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PubMed1998

Effect of surface properties on the antithrombogenicity of silk fibroin/S-carboxymethyl kerateine blend films.

Polymeric blends of silk fibroin (SF) and S-carboxymethyl kerateine (SCMK) were prepared by the solvent casting method to study the effect of surface properties on the antithrombogenicity. The films of SF/SCMK showed better antithrombogenic properties than SF or SCMK alone. Among them, the film containing 50 wt% SCMK showed the best antithrombogenicity. When the SF/SCMK films were treated with methanol, the antithrombogenicity of the films was scarcely affected except the SF-rich ones. The enhanced antithrombogenic properties were explained in terms of polarity of the surface. The blend films showed an enhancement of polar contribution to surface free energy (gamma(P)S and polar stabilization energy (I(SW)). SF-rich films showed high gamma(P)S and I(SW) values when treated with methanol. This change of surface properties was considered to be due to the fact that the conformational transition from random coil structure to beta-structure of proteins may have affected the surface properties, especially the polar properties.

Amino Acids↗

PubMed1984

Some ultrastructural aspects of genetic activity in eukaryotes.

A brief overview is given of the types of information that have been obtained by chromatin spreading methods regarding the ultrastructure and regulation of genetic units. The examples used are: ribosomal RNA genes of the spotted newt; the silk fibroin gene of Bombyx mori; chorion gene amplification in Drosophila melanogaster; RNA synthesis patterns during early embryogenesis of D. melanogaster; regulation of transcription on homologous non-ribosomal transcription units of D. melanogaster; specific nascent transcript cleavage in insects; and nascent polypeptides on insect polyribosomes. The conclusion is drawn that further innovations will be required before significant advances in the use of chromatin spreading techniques to study ultrastructural aspects of genetic activity can occur.

Animals↗

PubMed1993

[Gene expression and cellular function].

During the development of vertebrates, differentiated cells are already programmed to synthesize functional proteins to express their specified cellular functions by receiving several kinds of extracellular signals. By use of the in vitro and in vivo systems for analyzing the molecular mechanisms of antibody synthesis in B-lymphocytes, I have found that an activation of immunoglobulin gene could be involved in the activation of antibody synthesis induced by antigen signals. By developing the cell-free transcriptional systems of silk fibroin gene, I have subsequently found that the gene activation could mainly be achieved by the interactions between the defined nucleotide sequences located upstream from the TATA box of the gene and transcriptional factors which can specifically bind to these upstream sequences. To understand an exact role of neuronal cells in the generation of synaptic plasticity, I am now investigating the genetical responses of neuronal cells activated by synaptic transmission. Elucidation of the gene products whose expression could be affected by the synaptic transmission would reveal functional aspects of neuronal cells when they receive several kinds of synaptic inputs. By use of the primary culture of mouse cerebellar granule cells, we have already revealed several aspects of intracellular mechanisms responsible for the glutamatergic responses of the neuronal cells, focusing on an activation of transcriptional factors whose expression could be immediately induced by the glutamatergic inputs. This type of experiments would provide a valuable insight for understanding the cellular and the molecular mechanisms of synaptic plasticity.

Animals↗

PubMed1990

Transient expression of chimeric CAT genes injected into early embryos of the domesticated silkworm Bombyx mori.

In order to establish a transient expression system for genes introduced into early embryos of the silkworm, Bombyx mori, we tested various promoters ligated with CAT reporter genes. The embryos into which we injected supercoiled plasmid DNA of pFb(-860/+10)CAT containing the Bombyx fibroin promoter region ligated to the CAT gene showed a reasonably high CAT activity beginning around 30 h after oviposition. This high activity was observed only when the plasmid was injected before termination of the early nuclear cleavage stage, which was about 8 h after oviposition, but not after this stage. This means that the expression of injected DNA is closely related to the presence of cleavage nuclei in early embryos. Promoters originating from insect genes, like the Bombyx sericin-1 gene, Drosophila hsp70 and Drosophila copia LTR, functioned as strong promoters in the embryos. On the contrary, promoters from mammalian virus genes, such as the SV40 early and Moloney murine leukemia virus LTR genes, functioned as weak promoters. Moreover, linearized DNAs showed no or weak activity of expression in embryos. From these results, we conclude that the silkworm embryo transient expression system is a useful tool for studying the mechanism of regulation of insect genes.

Animals↗

PubMed2005

Characterization of historic silk by polarized attenuated total reflectance Fourier transform infrared spectroscopy for informed conservation.

When assessing historic textiles and considering appropriate conservation, display, and storage strategies, characterizing the physical condition of the textiles is essential. Our work has concentrated on developing nondestructive or micro-destructive methodologies that will permit this. Previously, we have demonstrated a correlation between the physical deterioration of unweighted and "pink" tin (IV) chloride weighted silk and certain measurable spectroscopic and chromatographic signatures, derived from polarized Fourier transform infrared attenuated total reflectance (FTIR-ATR) spectroscopy (Pol-ATR) and high-performance liquid chromatography (HPLC) microsampling analyses. The application of the Pol-ATR technique to aged silk characterization has now been extended to include a more comprehensive range of weighting methods and aging regimes. This was intended to replicate the full spectrum of states of deterioration observed in silk textiles, from pristine to heavily degraded. Breaking strength was employed as a measure of the physical integrity of the fibers, and, as expected, decreased with aging. An orientational crystallinity parameter, reflecting the microstructural ordering of the fibroin polymer within the fibers, was derived from the Pol-ATR spectra. A good correlation was observed between the breaking strength of the variety of fibers and this parameter. This suggests that the physical state of historic silk fabrics might be adequately characterized for conservation purposes by such indirect micromethodology.

Animals↗

PubMed2005

[Study on the conditions of high density fermentation for the engineering bacterial strain with RGD spider silk protein gene].

Spider silk becomes the protein fibroin with excellent character in the natural world because of its unique mechanical properties. The genetic engineering technique is an effective method to procure the spider silk protein. In order to obtain RGD-spider silk protein gene recombinant engineering strain pNSR-16 on a regular scale, we determine the optimal conditions of the growth and expression of pNSR-16 through culture in laboratory, and based on these, study the fed-batch high density culture. We control the flow of carbon-nitrogen, the dissolved oxygen concentration and the cell specific growth rate, which make the final cell density and concentration of recombinant protein of total protein arrive at OD600 57.15 and 20.8%, respectively.

Animals↗

PubMed1990

Codon usage and secondary structure of mRNA.

The specific codon usage pattern of the repetitive unit nucleotide sequence of silk fibroin mRNA suggests that selection has operated on the codon usage to optimize the secondary structure characteristic of the mRNA. The correlation between the stability map of local secondary structure of type I collagen mRNA and the codon usage pattern and the translation rate of the collagen is also implied.

Animals↗

PubMed1998

Identification of cystatin as a component of carp chorion.

An ovary-specific cystatin is immunocytochemically demonstrated to be localized in the chorions, cortical granules, and yolk granules of carp oocytes, as well as in the follicle cells surrounding oocytes. During cortical reaction, cystatin is exocytosed from cortical granules into the perivitelline space. In situ hybridization confirms that cystatin is synthesized by oocytes and follicle cells. Western blotting reveals that chorion cystatin appears in multiple bands of high molecular weight (from 65 kDa to larger than 200 kDa). No cystatin monomer of 14 kDa is found. These results indicate that chorion cystatin is conjugated with other chorion components. Two forms of conjugates are found. In one form, cystatin, ZP2, fibroin-like substance (FLS), and cathepsin-like substance (CLS) are conjugated, which is extracted by sodium dodecyl sulfate. In the other form, cystatin, FLS, and CLS are conjugated, which is extracted by guanidine thiocyanate (GTC). Most chorion cystatin of oocytes and ovulated eggs is solubilized by GTC, while a large amount of cystatin remains in the fertilization envelope of cortical reacted eggs after extraction by GTC.

Animals↗

PubMed2001

Spider flagelliform silk: lessons in protein design, gene structure, and molecular evolution.

Spiders spin multiple types of silks that are renowned for their superb mechanical properties. Flagelliform silk, used in the capture spiral of an orb-web, is one of the few silks characterized by both cDNA and genomic DNA data. This fibroin is composed of repeating ensembles of three types of amino acid sequence motifs. The predominant subrepeat, GPGGX, likely forms a beta-turn, and tandem arrays of these turns are thought to create beta-spirals. These spring-like helices may be critical for the exceptional ability of capture silk to stretch and recoil. Each ensemble of motifs was found to correspond to a different exon within the flagelliform gene. The pattern of sequence similarity among exons indicates intragenic concerted evolution. Surprisingly, the introns between the iterated exons are also homogenized with each other. This unusual molecular architecture in the flagelliform silk gene has implications for the evolution and maintenance of spider silk proteins.

Amino Acid Sequence↗

PubMed2003

Deposition of bone-like apatite on silk fiber in a solution that mimics extracellular fluid.

The fabrication of apatite-organic polymer hybrids is one of several attractive methods for the development of biomaterials as a substitute for bone. Such materials have both bone-bonding ability and mechanical properties analogous to natural bone. The biomimetic process has focused attention on fabricating such hybrids, where bone-like apatite is deposited on an organic polymer surface in solutions that mimic physiological conditions. In this process, a bone-like apatite layer can be coated onto organic substrates either by using a simulated body fluid (SBF) with ion concentrations nearly equal to those of human extracellular fluid, or by using fluids that are supersaturated with respect to apatite at ambient conditions. In this study, we investigated the ability of natural silk and its related materials to facilitate apatite deposition under biomimetic conditions. Cloths made of raw silk or normal silk fibers were soaked in 1.5SBF, which has 1.5 times the ion concentration of SBF. Sericin film, which is made from an extract of degummed raw silk, was soaked in 1.5SBF. The cloth and the film soaked in 1.5SBF then were characterized by scanning electron microscopic (SEM) observation, energy dispersive X-ray microanalysis (EDX), and thin-film X-ray diffraction (TF-XRD). Apatite deposition was observed on the surface of cloth made from raw silk fiber after it was soaked in 1.5SBF, but it was not observed on cloth made from normal silk fibers. The apatite deposition on the raw silk fiber cloth was accelerated when the fibers were subjected to treatment with CaCl(2) solution at a concentration of at least 1 kmol/m(3) before immersion in 1.5SBF. Apatite deposition also was observed on the sericin film after the film was soaked in 1.5SBF for 7 days. These results indicate that apatite deposition on raw silk cloth is attributable to the catalytic effect of sericin because the surface of raw silk consists of sericin whereas that of normal silk contains fibroin. The deposition of the apatite and its crystal growth are accelerated by the presence of calcium ions on the sericin after treatment with CaCl(2) solution. Thus, sericin on natural silk fiber has the potential to facilitate apatite deposition and can be useful as a polymer material in the fabrication of hybrid materials analogous to bone through biomimetic processes.

Apatites↗

PubMed1995

Flagelliform or coronata glands of Nephila clavipes.

The flagelliform or coronata glands of the orb-web spider, Nephila clavipes, have been studied and compared to other silk-producing glands from the organism. The glands, which produce silk for the double filament of the core thread in the sticky spiral, exhibit three distinct morphological areas: tail, sac, and duct. Electrophoretic separation of the solubilized contents of the glands yields an uppermost diffuse band of high molecular size, preceded by a stepladder of well-defined peptides, which have been shown to be products of discontinuous translation in three other sets of glands. The luminal contents do not migrate as a discrete and well-defined band as those of the other glands, but rather as a diffuse area, typical of glycosylated proteins. Fibroin synthesis is stimulated by the mechanical depletion of the organism's stored silks, as in other Nephila glands, judged by the increased intensity of the bands and also by the structural alterations seen in cross sections of the glands' tails.

Animals↗

PubMed2002

Cross-linking of ZP2 and ZP3 by transglutaminase is required for the formation of the outer layer of fertilization envelope of carp egg.

A transglutaminase (TGase) cDNA was cloned from carp ovary. It was highly homologous to zebrafish TGase. Immunoblot and enzymatical assay showed that TGase was present on the chorion and in the cytoplasm of carp eggs. Addition of TGase inhibitor, cadaverine or ethylene diaminetetracetic acid (EDTA) to the cortical reaction medium impaired the formation of the outer layer of fertilization envelope (FE(o)), the adhesive structure of carp egg. Fibroin-like substance (FLS), cystatin, cathepsin-like substance (CLS), and FEO-1 were the components of FE(o), wherein the majority of the former three were conjugated to form macromolecules of 90-205 kDa while the latter one was present in monomer of 22 kDa. Cadaverine interfered slightly the discharge of FLS conjugates out of the perivitelline space (PVS) but affected profoundly the recruitment of FLS conjugates to FE, whereas EDTA completely inhibited both the release and the recruitment of FLS conjugates to FE. Both EDTA and cadaverine did not inhibit the discharge of FEO-1 out of PVS but could inhibit the recruitment of FEO-1 to FE. The mechanism was studied. ZP2 and ZP3, the major constituents of inner layer of FE, were cross-linked during cortical reaction, which rendered FE hardened. In the presence of EDTA, the cross-linking of ZP2 and ZP3 were inhibited, thus FE remained soft. The PVS of an egg with a hardened FE was less expanded than an egg with a soft FE. It was assumed that a less expanded PVS would generate a higher fluid pressure than a more expanded PVS did. Therefore, the transportation of the macromolecules such as the FLS-cystatin-CLS conjugates out of PVS was facilitated in control and cadaverine-treated eggs whose FE were hardened but was blocked in EDTA-treated eggs whose FE were unhardened. On the other hand, the transportation of small molecules such as FEO-1 out of FE was not restrained, so they were discharged out of the PVS of the control and TGase inhibitor-treated eggs. In addition, TGase activity was also required for the recruitment of FLS conjugates to FE.

Animals↗

PubMed1992

Brain factor from Galleria mellonella (Lepidoptera) stimulating silk gland activity.

Brain extracts from day 1-4 last instar larvae of Galleria mellonella (Lepidoptera) stimulate RNA synthesis in cultured silk glands from day 3 last instar larvae. When the fibroin-synthesizing posterior parts of silk glands were incubated for 3 h in vitro in the presence of brain extract (0.1 brain equivalent), [3H]-uridine incorporation into RNA was stimulated more than twofold. The stimulating effect of brain extract showed a dose response relationship. It is suggested that the heat-resistant and protease-sensitive brain factor is a peptide.

Animals↗

PubMed2001

Microstructural homogeneity of support silk spun by Eriophora fuliginea (C.L. Koch) determined by scanning X-ray microdiffraction.

Scanning X-ray microdiffraction (SXD) permits the 'imaging' in-situ of crystalline phases, crystallinity and texture in whole biopolymer samples on the micrometre scale. SXD complements transmission electron microscopy (TEM) techniques, which reach sub-nanometre lateral resolution but require thin sections and a vacuum environment. This is demonstrated using a support thread from a web spun by the orb-weaving spider Eriophora fuliginea (C.L. Koch). Scanning electron microscopy (SEM) shows a central thread composed of two fibres to which thinner fibres are loosely attached. SXD of a piece of support thread approximately 60 microns long shows in addition the presence of nanometre-sized crystallites with the beta-poly(L-alanine) structure in all fibres. The crystallinity of the thin fibres appears to be higher than that of the central thread, which probably reflects a higher polyalanine content of the fibroins. The molecular axis of the polymer chains in the central thread is orientated parallel to the macroscopic fibre axis, but in the thin fibres the molecular axis is tilted by about 71 degrees to the macroscopic fibre axis. A helical model is tentatively proposed to describe this morphology. The central thread has a homogeneous distribution of crystallinity along the macroscopic fibre axis.

Animals↗

PubMed2001

Spider silk has an ice nucleation activity.

Several ice nucleating substances have been identified, which exist in vivo or can be extracted from biological materials. Spider silk, which has a strong ability for water condensation, has also been found to possess an ice nucleation activity. The freezing temperature of water droplets was higher in the presence than in the absence of spider silk. Moreover, by means of environmental scanning electron microscopy, it was observed that the activity is not due to foreign matter attached to the silk but to the silk fibroin itself.

Animals↗

PubMed2006

Production of poly(L-lactide)-degrading enzyme by Amycolatopsis orientalis for biological recycling of poly(L-lactide).

Efficient production of poly(L-lactide)(PLA)-degrading enzyme was achieved by addition of 0.1% (w/v) silk fibroin powder into a liquid culture medium of an actinomycete, Amycolatopsis orientalis, without other complex nitrogen sources, such as yeast extract and peptone. Scaled-up production of the enzyme in a 5-l jar fermenter showed the possibility of producing this enzyme on an industrial scale at low production cost. The extracellular PLA-degrading enzyme showed potent degrading activity, which is effective for biological recycling of PLA, i.e., 2,000 mg/l of PLA powder was completely degraded within 8 h at 40 degrees C using 20 mg/l purified enzyme. An optically active L-lactic acid with 600 mg/l was obtained as degradation product of PLA without undesirable racemization.

Actinomycetales↗

PubMed2006

Biodegradability and biodegradation of poly(lactide).

Poly(lactide) (PLA) has been developed and made commercially available in recent years. One of the major tasks to be taken before the widespread application of PLA is the fundamental understanding of its biodegradation mechanisms. This paper provides a short overview on the biodegradability and biodegradation of PLA. Emphasis is focused mainly on microbial and enzymatic degradation. Most of the PLA-degrading microorganisms phylogenetically belong to the family of Pseudonocardiaceae and related genera such as Amycolatopsis, Lentzea, Kibdelosporangium, Streptoalloteichus, and Saccharothrix. Several proteinous materials such as silk fibroin, elastin, gelatin, and some peptides and amino acids were found to stimulate the production of enzymes from PLA-degrading microorganisms. In addition to proteinase K from Tritirachium album, subtilisin, a microbial serine protease and some mammalian serine proteases such as alpha-chymotrypsin, trypsin, and elastase could also degrade PLA.

Bacteria↗

PubMed2005

A sericin-derived peptide protects sf9 insect cells from death caused by acute serum deprivation.

Sericin is the silk protein enveloping fibroin fibers in cocoons. Sericin hydrolysate protects cultured Sf9 insect cells from death caused by serum deprivation; the activity depends on the repeats of 38 amino acids. A partial peptide from the 38 residues, SGGSSTYGYS, inhibited serum-deprivation death as well. Cell viabilities in the presence of 10% (v/v) foetal calf serum, no additives and 1 mM: SGGSSTYGYS were 96, 12 and 31% on the third day after inoculation, respectively. Aromatic residues seemed to be important because SGGSSTWGWS had the same activity as SGGSSTYGYS but SGGSSTAGAS had no activity.

Amino Acid Sequence↗