Nucleation of hydroxyapatite crystals by self-assembled Bombyx mori silk fibroin

Bombyx mori silk fibroin (SF) is known to be capable of facilitating nucleation of the hydroxyapatite crystals (HAps). To find out how SF mediates the nucleation of HAps, self-assembly of SF in 1.5 simulated body fluid (SBF) was observed in this study through design of a co-solution of SF and 1.5-times SBF (SF/1.5 SBF). After the co-solution of SF/1.5 SBF was incubated at 37.2 °C up to 7 days, SEM, X-ray, and Fourier transform infrared (FTIR) observations indicated that nucleation of HAps was increased. In addition, the structure of SF was transited from random coil into β-sheet indicated by FTIR spectra. The β-sheet assembly of SF in 1.5 SBF was also supported by CD spectra. Atomic force microscopy provided detailed progress of the self-assembly that SF incubated in 1.5 SBF was self-assembled in the form from dot, through rod to final net. Therefore, this study suggested that nucleation of HAps of SF was controlled by its molecular self-assembly. © 2013 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2013     

Structure elucidation of deoxylutein II isomers by on-line capillary high performance liquid chromatography-(1)H nuclear magnetic resonance spectroscopy

Thermal and iodine-catalyzed photochemical (Z/E)-isomerization of deoxylutein II [(3R,6'R)-3-hydroxy-3',4'-didehydro-beta,gamma-carotene, anhydrolutein I] (2), the dehydration product of lutein [(3R,3'R,6'R)-beta,varepsilon-carotene-3,3'-diol] (4), yielded multi-component mixtures of (Z)-isomers. By I(2)-catalyzed photoisomerization, (9Z)-2, (9'Z)-2, (13Z)-2, (13'Z)-2 and (15Z)-2 are generated as main products. In addition, this thermodynamic-equilibrium mixture contains traces of (9Z,9'Z)-2 and other (di-Z)-isomers in minor concentrations. Thirteen isomers are chromatographically separated and detected on-line by UV-vis and mass spectrometry. (all-E)-Deoxylutein II (2) and six of its (Z)-configured isomers are separated by capillary HPLC (acetone-d(6)/D(2)O = 85:15) and detected on-line by (1)H NMR spectroscopy in a microprobe. With the microprobe and the active detection volume of 1.5 microl, it is possible to perform structure elucidation with very small amounts available for various (Z)-isomers of deoxylutein II (2) in the isomerization mixture.     

The application of two-dimensional nuclear magnetic resonance spectroscopy in computer-assisted structure elucidation

One of the most powerful tools for the determination of an unknown organic structure is two dimensional NMR spectroscopy. In some cases, this information quickly leads to a unique structure, but in many cases there remains a larger number of structural possibilities. A linked system of computer programs has been developed which uses several types of 2-D NMR data and generates all possible topological structures consistent with these data and user-input constraints. When symmetry is present, algorithms based on group theory limit the structures to those consistent with all the different symmetries that are possible. Applications of these programs to problems described in the literature, in which 2-D NMR was used to determine the structures of natural products, are described.     

Dissolution and regeneration of Bombyx mori silk fibroin using ionic liquids

In this work, the suitability of imidazolium-based ionic liquid solvents is investigated for the dissolution and regeneration of silkworm (Bombyx mori) silk. Within an ionic liquid the anion plays a larger role in dictating the ultimate solubility of the silk. The dissolution of the silk in the ionic liquid is confirmed using wide-angle X-ray scattering. The dissolved silk is also processed into 100 mum-thick, two-dimensional films, and the structure of these films is examined. The rinse solvent, acetonitrile or methanol, has a profound impact on both the topography of the films and the secondary structure of the silk protein. The image depicts a silkworm cocoon dissolved in 1-butyl-3-methylimidazolium chloride and then regenerated as a film with birefringence.     

Self-assembly of a peptide amphiphile based on hydrolysed Bombyx mori silk fibroin

An octapeptide, GAGAGAGY, was obtained by a novel method, i.e. hydrolysing Bombyx mori silk fibroin. Afterward, a dodecanoic acid-peptide conjugation was synthesized. This amphiphile assembled into cylindrical nanofibers of planar β-sheets at pH 9 and twisted β-sheets at pH 4.     

Defense role of the cocoon in the silk worm Bombyx mori L

Silk from the domesticated silk worm Bombyx mori procures foreign body response naturally, so it has been utilized as a biomaterial for decades. In India the prime focus of the sericulture industry is to improve silk production with high quality silk. Naturally, the silk worm builds its cocoon not only with silk proteins, but also with antimicrobial proteins to avoid infection since the cocoon is non-motile and non-feeding. The aim of the present study is to elucidate the antimicrobial proteins that persist in the cocoon of the silk worm Bombyx mori. At the pupal stage, the silk worm cocoon shell extract was prepared from the day of pupation (P0) to the day of natural rupture of the cocoon for the eclosion of moth (NR). Using the cocoon shell extract a microbial susceptibility test was performed by the disc diffusion method against the microbes Escherchia coli, Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae. The development of a zone of inhibition against the microbes confirmed the presence of antimicrobial/immunogenic activity of the cocoon shell extract. For further analysis, the cocoon shell extract was subjected to 7-15% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The protein profile of the cocoon extract revealed the coomassie blue stained bands resolved from the 150-15 kDa molecular range. Interestingly, a polypeptide localized at around 29 kDa showed remarkable expressional changes during the development of pupa. To characterize the 29 kDa protein, it was eluted from the gel, digested with trypsin and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The trypsin-digested peptide peaks were analyzed through MASCOT and peptides were matched with the NCBI nr database. The peptides were very well matched with the 18 wheeler protein, which is reported to be responsible for innate immunity, belonging to the Toll family in insects and responsible for cellular mediated immunity.     

Heterogeneous structure of silk fibers from Bombyx mori resolved by 13C solid-state NMR spectroscopy

The molecular conformation of silk fibrion is characterized by solid-state 13C NMR before spinning (silk I structure) and after spinning (silk II structure). We compare native silk fibers with the quasi-crystalline Cp-fraction and a synthetic model peptide (Ala-Gly)15, both of which can be converted either into silk I by dialysis from 9 M LiBr or into silk II by treatment with formic acid. Our results demonstrate that silk II fibers are intrinsically heterogeneous, consisting of beta-sheets, distorted beta-turns, and distorted beta-sheets. This higher-order heterogeneity is revealed by the 13C-NMR Cbeta-peak of Ala, indicating that the Ala side chains are stacked partially in parallel and partially face-to-face, at a ratio of 1:2.     

Conformational studies of cyclo(L-Phe-L-Pro-Gly-L-Pro)2 by 13C nuclear magnetic resonance

The 13C-NMR spectrum (Fig. 2,1) of cyclooctapeptide cyclo(L-phe-L-Pro-Gly-L-Pro)2 (A) in CDC13 suggested that its conformation involved the coexistence of two kinds of C2-symmetric conformation with trans-trans-trans-trans and cis-trans-trans-trans forms. Adding 0.5 equivalent of CsSCN or one equivalent of DL-Phe-OMe.HCl to the solution of cyclopeptide (A) in CDC13 yielded 13C-NMR spectra (Fig. 2,2 and Table I) which suggested a single C2-symmetric conformation with trans-trans-trans-trans form, resulting from the formation of complexes with CsSCN or DL-Phe-OMe.HCl. The 13C-NMR spectrum of complexes of A with DL-Phe-OMe.HCl displayed separate resonances for C(gamma), C(o), C(m), C(alpha), and C(beta) of D-Phe-OMe.HCl and L-Phe-OMe.HCl (Table I).     

用桑蚕丝素蛋白固定脲酶的研究及脲酶电极的制备

提出了一种新的酶固定化方法, 即通过甲醇处理, 使蚕丝素蛋白膜的构象由random coil向β-sheet发生根本性的变化, 从而将酶固定在β-sheet所特有的分子间氢键中。利用此方法所制成的脲酶电极, 在合适的操作条件下, 各项响应指标均令人满意, 并且脲酶的耐温性能被大大提高, 电极的有效使用寿命长达三个月以上。此种酶固定化方法原则上能够应用于其他不破坏蚕丝素蛋白分子结构的可溶性酶。     

29Si nuclear magnetic resonance of poly(methylphenyl)silane

A Cl-terminated poly(methylphenyl)silane is synthesized and analyzed by ²⁹Si-NMR spectroscopy. Assignments are proposed for the different peaks observed. A modification of the chemical shifts of the peaks relative to the main chain atoms is observed upon heating. From these experimental results and computational calculations on model compounds, the assignments of the three main peaks to configurationally different Si atoms are confirmed. © 1996 John Wiley & Sons, Inc.     

Effect of metallic ions on silk formation in the Mulberry silkworm, Bombyx mori

A protein conformation transition from random coil and/or helical conformation to beta-sheet is known to be central to the process used by silk-spinning spiders and insects to convert concentrated protein solutions to tough insoluble threads. Several factors including pH, metallic ions, shear force, and/or elongational flow can initiate this transition in both spiders and silkworms. Here, we report the use of proton induced X-ray emission (PIXE), inductively coupled plasma mass spectroscopy (ICP-MS) and atomic adsorption spectroscopy (AAS) to investigate the concentrations of six metal elements (Na, K, Mg, Ca, Cu, and Zn) at different stages in the silk secretory pathway in the Bombyx mori silkworm. We also report the use of Raman spectra to monitor the effects of these six metallic ions on the conformation transition of natural silk fibroin dope and concentrated regenerated silk fibroin solution at concentrations similar to the natural dope. The results showed that the metal element contents increased from the posterior part to the anterior part of silk gland with the exception of Ca which decreased significantly in the anterior part. We show that these changes in composition can be correlated with (i) the ability of Mg2+, Cu2+, and Zn2+ to induce the conformation transition of silk fibroin to beta-sheet, (ii) the effect of Ca2+ in forming a stable protein network (gel), and (iii) the ability of Na+ and K+ to break down the protein network.     

Anionic-nonionic surfactant interaction by nuclear magnetic resonance

Summary The interaction between anionic (sodium dodecyl benzene sulfonate) surfactant and nonionic (Tri and Tetra propylene glycol monomethyl ether) surfactant was studied using nuclear magnetic resonance measurement. It was observed that the addition of sodium dodecyl benzene sulfonate to the solution of nonionic surfactant (Tri and Tetra propylene glycol mono methyl ether) caused an upfield shift of the central protons of the nonionic surfactants. The aromatic protons of sodium-dodecyl benzene sulfonate undergo a very small, almost negligible, downfield shift. The changes in the chemical shift values and the integration values of the polypropylene protons and benzene protons was interpreted in terms of mixed micelle formation with the simultaneous presence of highly fluid mixed micelles of varying compositions.With 2 figures and 2 tables     

Microstructure of glycidylmethacrylate/vinyl acetate copolymers by two‐dimensional nuclear magnetic resonance spectroscopy

Glycidylmethacrylate/vinyl acetate copolymers were prepared by solution polymerization with benzene as a solvent and benzoyl peroxide as an initiator. Copolymer compositions were determined from ¹H NMR spectra, and comonomer reactivity ratios were determined by the Kelen–Tudos (KT) method and the nonlinear least‐squares error‐in‐variable method (EVM). The reactivity ratios obtained from KT and EVM were r_G = 37.4 ± 12.0 and r_V = 0.036 ± 0.019 and r_G = 35.2 and r_V = 0.03, respectively. Complete spectral assignments of ¹³C and ¹H NMR spectra were done with the help of distortionless enhancement by polarization transfer and two‐dimensional ¹³C–¹H heteronuclear single quantum coherence and total correlation spectroscopy. The methyl, methine, and methylene carbon resonance showed both stereochemical and compositional sensitivity. © 2001 John Wiley & Sons, Inc. J Polym Sci Part A: Polym Chem 39: 4051–4060, 2001     

Quantification of organic acids in beer by nuclear magnetic resonance (NMR)-based methods

The organic acids present in beer provide important information on the product's quality and history, determining organoleptic properties and being useful indicators of fermentation performance. NMR spectroscopy may be used for rapid quantification of organic acids in beer and different NMR-based methodologies are hereby compared for the six main acids found in beer (acetic, citric, lactic, malic, pyruvic and succinic). The use of partial least squares (PLS) regression enables faster quantification, compared to traditional integration methods, and the performance of PLS models built using different reference methods (capillary electrophoresis (CE), both with direct and indirect UV detection, and enzymatic essays) was investigated. The best multivariate models were obtained using CE/indirect detection and enzymatic essays as reference and their response was compared with NMR integration, either using an internal reference or an electrical reference signal (Electronic REference To access In vivo Concentrations, ERETIC). NMR integration results generally agree with those obtained by PLS, with some overestimation for malic and pyruvic acids, probably due to peak overlap and subsequent integral errors, and an apparent relative underestimation for citric acid. Overall, these results make the PLS-NMR method an interesting choice for organic acid quantification in beer.     

Atomic force microscopy: Bombyx mori silk fibroin molecules and their higher order structure

We performed atomic force microscopy measurements on fibroin molecules from the domestic silkworm, Bombyx mori. At low concentrations, we could observe single protein molecules. The shape of the observed molecules is a rod with long smaller chains extending from the ends. The size of the rod is 60 nm in length and 15 nm in width. At high concentrations, we observed long threadlike aggregates of fibroin molecules, their chains entangled with one another. These results provide insight into the microscopic mechanism of silk‐fiber formation. © 2000 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 38: 1436–1439, 2000