Preparation of silk fibroin and polyallylamine composites

Composite films made of silk fibroin (SF) and polyallylamine (PAA) are prepared that contain various compositional ratios. These materials are analyzed to elucidate the resulting physical properties and to assess their potential toward advanced applications as industrial materials. The composite films are obtained from a SF and PAA binary system by dry casting from aqueous solution. These composite films exhibit excellent processability such as film forming capabilities, and the elongation at break is increased in the wet state. The differential scanning calorimetry (DSC) curves of the composites suggest that a mutual interaction takes place between the SF and PAA. This interaction is believed to occur because the endothermic peak, corresponding to the individual polymer, shifts with increasing SF content. The random coil conformation of the SF is present, regardless of the PAA blending, as confirmed by FTIR and DSC measurements. Additionally, living cells from Antheraea pernyi and Bombyx mori insect tissues are shown to grow effectively on the composite films. Maximum growth levels occur when the cultivation flask is coated with the material in SF/PAA ratios of 75:25 to 25:75. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 84: 1963–1970, 2002; DOI 10.1002/app.10491     

Production of antimicrobially active silk proteins by use of metal‐containing dyestuffs

The work presented here discusses a new technique for preparing silk fibers and films with persistent antimicrobial activity through use of metallic dyestuffs during the fiber dyeing process. The length of the silk fibers investigated contracted when the fibers were immersed in concentrated neutral salt solutions, such as calcium or potassium nitrate, at elevated temperature levels. The birefringence and molecular orientation of the silk fibroin molecules became less ordered by the action of the neutral salt solutions, resulting in increased dyestuff absorption. Subsequently, contracted silk fibers were dyed with metallic dyestuffs containing Cr or Cu for the purpose of obtaining silk fibers with antimicrobial activity. Silk fibers dyed with metallic dyestuffs showed significant antimicrobial activity against the plant pathogen Cornebacterium and the human pathogen Coli bacillus. Tensile strength of the silk fibers after the salt shrinking and dyeing processes did not show a significant change, whereas the elongation at break was increased slightly. The techniques described here for preparing significantly active antimicrobial silk fibers are effective and economic ways of providing new materials for industrial and biomedical applications. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 1181–1188, 2002     

An absolute requirement of fructose 1,6-bisphosphate for the Lactobacillus caseiL-lactate dehydrogenase activity induced by a single amino acid substitution

Lactobacillus casei allosteric L-lactate dehydrogenase (L-LDH)absolutely requires fructose 1,6-bisphosphate [Fru(1,6)P2] forits catalytic activity under neutral conditions, but exhibitsmarked catalytic activity in the absence of Fru(1,6)P₂ underacidic conditions through the homotropic activation effect ofsubstrate pyruvate. In this enzyme, a single amino acid replacement,i.e. that of His205 conserved in the Fru(1,6)P₂-binding siteof certain allosteric L-LDHs of lactic acid bacteria with Thr,did not induce a marked loss of the activation effect of Fru(1,6)P₂or divalent metal ions, which are potent activators that improvethe activation function of Fru(1,6)P₂ under neutral conditions.However, this replacement induced a great loss of the Fru(1,6)P₂-independentactivation effect of pyruvate or pyruvate analogs under acidicconditions, consequently indicating an absolute Fru(1,6)P₂ requirementfor the enzyme activity. The replacement also induced a significantreduction in the pH-dependent sensitivity of the enzyme to Fru(1,6)P₂,through a slight decrease and increase of the Fru(1,6)P₂ sensitivityunder acidic and neutral conditions, respectively, indicatingthat His205 is also largely involved in the pH-dependent sensitivityof L.casei L-LDH to Fru(1,6)P₂. The role of His205 in the allostericregulation of the enzyme is discussed on the basis of the knowncrystal structures of L-LDHs.     

Grafting vinyl monomers onto silk (Bombyx mori) using different initiators: Properties of grafted silk

The graft–copolymerization of silk with methacrylamide (MAA), 2‐hydroxyethyl methacrylate (HEMA), and methyl methacrylate (MMA) was studied using three different free‐radical initiators: an inorganic peroxide [ammonium persulfate (APS)] and two azo compounds [2,2′‐azobisisobutyronitrile (AIBN) and 2,2′‐azobis(2‐methylpropionamidine) dihydrochloride (ADC)]. The rate and yield of grafting followed the order: APS > AIBN > ADC. The performance of AIBN was close to that of APS in terms of weight gain attained. The degree of yellowing of grafted silk varied as a function of the initiator–monomer system used. APS induced the highest degree of yellowing, regardless of which monomer was used, whereas silk grafted with the MAA/AIBN system displayed the lowest level of yellowing. Moisture regain of grafted silk changed as a function of the hydrophilic/hydrophobic character of the grafted polymer, regardless of the kind of initiator used. Accordingly, tensile properties showed a tendency to decrease with increasing weight gain of grafted silk, the extent of which was independent of the kind of monomer and initiator used. The different initiators did not induce any appreciable change in the fine structure of silk, as demonstrated by optical measurements. Uneven surface deposition of homopolymer was detected to variable extent with MMA and HEMA grafting, whereas the surface of MAA‐grafted silk was completely free of any foreign deposit, independently of the initiator used. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 81: 1401–1409, 2001     

Hydrostatic Pressure Influences HIF-2 Alpha Expression in Chondrocytes

Hypoxia-inducible factor (HIF)-2α is considered to play a major role in the progression of osteoarthritis. Recently, it was reported that pressure amplitude influences HIF-2α expression in murine endothelial cells. We examined whether hydrostatic pressure is involved in expression of HIF-2α in articular chondrocytes. Chondrocytes were cultured and stimulated by inflammation or hydrostatic pressure of 0, 5, 10, or 50 MPa. After stimulation, heat shock protein (HSP) 70, HIF-2α, nuclear factor kappa B (NF-κB), matrix metalloproteinase (MMP)-13, MMP-3, and vascular endothelial growth factor (VEGF) gene expression were evaluated. The levels of all gene expression were increased by inflammatory stress. When chondrocytes were exposed to a hydrostatic pressure of 5 MPa, HIF-2α, MMP-13, and MMP-3 gene expression increased significantly although those of HSP70 and NF-κB were not significantly different from the control group. In contrast, HIF-2α gene expression did not increase under a hydrostatic pressure of 50 MPa although HSP70 and NF-κB expression increased significantly compared to control. We considered that hydrostatic pressure of 5 MPa could regulate HIF-2α independent of NF-κB, because the level of HIF-2α gene expression increased significantly without upregulation of NF-κB expression at 5 MPa. Hydrostatic pressure may influence cartilage degeneration, inducing MMP-13 and MMP-3 expression through HIF-2α.     

Hydrogenation of nitro compounds with supported platinum catalyst in supercritical carbon dioxide

Hydrogenation of a series of substituted nitro compounds such as 2-,3-,4-nitroanisole, 2-,3-,4-nitrotoluene, 2,4-dinitrobenzene and 2,4-dinitrotoluene has been studied in supercritical carbon dioxide, scCO₂ (two phases), and ethanol (three phases) with a 5 wt.% carbon supported platinum catalyst. The solubility of these compounds in scCO₂ has also been examined in the presence and absence of hydrogen. The solubility of those nitro compounds increases with increasing CO₂ pressure but decreases with the presence of hydrogen. The solubility is in the order of nitrotoluene > nitroanisole > dinitrotoluene, dinitrobenzene. Although the total conversion obtained with hydrogenation in scCO₂ is similar to that in ethanol, the selectivity to amino products is higher in the former reaction medium, indicating that scCO₂ is an ideal medium for the production of amino compounds with hydrogenation of nitro substrates using conventional supported metal catalysts.     

Absorption of metal cations by modified B. mori silk and preparation of fabrics with antimicrobial activity

Silk fabrics were modified by treatment with tannic acid (TA) solution or by acylation with ethylenediaminetetraacetic (EDTA) dianhydride. Kinetics of modification with TA and acylation with EDTA–dianhydride was investigated. The physico‐mechanical properties of silk fabrics acylated with EDTA–dianhydride remained unchanged regardless of chemical modification. The absorption of metal cations (Ag⁺, Cu²⁺) by untreated and modified silk fabrics was studied as a function of the kind of modifying agent, weight gain, and pH of the metal solution. The absorption of Cu²⁺ at alkaline pH was not significantly influenced by chemical modification of the silk substrate. The absorption of Ag⁺ by acylated silk remained at a level as low as untreated silk, while was enhanced by TA. The higher the content of TA, the higher the absorption of Ag⁺. With respect to the pH of the metal solution, the acylation with EDTA–dianhydride enabled silk to absorb and bind metal cations even in the acidic and neutral pH range, where tannic acid had no effect. Medium to high levels of metal desorption were exhibited by untreated and modified silk fabrics towards the metal cations, with the only exception of the silk–tannic acid–Ag complex, which displayed an extraordinary stability. All metal‐containing silks exhibited significant antibacterial activity. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 80: 297–303, 2001     

A Comprehensive Study of the Interaction between Peptidoglycan Fragments and the Extracellular Domain of Mycobacterium tuberculosis Ser/Thr Kinase PknB

The Mycobacterium tuberculosis Ser/Thr kinase PknB is implicated in the regulation of bacterial cell growth and cell division. The intracellular kinase function of PknB is thought to be triggered by peptidoglycan (PGN) fragments that are recognized by the extracytoplasmic domain of PknB. The PGN in the cell wall of M. tuberculosis has several unusual modifications, including the presence of N-glycolyl groups (in addition to N-acetyl groups) in the muramic acid residues and amidation of d -Glu in the peptide chains. Using synthetic PGN fragments incorporating these diverse PGN structures, we analyzed their binding characters through biolayer interferometry (BLI), NMR spectroscopy, and native mass spectrometry (nMS) techniques. The results of BLI showed that muropeptides containing 1,6-anhydro-MurNAc and longer glycan chains exhibited higher binding potency and that the fourth amino acid of the peptide stem, d -Ala, was crucial for protein recognition. Saturation transfer difference (STD) NMR spectroscopy indicated the major involvement of the stem peptide region in the PASTA-PGN fragment binding. nMS suggested that the binding stoichiometry was 1:1. The data provide the first molecular basis for the specific interaction of PGN with PknB and firmly establish PGNs as the effective ligands of PknB.     

Responsiveness of electric resistance of polymer-grafted carbon black/alumina gel composite against solvent vapor and solute in solution

The carbon black/alumina gel composites were prepared by sol-gel reaction of aluminum isopropoxide in the presence of polymer-grafted carbon black. The electric resistance of the alumina gel composite from polymer-grafted carbon black was very sensitive to vapor of good solvent for grafted polymer on carbon black: the electric resistance of the alumina gel composite suddenly decreased in solvent vapor and returned to initial resistance when it was transferred into dry air. The effect of surface area and particle size of carbon black on the responsiveness was also investigated. In addition, the electric resistance of the alumina gel composite was found to respond to water and methanol in n-hexane and diethyl ether.