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]
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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.
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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.
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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.
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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.
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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]
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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