of 1.0 x 10(-7)M and with response time of less than 40 s.

Biosensing Techniques↗

PubMed1985

Molecular cloning of the fibroin light chain complementary DNA and its use in the study of the expression of the light chain gene in the posterior silk gland of Bombyx mori.

Fibroin light chain (L-chain) mRNA (mol. wt 4.0 X 10(5) daltons) was purified from the posterior silk gland of the silkworm, Bombyx mori (J-131 strain). Double-stranded complementary DNA was synthesized and inserted into the PstI site of pBR322 employing the oligo(dC)-oligo(dG) tailing method. Several recombinant plasmids containing the inserts of about 800 base pairs were isolated. Hybridization-translation assay demonstrated that these clones hybridized specifically with the fibroin L-chain mRNA. One of these clones (pLA23) was used as a probe to investigate relative concentrations of the fibroin L-chain gene and mRNA in the posterior silk glands at different stages of late larval development.

Animals↗

PubMed2003

The expression of GFP under the control of fibroin promotor in primary ovarian cells of Antheraea pernyi.

The fibroin promoter can stably express foreign gene in lepidopteran cells. Total RNA was extracted from the gland of silkworm, Antheraea pernyi and the transcription initiation site of fibroin gene of A. pernyi was identified by RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). The expression vector (pGFP-N2/Fib) was constructed by use of replacing the CMV promoter with the fibroin promoter. The results of visual screening under a fluorescent inverted microscope and Western blot analysis indicated that the GFP gene was expressed in the primary cells of ovary origins from A. pernyi.

Animals↗