the silkworm

This blog covers the entire domain of sericulture. It is designed for providing a common platform for discussion between scientists, policy makers and students in the field. reproduction of content from this blog with due acknowledgement is encouraged.

Tuesday, 1 January 2008

Research updates 04

Nine assorted papers on mulberry, silkworm physiology, molecular biology and seri medicine.

1. Micromorphological Characterization of Ten Mulberry Cultivars (Morus spp)
Magda Biasiolo, Maria Teresa Da Canal, and Noemi Tornadore
Economic Botany Volume 58, Issue 4 (December 2004) pp. 639–646
The micromorphological features of the vegetative and reproductive structures of ten mulberry cultivars grown at the Specialized Sericultural Section of the Agricultural Zoology Experimental Institute of Padua, northeastern Italy, were examined by SEM in order to determine the charactetistics that were the most valuable taxonomically. The observed specimens (leaves, flowers, seeds, and pollen grains) showed micromorphological differences regarding leaf hairiness, quantity of waxes, quality of epidermis cuticle and tepal hairiness. The effects of differing environments in altering the floral sex ratios of this basically monoecious group of plants were also investigated. However, no significant differences between the micromorphology of the seeds and the pollen grains of these selected cultivars were detected. The authors are hopeful that the information gained in this study may prove useful in the future creation of an exhaustive and final catalogue “descriptor” of cultivated varieties belonging to the genus Morus L.
2. Developmental Profile of Annexin IX and its Possible Role in Programmed Cell Death of the Bombyx mori Anterior Silk Gland
Yu Kaneko, Keiko Takaki, Masafumi Iwami, and Sho Sakurai
Zoological Science Volume 23, Issue 6 (June 2006) pp. 533–542
During pupal metamorphosis, the anterior silk gland (ASG) of the silkworm, Bombyx mori, undergoes programmed cell death (PCD), which is triggered by 20-hydroxyecdysone (20E). Annexin IX (ANX IX) has been identified as a 20E-inducible gene in dying ASGs, and we show here that its expression is down-regulated in tissues destined to die but not in tissues that survive pupal metamorphosis. ANX IX expression was high in the ASGs during the feeding period, when the ecdysteroid titer was low, and decreased in response to the rising ecdysteroid titer that triggered pupal metamorphosis. Before gut purge, in vitro exposure of the ASGs to 20E levels corresponding to the ecdysteroid concentration present at the time of gut purge caused a decrease in ANX IX messenger RNA levels. Expression profiles of EcR and USP, and the 20E concentration-responses of these genes, indicate the importance of the relative abundance of EcR-A and EcR-B1 isoforms in ANX IX regulation. These results suggest an involvement of ANX IX in the determination of PCD timing by delaying or suppressing the response to the increase in hemolymph ecdysteroid concentration during the prepupal period.

3. Release of Ecdysteroid-Phosphates from Egg Yolk Granules and Their Dephosphorylation during Early Embryonic Development in Silkworm, Bombyx mori
Ryouichi Yamada, Yumi Yamahama, and Haruyuki Sonobe
Zoological Science Volume 22, Issue 2 (February 2005) pp. 187–198
Newly laid eggs of many insect species store maternal ecdysteroids as physiologically inactive phosphoric esters. In the silkworm Bombyx mori, we previously reported the presence of a specific enzyme, called ecdysteroid-phosphate phosphatase (EPPase), which catalyzes the dephosphorylation of ecdysteroid-phosphates to increase the amount of free ecdysteroids during early embryonic development. In this study, we demonstrated that (1) EPPase is found in the cytosol of yolk cells, (2) ecdysteroid-phosphates are localized in yolk granules, being bound to the yolk protein vitellin (Vn), and (3) Vn-bound ecdysteroid-phosphates are scarcely hydrolyzed by EPPase, although free ecdysteroid-phosphates are completely hydrolyzed by EPPase. Thus, we investigated the mechanism by which ecdysteroid-phosphates dissociate from the Vn-ecdysteroid-phosphate complex, and indicated that the acidification of yolk granules causes the dissociation of ecdysteroid-phosphates from the Vn-ecdysteroid-phosphate complex and thereby ecdysteroid-phosphates are released from yolk granules into the cytosol. Indeed, the presence of vacuolar-type proton-translocating ATPase in the membrane fraction of yolk granules was also verified by Western blot analysis. Our experiments revealed that Vn functions as a reservoir of maternal ovarian ecdysteroid-phosphates as well as a nutritional source during embryonic development. This is the first report showing the biochemical mechanism by which maternal Vn-bound ecdysteroid-phosphates function during early embryonic development.

4. Establishment of a Sandwich ELISA System to Detect Diapause Hormone, and Developmental Profile of Hormone Levels in Egg and Subesophageal Ganglion of the Silkworm, Bombyx mori
Norio Kitagawa, Kunihiro Shiomi, Kunio Imai, Teruyuki Niimi, Toshinobu Yaginuma, and Okitsugu Yamashita
Zoological Science Volume 22, Issue 2 (February 2005) pp. 213–221
In the silkworm Bombyx mori, diapause hormone (DH) is produced in the female subesophageal ganglion (SG) and induces embryonic diapause by targeting developing ovaries. DH is processed from a precursor protein consisting of DH, pheromone biosynthesis activating neuropeptide (PBAN) and three other neuropeptides (SGNPs). Because these five neuropeptides share a common sequence, FXPRLamide, at the C-terminus, a direct and specific assay for DH itself is required in order to understand the profile of concentration changes. In this study, we produced a mouse monoclonal antibody (anti-DH[N] mAb) against the N-terminal region of DH and developed a sandwich enzyme-linked immunosorbent assay using the anti-DH[N] mAb and a rabbit polyclonal antibody against the C-terminus of DH. This procedure enabled us to specifically quantify the DH molecule at femtomolar levels (equivalent to 1/10 of SG). We then plotted DH levels in eggs and SGs during embryonic and post-embryonic development. DH was present in late-stage embryos that had been destined for the production of both diapause and nondiapause eggs. DH levels in SG gradually increased in both types during larval development and peaked at the early pupal stage. At the middle pupal stage, DH levels in SG and SG-brain complex decreased markedly in the diapause-egg producing type, thus indicating active release of DH into the hemolymph. From 5th instar larva to adult, no sexual differences in DH levels were observed in SGs or SG-brain complexes from diapause and nondiapause egg-producing types.

5. Functional analysis of four Gloverin-like genes in the silkworm, Bombyx mori. Kawaoka S, Katsuma S, Daimon T, Isono R, Omuro N, Mita K, Shimada T.
Arch Insect Biochem Physiol. 2007 Dec 12; [Epub ahead of print]
To identify genes involved in the innate immunity of the silkworm Bombyx mori, we constructed a cDNA library from the fat body of Escherichia coli-challenged B. mori larvae. Based on the expressed sequence tag (EST) data and whole genome shotgun sequence analysis, we found four Gloverin-like genes, BmGlov1-4, in the Bombyx genome. Northern blot and RT-PCR analysis showed that BmGlov1-4 were induced in the larval fat body after an immune challenge by the injection of E. coli; however, less induction was observed after the injection of a yeast Candida albicans. In silico sequence analysis revealed the presence of a motif homologous to NF-kappaB binding site in the upstream region of each BmGlov gene. Moreover, we expressed recombinant BmGlov1-4 proteins using the baculovirus expression system, and found that all the recombinant BmGlov1-4 significantly inhibited the growth of E. coli. Arch. Insect Biochem. Physiol. 2007. (c) 2007 Wiley-Liss, Inc.

6. Solubilization of the Ecdysone Binding Protein from Anterior Silk Gland Cell Membranes of the Silkworm, Bombyx mori.
M Elmogy, M Iwami, and S Sakurai
Zoolog Sci. 2007; 24: 971
We previously provided preliminary evidence for the presence of a putative membrane ecdysone receptor (mEcR) anchored in the plasma membranes of anterior silk glands (ASGs) in Bombyx mori. This receptor may act in concert with the conventional EcR in 20E-dependent programmed cell death of these glands. We report here, for the first time, the solubilization of mEcR from ASG membranes using the zwitterionic detergent CHAPS in the presence of NaCl. Our results show by ligand binding assay that mEcR solubilized this way is functionally active and retains 75% of its native binding activity. We also defined experimental conditions that yielded protein/detergent complexes with partial binding activity, which makes it possible to purify the membrane-bound ecdysone binding protein.

7. A silkworm baculovirus model for assessing the therapeutic effects of antiviral compounds: characterization and application to the isolation of antivirals from traditional medicines.
Y Orihara, H Hamamoto, H Kasuga, T Shimada, Y Kawaguchi, and K Sekimizu
J Gen Virol. 2008; 89: 188
Ganciclovir, foscarnet, vidarabine and ribavirin, which are used to treat viral infections in humans, inhibited the proliferation of a baculovirus (Bombyx mori nucleopolyhedrovirus) in BmN4 cells, a cultured silkworm cell line. These antiviral agents inhibited the proliferation of baculovirus in silkworm body fluid and had therapeutic effects. Using the silkworm infection model, the antiviral activity of Kampo medicines was screened and it was found that cinnamon bark, a component of the traditional Japanese medicine Mao-to, had a therapeutic effect. Based on the therapeutic activity, the antiviral substance was purified. Nuclear magnetic resonance analysis of the purified fraction revealed that the antiviral activity was due to cinnzeylanine, which has previously been isolated from Cinnamomum zeylanicum. Cinnzeylanine inhibits the proliferation of herpes simplex virus type 1 in Vero cells. These results suggest that the silkworm-baculovirus infection model is useful for screening antiviral agents that are effective for treating humans infected with DNA viruses.

8. Antidiabetic Properties of 2,5-Dihydroxy-4,3'-Di(beta-D-Glucopyranosyloxy)-trans-Stilbene from Mulberry (Morus bombycis Koidzumi) Root in Streptozotocin-Induced Diabetic Rats.
Heo SI, Jin YS, Jung MJ, Wang MH.
J Med Food. 2007 Dec;10(4):602-7.
We investigated the antidiabetic properties of 2,5-dihydroxy-4,3-di(beta-D-glucopyranosyloxy)-trans-stilbene (DGTS) isolated from Morus bombycis Koidzumi in streptozotocin (STZ)-induced diabetic rats. The DGTS prevented the increase in aspartate aminotransferase, alanine aminotransferase, and blood urea nitrogen levels in serum of diabetic rats. At doses of 200-800 mg/kg, DGTS improved hyperglycemia in the rats, and the hypoglycemic effect of DGTS was comparable to that of tolbutamide. The histological observations showed that DGTS prevented atrophy of pancreatic beta-cells and vascular degenerative changes in the islets. DGTS reversed STZ-induced diabetes and had antioxidant activity in assays of FeCl(2)/ascorbic acid-induced lipid peroxidation in the rats. Levels of cytochrome P450 2E1 mRNA, as measured by reverse transcription-polymerase chain reaction, were lower in the livers of the DGTS-treated rats than those of the control group. These results suggest that DGTS might be beneficial in the treatment of type 1 diabetes

9. Structural Disorder in Silk Proteins Reveals the Emergence of Elastomericity. Dicko C, Porter D, Bond J, Kenney JM, Vollrath F.
Biomacromolecules. 2007 Dec 14; [Epub ahead of print]
Spider silks combine basic amino acids into strong and versatile fibers where the quality of the elastomer is attributed to the interaction of highly adapted protein motifs with a complex spinning process. The evaluation, however, of the interaction has remained elusive. Here, we present a novel analysis to study silk formation by examining the secondary structures of silk proteins in solution. Using the seven different silks of Nephila edulis as a benchmark system, we define a structural disorder parameter (the folding index, gamma). We found that gamma is highly correlated with the ratio of glycine present. Testing the correlation between glycine content and the folding index (gamma) against a selected range of silks, we find quantitatively that, in order to achieve specialization with changes in mechanical performance, the spider's silks require higher structural flexibility at the expense of reduced stability and consequently an increased conversion-energy cost. Taken together, our biophysical and evolutionary findings reveal that silk elastomericity evolved in tandem with specializations in the process of silk spinning.
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Monday, 10 December 2007

SILK SOCKS, A NEW BIOTECH PRODUCT



It is reported by The “Times on line” that researchers at Shinshu University have succeeded in creating a silk thread that is stronger, softer and more durable than conventional silk by genetically modifying silkworms with spider genes. It is also reported that a Japanese manufacturer is already experimenting with the thread, and spider socks, stockings and even fishing lines are expected to appear on the market within a few years. The news item claims that the fiber is going to have far reaching consequences in the textile industry: “It is a miracle substance - lighter than feathers, stronger than steel, one of the toughest fibres found in nature. Now scientists in Japan have found a way of harnessing the remarkable power of spiders’ webs to make anything from tights and fishing nets to bulletproof vests”. The genetically modified silkworm caterpillars spin cocoons, 10 per cent of which consist of spider proteins. The team is hopeful to increase the proportion of spider thread material to 50 per cent. The spider used as gene donor is Nephila clavata, the golden orb spider.

The research team is led by Masao Nakagaki, a professor of insect genetics (Faculty of Textile Science and Technology "Silkworm Genetics and Pathology, Department pf Applied Biology, Shinshu University "). Efforts to harness spider silk were afoot for a number of years through genetically modified goats by extruding it from the udders of female goats. However silkworm is the most apropriate bio reactor for this purpose, given its five thousand years old relation with man and the unique genetic make up of the organism enabling it to be the supplest creature for genetic manipulations. Though the silkworm was heralded as a most important laboratory tool by such veteran genetists as Prof. Y. Tazima, it was yet to be used as one. The creature was named by Carl Linnaeus himself, studied by stalwarts such as Darwin, Pasteur and Malphigi. In fact the ‘Malphigion tubules’ were first identified in silkworms.
Fritz Vollrath, a German zoologist is an authority on ‘nephilia’ silk. In “Discovery” site you can read about his findings on ‘nephilia’ silk properties. (Link: ttp://discovermagazine.com/2001/sep/featbiology/) Also his detailed account on micro scopic analy sis and surface properties of ‘nephilia’ silk can be read from www.blackwell-synergy.com/doi/pdf/10.1046/j.1365-2818.1998.00285.x
A large collection of ‘Nephila’ photographs is available at the site: Photographers direct http://www.photographersdirect.com/stockimages/n/nephila.asp
Stress–strain curves of washed and degummed single-filament silkworm silk and ‘Nephila ‘spider dragline silk are compared to illustrate the extra stress bearing capacity of ‘nephilia’ silk (See the link: http://images.google.co.in/imgres?imgurl=http://www.nature.com/nature/journal/v418/n6899/images/418741a-f2.2.jpg&imgrefurl=http://www.nature.com/nature/journal/v418/n6899/fig_tab/418741a_F2.html&h=621&w=600&sz=72&hl=en&start=10&um=1&tbnid=6CEyhfaSIg4POM:&tbnh=136&tbnw=131&prev=/images%3Fq%3Dsilkworm%26svnum%3D10%26um%3D1%26hl%3Den%26sa%3DN )
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Tuesday, 4 December 2007

Research updates 03

Seven papers on silkworm molecular biology, silk nanotechnology and biomaterial properties.
Inactivation of pyocyanin synthesis genes has no effect on the virulence of Pseudomonas aeruginosa PAO1 toward the silkworm, Bombyx mori.
Chieda Y, Iiyama K, Lee JM, Kusakabe T, Yasunaga-Aoki C, Shimizu . S (Laboratory of Insect Pathology and Microbial Control, Institute of Biological Control, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.)
FEMS Microbiol Lett. 2007 Nov 19; [Epub ahead of print]
The contribution of pyocyanin to the virulence of Pseudomonas aeruginosa against the silkworm Bombyx mori was studied. First, purified pyocyanin was injected into the hemocoel of B. mori. Acute toxicity was observed only when a high dose of pyocyanin was injected. The lethal dose 50% value of pyocyanin was found to be 9.52 mug per larva. Next, mutant strains of phzM and phzS, which encode putative phenazine-specific methytransferase and flavin-containing monooxygenase, respectively, were created, and their virulence was compared with that of the PAO1 parent strain. Although the ability to produce pyocyanin was completely lost in the phz-mutant strains, they maintained the same level of virulence as the PAO1 parent strain. In addition, the complementation of the corresponding gene in trans in the mutant strains did not have any effect on the virulence of those mutant strains. These results indicated that pyocyanin does not act as a virulence factor in B. mori after invasion, which was different from the results obtained in other Lepidopteran host models.
PMID: 18031534 [PubMed - as supplied by publisher]
Related Links
Effect of superoxide dismutase gene inactivation on virulence of Pseudomonas aeruginosa PAO1 toward the silkworm, Bombyx mori. [Appl Environ Microbiol. 2007] PMID: 17220257
Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1. [J Bacteriol. 2001] PMID: 11591691
Pathogenicity of gacA mutant of Pseudomonas aeruginosa PA01 in the silkworm, Bombyx mori. [FEMS Microbiol Lett. 2005] PMID: 15727838
Effect of rpoS mutation on the stress response and expression of virulence factors in Pseudomonas aeruginosa. [J Bacteriol. 1999] PMID: 10383954
Structural and functional analysis of the pyocyanin biosynthetic protein PhzM from Pseudomonas aeruginosa. [Biochemistry. 2007] PMID: 17253782

Silver nanoparticles incorporated electrospun silk fibers.
M Kang, R Jung, HS Kim, JH Youk, and HJ Jin
J Nanosci Nanotechnol. 2007; 7: 3888.
We present a simple and mass-producible method of incorporating silver nanoparticles on the surface of electrospun silk non-woven membranes for the fabrication of antimicrobial wound dressings. Nanofibrous silk membranes with fiber diameters of 460 +/- 40 nm were electrospun from an aqueous Bombyx mori fibroin solution. The electrospun membranes incorporating silver nanoparticles were prepared by dipping the membranes in aqueous silver nitrate (AgNO3) solution (0.5 or 1.0 wt%) followed by photoreduction. Field emission scanning and transmission electron microscopy showed that silver nanoparticles were generated on the electrospun silk fibroin nanofibers as well as inside them. The interaction between the silver nanoparticles and amide groups in the silk fibroin molecules was characterized using X-ray photoelectron spectroscopy.
Link: http://highwire.stanford.edu/cgi/medline/pmid;18047081

Controlled release from multilayer silk biomaterial coatings to modulate vascular cell responses.
Wang X, Zhang X, Castellot J, Herman I, Iafrati M, Kaplan DL. (Department of Biomedical Engineering, Tufts University, Medford, MA 02155, USA.)
Biomaterials. 2007 Nov 27; [Epub ahead of print]
A multilayered silk fibroin protein coating system was employed as a drug carrier and delivery system to evaluate vascular cell responses to heparin, paclitaxel, and clopidogrel. The results demonstrated that the silk coating system was an effective system for drug-eluting coatings, such as for stent applications, based on its useful micromechanical properties and biological outcomes. Cell attachment and viability studies with human aortic endothelial cells (HAECs) and human coronary artery smooth muscle cells (HCASMCs) on the drug-incorporated silk coatings demonstrated that paclitaxel and clopidogrel inhibited smooth muscle cell (SMC) proliferation and retarded endothelial cell proliferation. Heparin-loaded silk multilayers promoted HAEC proliferation while inhibiting HCASMC proliferation, desired outcomes for the prevention of restenosis. The preservation of the phenotype of endothelial cells on silk and heparin-loaded silk coatings was confirmed with the presence of endothelial markers CD-31, CD-146, vWF and VE-Cadherin using immunocytochemistry assays. A preliminary in-vivo study in a porcine aorta showed integrity of the silk coatings after implantation and the reduction of platelet adhesion on the heparin-loaded silk coatings.
Related Links
Silk coatings on PLGA and alginate microspheres for protein delivery. [Biomaterials. 2007] PMID: 17583788
Platelet-derived growth factor receptor antagonist STI571 (imatinib mesylate) inhibits human vascular smooth muscle proliferation and migration in vitro but not in vivo. [J Invasive Cardiol. 2007] PMID: 17541129
Vascular endothelial growth factor and heparin in a biologic glue promotes human aortic endothelial cell proliferation with aortic smooth muscle cell inhibition. [Surgery. 1996] PMID: 8751615
Nanolayer biomaterial coatings of silk fibroin for controlled release. [J Control Release. 2007] PMID: 17628161
Survival of endothelial cells in vitro on Paclitaxel-loaded coronary stents. [J Biomater Appl. 2005] PMID: 15788425
Performance evaluation of a silk protein-based matrix for the enzymatic conversion of tyrosine to L-DOPA.
Acharya C, Kumar V, Sen R, Kundu SC. (Department of Biotechnology, Indian Institute of Technology, Kharagpur, India.)
Biotechnol J. 2007 Nov 22; [Epub ahead of print]
L-DOPA (3,4-dihydroxyphenyl-L-alanine), one of the most important intermediates in the melanin biosynthesis pathway, is used for the treatment of Parkinson's disease. With a view of developing a cheaper and more effective method for the bioconversion of tyrosine to L-DOPA, the potential and performance of a novel fibrous matrix prepared from Bombyx mori silk protein fibroin were evaluated for the immobilization of tyrosinase. Cross-linkage between fibroin and tyrosinase using glutaraldehyde was evident from Fourier transform infra red spectroscopy. Maximum product formation occurred when 1000 U enzyme was immobilized on 20 mg fibroin. The optimum conditions for maximal L-DOPA production using immobilized tyrosinase were 40 degrees C and pH 5.5, conditions that caused a 50% loss of free enzyme activity. Immobilized tyrosinase also showed to have a higher degree of stability during storage and it retained 80% of its original activity after repeated reuses. The efficiency of this immobilized tyrosinase system to produce L-DOPA was high, as evident from a high effectiveness factor, between 0.7 and 0.8, thereby making this method feasible for the large-scale production of L-DOPA.
Related Links
Production of L-DOPA by tyrosinase immobilized on modified polystyrene. [Appl Biochem Biotechnol. 2003] PMID: 14665734
Tyrosinase-catalyzed modification of Bombyx mori silk fibroin: grafting of chitosan under heterogeneous reaction conditions. [J Biotechnol. 2006] PMID: 16621091
L-DOPA production by immobilized tyrosinase. [Appl Biochem Biotechnol. 2000] PMID: 10849837
Innovative effect of illite on improved microbiological conversion of L-tyrosine to 3,4 dihydroxy phenyl L-alanine (L-DOPA) by Aspergillus oryzae ME2 under acidic reaction conditions. [Curr Microbiol. 2006] PMID: 17039388
Fast enzymatic preparation of L-dopa from tyrosine and molecular oxygen: a potential method for preparing [15O]L-dopa. [Int J Rad Appl Instrum [A]. 1990] PMID: 2176194
Silkworm powder containing manganese superoxide dismutase regulated the immunity and inhibited the growth of Hepatoma 22 cell in mice.
Yue WF, Deng W, Li XH, Roy B, Li GL, Liu JM, Wu XF, Sun HX, Yao ML, David WC, Miao YG. (Institute of Sericulture and Apiculture, College of Animal Sciences, Zhejiang University, Hangzhou, 310029, P.R. China, miaoyg@zju.edu.cn.)
Mol Biol Rep. 2007 Nov 22; [Epub ahead of print]
The effects of SOD contained silkworm powder on immune regulation and inhibition against Hepatoma 22 tumor cells in vivo were investigated. The activity of natural killer cell (NK) and the ConA-stimulated spleen proliferation were measured. The results found that the SOD-contained silkworm powder caused an enhancement on NK cell activity, which implied this material modulated the immune system in mice in vivo. The NK cell activities of Hepatoma 22 tumor modeled mice treated with silkworm powder including SOD were increased significantly compared to a modeled control and silkworm powder without SOD, reaching 36.18%. In addition, the ConA-stimulated spleen proliferation of SOD treated mice was higher than that of the controls. The treatment of SOD contained silkworm powder presented 40.3% of average inhibition rate to Hepatoma 22 tumor, showing stronger inhibition against tumor. There were no significant difference in body weight between modeled control and SOD silkworm powder feeding in Hepatoma 22 tumor modeled mice, suggesting the SOD silkworm powder is safety as an inhibitant to tumor. In conclusion, these findings demonstrate that administration of silkworm powder containing SOD results in activation of NK cells and immunity, suggesting the silkworm powder containing SOD plays a positive role in tumor inhibition.
Related Links
Manganese superoxide dismutase expressed in silkworm larvae, Bombyx mori L enhances the NK activity and splenocyte proliferation against Sarcoma 180 tumor cells in vivo. [Mol Biol Rep. 2007] PMID: 17934870
Anti-oxidation and immune responses in mice upon exposure to manganese superoxide dismutase expressed in silkworm larvae, Bombyx mori L. [Cell Biol Int. 2007] PMID: 17452112
Immunity promotion and proteomic identification in mice upon exposure to manganese superoxide dismutase expressed in silkworm larvae. [J Proteome Res. 2007] PMID: 17385907
Effects of silkworm larvae powder containing manganese superoxide dismutase on immune activity of mice. [Mol Biol Rep. 2007] PMID: 17605091
CpG oligodeoxynucleotides inhibit tumor growth and reverse the immunosuppression caused by the therapy with 5-fluorouracil in murine hepatoma. [World J Gastroenterol. 2005] PMID: 15754409
High-level expression of orange fluorescent protein in the silkworm larvae by the Bac-to-Bac system.
Liu JM, David WC, Ip DT, Li XH, Li GL, Wu XF, Yue WF, Zhang X, Miao YG. (Institute of Sericulture and Apiculture, College of Animal Sciences, Zhejiang University, Hangzhou, 310029, P.R. China, miaoyg@zju.edu.cn.)
Mol Biol Rep. 2007 Nov 23; [Epub ahead of print]
This novel orange fluorescent protein (OFP) emits brilliant orange fluorescent light. OFP has high fluorescence quantum yield, fast maturation rate, and stability, which imply this protein should be the most favorable biotechnological tools used to investigate the function of target gene by visualizing, monitoring, and quantifying in living cells. B. mori, silkworm has been used as an important bioreactor for the production of recombinant proteins through baculovirus expression system (BES). In this paper, we used infection technique which introduced the baculovirus DNA into silkworms using a cationic lipofectin reagent instead of directly injecting the virus, and demonstrated a high-level expression of the orange fluorescent protein (OFP) gene in the Bombyx mori, silkworm larvae. When recombinant rBacmid/BmNPV/OFP DNA ranging from 50-100 ng/larval was injected, a sufficient OFP expression in hemolymph was harvested. The recombinant viruses could be obtained from the hemolymph of infected larvae and stored as seed which could be used for the large-scale expression. This procedure omitted the costly and labor-consumed insect cell culture. Further investigation of OFP should provide us with more insight in unlocking the mystery of the mechanisms of autocatalytic bioluminescence and its utilization in biotechnology.
Related Links
Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system. [Appl Microbiol Biotechnol. 2006] PMID: 16158284
A highly efficient method for the generation of a recombinant Bombyx mori nuclear-polyhedrosis-virus Bacmid and large-scale expression of foreign proteins in silkworm (B. mori) larvae. [Biotechnol Appl Biochem. 2007] PMID: 17428194
Cloning and expression of manganese superoxide dismutase of the silkworm, Bombyx mori by Bac-to-Bac/BmNPV Baculovirus expression system. [Appl Microbiol Biotechnol. 2006] PMID: 16804693
A new technique for producing recombinant baculovirus directly in silkworm larvae. [Biotechnol Lett. 2007] PMID: 17091380
An innovative technique for inoculating recombinant baculovirus into the silkworm Bombyx mori using lipofectin. [Res Microbiol. 2004] PMID: 15249063
Sonication-induced gelation of silk fibroin for cell encapsulation.
Wang X, Kluge JA, Leisk GG, Kaplan DL. (Department of Biomedical Engineering, Tufts University, 4 Colby Street, Medford, MA 02155, USA).
Biomaterials. 2007 Nov 19; [Epub ahead of print]
Purified native silk fibroin forms beta-sheet-rich, physically cross-linked, hydrogels from aqueous solution, in a process influenced by environmental parameters. Previously we reported gelation times of days to weeks for aqueous native silk protein solutions, with high ionic strength and temperature and low pH responsible for increasing gelation kinetics. Here we report a novel method to accelerate the process and control silk fibroin gelation through ultrasonication. Depending on the sonication parameters, including power output and time, along with silk fibroin concentration, gelation could be controlled from minutes to hours, allowing the post-sonication addition of cells prior to final gel setting. Mechanistically, ultrasonication initiated the formation of beta-sheets by alteration in hydrophobic hydration, thus accelerating the formation of physical cross-links responsible for gel stabilization. K(+) at physiological concentrations and low pH promoted gelation, which was not observed in the presence of Ca(2+). The hydrogels were assessed for mechanical properties and proteolytic degradation; reported values matched or exceeded other cell-encapsulating gel material systems. Human bone marrow derived mesenchymal stem cells (hMSCs) were successfully incorporated into these silk fibroin hydrogels after sonication, followed by rapid gelation and sustained cell function. Sonicated silk fibroin solutions at 4%, 8%, and 12% (w/v), followed by mixing in hMSCs, gelled within 0.5-2h. The cells grew and proliferated in the 4% gels over 21 days, while survival was lower in the gels with higher protein content. Thus, sonication provides a useful new tool with which to initiate rapid sol-gel transitions, such as for cell encapsulation.
PMID: 18031805 [PubMed - as supplied by publisher]
Related Links
Structure and properties of silk hydrogels. [Biomacromolecules. 2004] PMID: 15132662
Mechanisms of silk fibroin sol-gel transitions. [J Phys Chem B. 2006] PMID: 17064118
Structure and properties of regenerated Antheraea pernyi silk fibroin in aqueous solution. [Int J Biol Macromol. 2007] PMID: 17173967
Conformation transition kinetics of Bombyx mori silk protein. [Proteins. 2007] PMID: 17436322 New oral dosage form for elderly patients: preparation and characterization of silk fibroin gel. [Chem Pharm Bull (Tokyo). 1995] PMID: 7728934
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Wednesday, 3 October 2007

RESEARCH UPDATES 02

9 papers on silkworm, 4 papers on silk proteins and 5 papers on medicinal applications of sericulture
A. SILKWORM
1. Determination of phosphorylated amino acid residues of Rab8 from Bombyx mori.
Uno T, Nakada T, Okamaoto S, Nakamura M, Matsubara M, Imaishi H, Yamagata H, Kanamaru K, Takagi M.
Arch Insect Biochem Physiol. 2007 Oct;66(2):89-97.
The Rab family of small GTPases are key regulators of membrane trafficking. Partially purified Rab8 from Bombyx mori (BRab8) was phosphorylated by protein kinase C in mammalian cells in vitro. To determine which of the seven serines and four threonines are phosphorylated, we generated deletion and site-directed mutants of BRab8, inserted them in Escherichia coli, partially purified the encoded fusion proteins by affinity chromatography, and examined their phosphorylation by protein kinase C in vitro. We found that Ser-132 of BRab8 was specifically phosphorylated by protein kinase C. In addition, Western blotting using an antiserum against BRab8 and in-gel staining for phosphorylated proteins revealed that BRab8 is phosphorylated in vivo.
2. The nicotinic acetylcholine receptor gene family of the silkworm, Bombyx mori.
Shao YM, Dong K, Zhang CX.
BMC Genomics 2007, 8:324doi:10.1186/1471-216
Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic cholinergic transmission in the insect central nervous system. The insect nAChR is the molecular target of a class of insecticides, neonicotinoids. Like mammalian nAChRs, insect nAChRs are considered to be made up of five subunits, coded by homologous genes belonging to the same family. The nAChR subunit genes of Drosophila melanogaster, Apis mellifera and Anopheles gambiae have been cloned previously based on their genome sequences. The silkworm Bombyx mori is a model insect of Lepidoptera, among which are many agricultural pests. Identification and characterization of B. mori nAChR genes could provide valuable basic information for this important family of receptor genes and for the study of the molecular mechanisms of neonicotinoid action and resistance. RESULTS: We searched the genome sequence database of B. mori with the fruit fly and honeybee nAChRs by tBlastn and cloned all putative silkworm nAChR cDNAs by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. B. mori appears to have the largest known insect nAChR gene family to date, including nine alpha-type subunits and three beta-type subunits. The silkworm possesses three genes having low identity with others, including one alpha and two beta subunits, alpha9, beta2 and beta3. Like the fruit fly and honeybee counterparts, silkworm nAChR gene alpha6 has RNA-editing sites, and alpha4, alpha6 and alpha8 undergo alternative splicing. In particular, alternative exon 7 of Bma8 may have arisen from a recent duplication event. Truncated transcripts were found for Bma4 and Bma5. CONCLUSIONS: B. mori possesses a largest known insect nAChR gene family characterized to date, including nine alpha-type subunits and three beta-type subunits. RNA-editing, alternative splicing and truncated transcripts were found in several subunit genes, which might enhance the diversity of the gene family.
3. A germline transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon.
Tomita M, Hino R, Ogawa S, Iizuka M, Adachi T, Shimizu K, Sotoshiro H, Yoshizato K. (Yoshizato Project, Cooperative Link of Unique Science and Technology for Economy Revitalization, Hiroshima Prefectural Institute of Industrial Science and Technology, 3-10-32 Kagamiyama, Higashihiroshima, Hiroshima, 739-0046, Japan.)
Transgenic Res. 2007 Aug;16(4):449-65. Epub 2007 Apr 6.
A silk thread of the silkworm, Bombyx mori, is composed of the insoluble inner fibroin and the hydrophilic outer sericin layer, which are synthesized in the posterior and middle silk gland (MSG), respectively. This study aimed to develop a novel sericin 1 gene (ser1) promoter-driven recombinant expression system using transgenic silkworms, in which recombinant proteins are synthesized in MSG and secreted into the sericin layer. To obtain a high level of gene expression, we tested whether a baculovirus-derived enhancer, hr3, and a trans-regulator, IE1, are capable of stimulating the transcriptional activity of the ser1 promoter, using a transient gene expression system. The results showed that hr3 and IE1 cooperatively increased the ser1 promoter activity more than 30-fold. Then, transgenic silkworms were generated which expressed the EGFP with the signal peptide in MSG under the control of the hr3-linked ser1 promoter and IE1 gene. The silkworms exclusively secreted the EGFP into the sericin layer of cocoons as predicted. The expressed EGFP was extractable from cocoons through a simple procedure with neutral pH buffer solution. The expression system developed in this study enables us to produce recombinant proteins in bulk that can be easily extracted and purified.
4. Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon: production of recombinant human serum albumin
Ogawa S, Tomita M, Shimizu K, Yoshizato K.(Yoshizato Project, Cooperative Link of Unique Science and Technology for Economy Revitalization, Hiroshima Prefectural Institute of Industrial Science and Technology, 3-10-32 Kagamiyama, Higashihiroshima, Hiroshima 739-0046, Japan.)
J Biotechnol. 2007 Feb 20;128(3):531-44. Epub 2006 Nov 17.
In this study we produced germline transgenic silkworms that spin cocoons containing recombinant human serum albumin (rHSA) in the sericin layer. A piggyBac-based transformation vector was constructed that carried HSA cDNA driven by sericin-1 gene promoter, viral enhancer hr3, and gene encoding viral trans-activator IE1. Isolated silk glands were bombarded with the vector and transplanted into host larvae. Three days later, the transplants were immunohistochemically analyzed, which showed that middle silk gland (MSG) cells expressed rHSA and secreted it into the MSG lumen. Then, silkworm eggs were injected with the vector and developed to larvae. The obtained transgenic silkworms spun silk threads whose sericin layers contained rHSA at 3.0microg/mg of cocoons. Most (83%) of the rHSA in cocoons was extracted with phosphate buffered saline, which was then subjected to ammonium sulfate precipitation and affinity chromatography. Finally, we obtained 2.8mg of 99%-pure rHSA from 2g of cocoons. Measurements of circular dichroism spectra of rHSA, and equilibrium dissociation constants of rHSA to warfarin and naproxen indicated that rHSA was conformationally and functionally identical to natural plasma HSA. Germline transgenic silkworms will be useful for producing various recombinant proteins in the sericin layer of cocoons.
5. Silk gland specific secretory expression of egfp gene in silkworm Bombyx mori with rAcMNPV system.
Guo XY, Guo TQ, Wang SP, Wang JY, Lu CD. (Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, PR China.)
Arch Virol. 2005 Jun;150(6):1151-60. Epub 2005 Feb 10.
To evaluate the possibility of establishing an in vivo baculovirus expression system in a silk gland specific secretory way, the recombinant Autographa californica nucleopolyhedrovirus (AcserpegfpDeltaEGT) carrying the reporter gene egfp downstream of silkworm ser1 promoter and signal peptide coding sequence was generated. The purified recombinant baculovirus AcserpegfpDeltaEGT was injected into the haemocoel of newly ecdysed 5thHendekl) instar silkworm larvae at the amount of 10(6) pfu per larva. At 5 days post injection, green fluorescence derived from EGFP could be observed with fluorescent microscope in only the silk gland but not other tissues after dissection of the silkworm. By making an opening on the silk gland wall, green fluorescence could be observed in the outflow of silk gland indicating the secretion of EGFP and the effectiveness of ser1 signal peptide. Western blotting assay confirmed that EGFP exists in the water-soluble part of cocoon silk too. We also established a simple protocol to purify EGFP from the secreted silk proteins.
6. Analysis of tissue-specific region in sericin 1 gene promoter of Bombyx mori.
Liu Y, Yu L, Guo X, Guo T, Wang S, Lu C. (College of Biomedical Engineering and Instrument Science, Zhejiang University, Hangzhou 310027, China.)
Biochem Biophys Res Commun. 2006 Mar 31;342(1):273-9. Epub 2006 Feb 6
The gene encoding sericin 1 (Ser1) of silkworm (Bombyx mori) is specifically expressed in the middle silk gland cells. To identify element involved in this transcription-dependent spatial restriction, truncation of the 5' terminal from the sericin 1 (Ser1) promoter is studied in vivo. A 209bp DNA sequence upstream of the transcriptional start site (-586 to -378) is found to be responsible for promoting tissue-specific transcription. Analysis of this 209bp region by overlapping deletion studies showed that a 25bp region (-500 to -476) suppresses
the ectopic expression of the Ser1 promoter. An unknown factor abundant in fat body nuclear extracts is shown to bind to this 25bp fragment. These results suggest that this 25bp region and the unknown factor are necessary for determining the tissue-specificity of the Ser1 promoter.
7. Expression in Escherichia coli and purification of bioactive antibacterial peptide ABP-CM4 from the Chinese silk worm, Bombyx mori.
Li BC, Zhang SQ, Dan WB, Chen YQ, Cao P. (Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Jiangsu, Nanjing, PR China.)
Biotechnol Lett. 2007 Jul;29(7):1031-6. Epub 2007 Mar 21.
The antibacterial peptide CM4 (ABP-CM4), isolated from Chinese Bombys mori, is a 35-residue cationic, amphipathic alpha-helical peptide that exhibits a broad range of antimicrobial activity. To explore a new approach for the expression of ABP-CM4 in E. coli, the gene ABP-CM4, obtained by recursive PCR (rPCR), was cloned into the vector pET32a to construct a fusion expression plasmid. The fusion protein Trx-CM4 was expressed in soluble form, purified by Ni(2+)-chelating chromatography, and cleaved by formic acid to release recombinant CM4. Purification of rCM4 was achieved by affinity chromatography and reverse-phase HPLC. The purified of recombinant peptide showed antimicrobial activities against E. coli K(12)D(31), Penicillium chrysogenum, Aspergillus niger and Gibberella saubinetii. According to the antimicrobial peptide database (http://aps.unmc.edu/AP/main.html), 116 peptides contain a Met residue, but only 5 peptides contain the AspPro site, indicating a broader application of formic acid than CNBr in cleaving fusion protein. The successful application to the expression of the ABP-CM4 indicates that the system is a low-cost, efficient way of producting milligram quantities of ABP-CM4 that is biologically active.
8. Expression of the antibacterial peptide CM4-like gene of Chinese silkworm Bombyx mori in Escherichia coli and its antibacterial activity analysis
Li BC, Chen YQ, Liu P, Zhang SQ. (The Life Science School of Nanjing Normal University, Nanjing 210097. jslbc@126.com)
Fen Zi Xi Bao Sheng Wu Xue Bao. 2007 Apr;40(2):98-102
[Article in Chinese] According to the amino acid sequence of CM4 and the bias for preferred condons of E. coli, the CM4-like gene was obtained by a recursive PCR (rPCR) strategy using two lapping oligonucleotides. The synthesized gene was coloned into the expression vector pET32(a) and transformed into E. coli BL21 (DE3). Recombinant CM4-like gene expression was driven by the T7 promoter on the vector upon addition of IPTG and high level of expression was achieved. The solube protein was purified by Ni-chelating agarose and treated with formic acid. After cleavege, the recombinant peptide was purified by another Ni(2+)-NTA-Agarose affinity chromatography and cation-exchange chromatography. Results of agarose diffuse assay and liquid turbidity analysis indicated that the recombinant peptide exhibited the antibacterial activity.
9. Structural Basis of Ligand Binding and Release in Insect Pheromone-binding Proteins: NMR Structure of Antheraea polyphemus PBP1 at pH 4.5.
Damberger FF, Ishida Y, Leal WS, Wuthrich K.
J Mol Biol. 2007 Aug 17; [Epub ahead of print]
The NMR structure of the Antheraea polyphemus pheromone-binding protein 1 at pH 4.5, ApolPBP1(A), was determined at 20 degrees C. The structure consists of six alpha-helices, which are arranged in a globular fold that encapsulates a central helix alpha7 formed by the C-terminal polypeptide segment 131-142. The 3D arrangement of these helices is anchored by the three disulfide bonds 19-54, 50-108 and 97-117, which were identified by NMR. Superposition of the ApolPBP1(A) structure with the structure of the homologous pheromone-binding protein of Bombyx mori at pH 4.5, BmorPBP(A), yielded an rmsd of 1.7 A calculated for the backbone heavy-atoms N, C(alpha) and C' of residues 10-142. In contrast, the present ApolPBP1(A) structure is different from a recently proposed molecular model for a low-pH form of ApolPBP1 that does not contain the central helix alpha7. ApolPBP1 exhibits a pH-dependent transition between two different globular conformations in slow exchange on the NMR chemical shift timescale similar to BmorPBP, suggesting that the two proteins use the same mechanism of ligand binding and ejection. The extensive sequence homology observed for pheromone-binding proteins from moth species further implies that the previously proposed mechanism of ligand ejection involving the insertion of a C-terminal helix into the pheromone-binding site is a general feature of pheromone signaling in moths.
B. SILK PROTEINS
1. Structure and properties of regenerated Antheraea pernyi silk fibroin in aqueous solution
Tao W, Li M, Zhao C.(School of Material Engineering, Stem Cell Research Laboratory of Jiangsu Province, Suzhou University, Campus Box 64, No. 178 East Gan-Jiang Road, Suzhou 215021, China.)
Int J Biol Macromol. 2007 Apr 10;40(5):472-8. Epub 2006 Nov 24.
Antheraea pernyi silk fibroin fibers were dissolved by aqueous lithium thiocyanate to obtain regenerated A. pernyi silk fibroin solution. By means of circular dichroism, (13)C NMR and Raman spectroscopy, the molecular conformation of regenerated A. pernyi silk fibroin in aqueous solution was investigated. The relationship of environmental factors and sol-gel transformation behavior of regenerated A. pernyi silk fibroin was also studied. The molecular conformations of regenerated A. pernyi silk fibroin mainly were alpha-helix and random coil in solution. There also existed a little beta-sheet conformation. It was obviously different with Bombyx mori silk fibroin, whose molecular conformation in solution was only random coil but no alpha-helix existence. With the increase of temperature and solution concentration and with the decrease of solution pH value, the gelation velocity of regenerated A. pernyi silk fibroin solution increased. Especially, it showed that A. pernyi silk fibroin was more sensitive to temperature than B. mori silk fibroin during the sol-gel transformation. The velocity increased obviously when the temperature was above 30 degrees C. During the sol-gel transformation, the molecular conformation of regenerated A. pernyi silk fibroin changed from random coil to beta-sheet structure. The results of these studies provided important insight into the preparation of new biomaterials by silk fibroin protein.
2. Tyrosinase-catalyzed modification of Bombyx mori silk fibroin: grafting of chitosan under heterogeneous reaction conditions.
Freddi G, Anghileri A, Sampaio S, Buchert J, Monti P, Taddei P. (Stazione Sperimentale per la Seta, via Giuseppe Colombo 83, Milano, Italy. freddi@ssiseta.it)
J Biotechnol. 2006 Sep 1;125(2):281-94. Epub 2006 Apr 18
The capability of mushroom tyrosinase to catalyze the oxidation of tyrosine residues of Bombyx mori silk fibroin was studied under heterogeneous reaction conditions, by using a series of silk substrates differing in surface and bulk morphology and structure, i.e. hydrated and insoluble gels, mechanically generated powder and fibre. Tyrosinase was able to oxidize 10-11% of the tyrosine residues of silk gels. The yield of the reaction was very low for the powder and undetectable for fibres. FT-Raman spectroscopy gave evidence of the oxidation reaction. New bands attributable to vibrations of oxidized tyrosine species (o-quinone) appeared, and the value of the I853/I829 intensity ratio of the tyrosine doublet changed following oxidation of tyrosine. The thermal behaviour of SF substrates was not affected by enzymatic oxidation. o-Quinones formed by tyrosinase onto gels and powder were able to undergo non-enzymatic coupling with chitosan. FT-IR and FT-Raman spectroscopy provided clear evidence of the formation of silk-chitosan bioconjugates under heterogeneous reaction conditions. Chitosan grafting caused a beta-sheet --> random coil conformational transition of silk fibroin and significant changes in the thermal behaviour. Chitosan grafting did not occur, or occurred at an undetectable level on silk fibres. The results reported in this study show the potential of the enzymatically initiated protein-polysaccharide grafting for the production of a new range of bio-based, environmentally friendly polymers.
3. Chemical and physical properties of sulfated silk fabrics.
Taddei P, Arosio C, Monti P, Tsukada M, Arai T, Freddi G.(Centro di Studio sulla Spettroscopia Raman, Dipartimento di Biochimica G. Moruzzi, Università di Bologna, via Belmeloro 8/2, Bologna 40126, Italy.)
Biomacromolecules. 2007 Apr;8(4):1200-8. Epub 2007 Mar 6.
Silk fabrics were treated with chlorosulphonic acid in pyridine for different times. The amount of sulfur bound to silk increased during the first 2 h of reaction and then reached a plateau. The amino acidic pattern of sulfated silk remained essentially unchanged for short reaction times (< or ="2"> or =2 h). Spectroscopic analyses performed by FT-IR and FT-Raman showed the appearance of new bands attributable to various vibrations of sulfated groups. The IR bands at 1049 and 1014 cm-1, due to organic sulfate salts, were particularly intense. Bands assigned to alkyl sulfates and sulfonamides appeared in the 1300-1180 cm-1 range. Organic covalent sulfates displayed a weak but distinct IR band at 1385 cm-1. Both IR and Raman spectra revealed that silk fibroin mainly bound sulfates through the hydroxyl groups of Ser and Tyr, while involvement of amines could not be proved. Changes observed in the amide I and II range indicated an increase of the degree of molecular disorder of sulfated silk. Accordingly, the I850/I830 intensity ratio between the two Tyr bands at 850-830 cm-1 increased from 1.41 to 1.52, indicating a more exposed state of Tyr residues in sulfated silk. TGA, DSC, and TG analyses showed that sulfated silk attained a higher thermal stability. A thermal transition attributable to sulfated silk fibroin fractions appeared at about 260 degrees C in the DSCthermograms.
4. A study on the flow stability of regenerated silk fibroin aqueous solution.
Wang H, Zhang Y, Shao H, Hu X.(State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Material Science and Engineering, Donghua University, Shanghai 200051, PR China).
Int J Biol Macromol. 2005 Jul;36(1-2):66-70
The flow stability of silk fibroin (SF) aqueous solutions with different concentrations under different temperatures was investigated. It was found that the flow stability decreased quickly with the increase of solution concentration and temperature. X-ray diffraction, Fourier transform infrared (FTIR) and Raman spectroscopy analysis showed that silk fibroin in aqueous solution was mainly in random coil and alpha-helix conformation. However, it turned into alpha-helix and beta-sheet conformation after gelation, and both silk I and silk II crystalline structures appeared accordingly. The investigation implies that the original dilute regenerated SF aqueous solution should be stored under low temperature and concentrated just before spinning.
C. MEDICINAL APPLICATIONS
1. Development and evaluation of silk fibroin-based nerve grafts used for peripheral nerve regeneration.
Yang Y, Ding F, Wu J, Hu W, Liu W, Liu J, Gu X.
Biomaterials. 2007 Sep 18; [Epub ahead of print]
Slk fibroin (SF), derived from natural silk long used as a textile material, has recently become an important biomaterial for tissue engineering applications. We have previously reported on good in vitro biocompatibility of SF fibers with peripheral nerve tissues and cells. In the present study, we developed a novel biomimetic design of the SF-based nerve graft (SF graft) which was composed of a SF-nerve guidance conduit (NGC) inserted with oriented SF filaments. The SF-NGC prepared via well-established procedures exhibits an eggshell-like microstructure that is responsible for its superior mechanical and permeable properties beneficial to nerve regeneration. The SF graft was used for bridge implantation across a 10-mm long sciatic nerve defect in rats, and the outcome of peripheral nerve repair at 6 months post-implantation was evaluated by a combination of electrophysiological assessment, FluoroGold retrograde tracing and histological investigation. The examined functional and morphological parameters show that SF grafts could promote peripheral nerve regeneration with effects approaching those elicited by nerve autografts which are generally considered as the gold standard for treating large peripheral nerve defects, thus raising a potential possibility of using these newly developed nerve grafts as a promising alternative to nerve autografts.
2. Silk-fibroin-coated liposomes for long-term and targeted drug delivery.
Gobin AS, Rhea R, Newman RA, Mathur AB. (University of Texas MD Anderson Cancer Center, Laboratory of Reparative Biology and Bioengineering, Plastic Surgery, Houston, TX 72230, USA.)
Int J Nanomedicine. 2006;1(1):81-7
Many barriers to drug delivery into a tumor site require careful consideration when designing a new drug. In this study, the adhesive targeting and drug specificity of modified liposomal vesicles on human-scar-producing cells, keloid fibroblasts, were investigated. Keloids express abundant levels of mucopolysaccharides and receptor tyrosine kinase (RTK). In this report, the structural properties, drug release kinetics, and therapeutic availability of silk-fibroin-coated, emodin-loaded liposomes (SF-ELP), compared with uncoated, emodin-loaded liposomes (ELP), were investigated. SF-ELP had a highly organized lamellae structure, which contributed to 55% of the liposomal diameter. This modified liposomal structure decreased emodin release rates by changing the release kinetics from a swelling and diffusional process to a purely diffusional process, probably due to steric hindrance. SF-ELP also increased adhesion targeting to keloid fibroblasts. Increased retention of SF-ELP is most likely due to the interaction of the fibrous protein coating around the ELP with the pericellular molecules around the cell. SF-ELP also decreased survival rate of keloids that expressed high levels of RTK. These results demonstrated that SF-ELP enhanced emodin delivery by improved diffusion kinetics and specific cell targeting.
3. Nanolayer biomaterial coatings of silk fibroin for controlled release
Wang X, Hu X, Daley A, Rabotyagova O, Cebe P, Kaplan DL. (Department of Biomedical Engineering, Tufts University, Medford, Massachusetts 02155, USA.)
J Control Release. 2007 Aug 28;121(3):190-9. Epub 2007 Jun 14
An all-aqueous, stepwise deposition process with silk fibroin protein for the assembly of nanoscale layered controlled release coatings was exploited. Model compounds, Rhodamine B, Even Blue and Azoalbumin, representing small molecule drugs and therapeutically relevant proteins were incorporated in the nanocoating process and their loading and release behavior was quantified. In addition, the structure and morphology of the coatings were characterized. Release studies in vitro showed that control of beta-sheet crystal content and the multilayer structure of the silk coatings correlated with the release properties of the incorporated compounds. In particular, higher crystallinity and a thicker silk capping layer suppressed the initial burst of release and prolonged the duration of release. These novel coatings and deposition approach provide a unique option to regulate structure and morphology, and thus release kinetics. The results also suggest these systems as a promising framework for surface engineering of biomaterials and medical devices to regulate the release of drugs, when considered with the all-aqueous process involved, the conformal nature of the coatings, the robust material properties of silk fibroin, and the degradability and biocompatibility of this family of protein.
4. Silk fibroin mediated delivery of liposomal emodin to breast cancer cells.
Cheema SK, Gobin AS, Rhea R, Lopez-Berestein G, Newman RA, Mathur AB. (University of Texas M.D. Anderson Cancer Center, Departments of Biomedical Engineering and Plastic Surgery, Unit 602, P.O. Box 301402, Houston, TX 72230-1402, United States.)
Int J Pharm. 2007 Aug 16;341(1-2):221-9. Epub 2007 Apr 3.
The efficacy of a drug is dependent on its mode of delivery and its potency at the tumor site. In this study, the drug delivery and efficacy of silk fibroin coated liposomes (SF-ELP), encapsulating a receptor tyrosine kinase inhibitor, emodin, on Her2/neu over-expressing breast cancer cells, was investigated. This study demonstrates that SF-ELP was more efficacious in suppressing the growth of Her2/neu over-expressing breast cancer cells MDA-MB-453 and BT-474 as compared to uncoated emodin loaded liposomes (ELP). Reduced levels of phosphorylated Her2/neu correlated with growth inhibition observed in the MDA-MB-453 cells, treated with both ELP and SF-ELP. ELP treatment of MDA-MB-453 breast cancer cells resulted in inhibition of the PI3K pathway whereas SF-ELP treatment inhibited both the PI3K and MAPK pathways, which contributed to the enhanced growth inhibitory effects of Her2/neu over-expressing breast cancer cells. Coating of ELP with silk fibroin did not alter the target specificity of emodin, on the other hand the emodin efficacy was enhanced. Higher uptake of emodin delivered by SF-ELP lead to increased cell death as compared to emodin delivery via ELP. Silk fibroin coating around the liposome imparts an extra layer that emodin has to extravasate in order to release from the encapsulating liposome. This increases retention of the drug in the cell for a longer time and protects emodin from quick release and metabolism. Longer intracellular retention may lead to the longer availability of emodin for down-modulation of various Her2/neu pathways. This study demonstrates that silk fibroin coating enhanced emodin delivery in Her2/neu over-expressing breast cancer cells thereby increasing the overall efficacy of the drug.
5. Food-grade mulberry powder enriched with 1-deoxynojirimycin suppresses the elevation of postprandial blood glucose in humans
Kimura T, Nakagawa K, Kubota H, Kojima Y, Goto Y, Yamagishi K, Oita S, Oikawa S, Miyazawa T. (Food & Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan. kmr@affrc.go.jp)
J. Agric. Food Chem., 55 (14), 5869 -5874, 2007. 10.1021
Mulberry 1-deoxynojirimycin (DNJ), a potent glucosidase inhibitor, has been hypothesized to be beneficial for the suppression of abnormally high blood glucose levels and thereby prevention of diabetes mellitus. However, DNJ contents in commercial mulberry products were as low as about 0.1% (100 mg/100 g of dry product), implying that the bioavailability of DNJ might not be expected. We carried out studies in two directions: (1) production of food-grade mulberry powder containing a maximally high DNJ content; (2) determination of the optimal dose of the DNJ-enriched powder for the suppression of the postprandial blood glucose through clinical trials. The following method was used: (1) DNJ concentrations in mulberry leaves from different cultivars, harvest seasons, and leaf locations were determined using hydrophilic interaction chromatography with evaporative light scattering detection. (2) Healthy volunteers received 0, 0.4, 0.8, and 1.2 g of DNJ-enriched powder (corresponding to 0, 6, 12, and 18 mg of DNJ, respectively), followed by 50 g of sucrose. Before and 30-180 min after the DNJ/sucrose administration, plasma glucose and insulin were determined. The following results were obtained: (1) Young mulberry leaves taken from the top part of the branches in summer contained the highest amount of DNJ. After optimization of the harvesting and drying processes for young mulberry leaves (Morus alba L. var. Shin ichinose), DNJ-enriched powder (1.5%) was produced. (2) A human study indicated that the single oral administration of 0.8 and 1.2 g of DNJ-enriched powder significantly suppressed the elevation of postprandial blood glucose and secretion of insulin, revealing the physiological impact of mulberry DNJ (effective dose and efficacy in humans). This study suggests that the newly developed DNJ-enriched powder can be used as a dietary supplement for preventing diabetes mellitus.
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