Flow behavior of regenerated wool-keratin proteins in different mediums

Keratin is abundantly present in nature and the major component of hair, wool, feather, nail and horns. Dissolution of keratin is often required when non-textile applications are demanded. However, the low solubility of keratin in water is the major problem. It becomes unstable and precipitated when stored for a long time. Therefore, it is necessary to find a good solvent that provides high stability and easy processibility. In this research, we used formic acid and dimethylformamide (DMF) to dissolve regenerated keratin protein films. It is shown that formic acid is a good solvent for regenerated keratin proteins for the purpose of storage. Transparent and stable regenerated keratin solution is obtained in formic acid.     

Structural characteristics and properties of the regenerated silk fibroin prepared from formic acid

Structural characteristics and thermal and solution properties of the regenerated silk fibroin (SF) prepared from formic acid (FU) were compared with those of SF from water (AU). According to the turbidity and shear viscosity measurement, SF formic acid solution was stable and transparent, no molecular aggregations occurred. The sample FU exhibited the beta-sheet structure, while AU random coil conformation using Fourier transform infrared (FTIR), X-ray diffraction (XRD), and differential scanning calorimetry. The effects of methanol treatment on samples were also examined. According to the measurement of crystallinity (XRD) and crystallinity index (FTIR), the concept of long/short-range ordered structure formation was proposed. Long-range ordered crystallites are predominantly formed for methanol treated SF film while SF film cast from formic acid favors the formation of short-range ordered structure. The relaxation temperatures of SF films measured by dynamic thermomechanical analysis supported the above mechanism due to the sensitivity of relaxation temperature on the short-range order.     

Biodegradable materials based on silk fibroin and keratin

Wool and silk were dissolved and used for the preparation of blended films. Two systems are proposed: (1) blend films of silk fibroin and keratin aqueous solutions and (2) silk fibroin and keratin dissolved in formic acid. The FTIR spectra of pure films cast from aqueous solutions indicated that the keratin secondary structure mainly consists of alpha-helix and random coil conformations. The IR spectrum of pure SF is characteristic of films with prevalently amorphous structure (random coil conformation). Pure keratin film cast from formic acid shows an increase in the amount of beta-sheet and disordered keratin structures. The FTIR pattern of SF dissolved in formic acid is characteristic of films with prevalently beta-sheet conformations with beta-sheet crystallites embedded in an amorphous matrix. The thermal behavior of the blends confirmed the FTIR results. DSC curve of pure SF is typical of amorphous SF and the curve of pure keratin show the characteristic melting peak of alpha-helices for the aqueous system. These patterns are no longer observed in the films cast from formic acid due to the ability of formic acid to induce crystallization of SF and to increase the amount of beta-sheet structures on keratin. The nonlinear trend of the different parameters obtained from FTIR analysis and DSC curves of both SF/keratin systems indicate that when proteins are mixed they do not follow additives rules but are able to establish intermolecular interactions. Degradable polymeric biomaterials are preferred candidates for medical applications. It was investigated the degradation behavior of both SF/keratin systems by in vitro enzymatic incubation with trypsin. The SF/keratin films cast from water underwent a slower biological degradation than the films cast from formic acid. The weight loss obtained is a function of the amount of keratin in the blend. This study encourages the further investigation of the type of matrices presented here to be applied whether in scaffolds for tissue engineering or as controlled release drug delivery vehicles.     

Study on cast membranes and electrospun nanofibers made from keratin/fibroin blends

Keratin regenerated from wool and fibroin regenerated from silk were mixed in different proportions using formic acid as the common solvent. Both solutions were cast to obtain films and electrospun to produce nanofibers. Scanning electron microscopy investigation showed that, for all electrospun blends (except for 100% keratin where bead defects are present), the fiber diameter of the mats ranged from 900 (pure fibroin) to 160 nm (pure keratin). FTIR and DSC analysis showed that the secondary structure of the proteins was influenced by the blend ratios and the process used (casting or electrospinning). Prevalence of beta-sheet supramolecular structures was observed in the films, while proteins assembled in alpha-helix/random coil structures were observed in nanofibers. Higher solution viscosity, thinner filaments, and differences in the thermal and structural properties were observed for the 50/50 blend because of the enhanced interactions between the proteins.     

Molecular weight distribution and solution properties of silk fibroins with different dissolution conditions

Four regenerated silk fibroin (SF) samples were prepared under different dissolution conditions and their molecular weight (MW) distributions and solution properties in water and formic acid were examined. SFL, produced by dissolving in LiBr aqueous solution for 6h, showed the highest MW level. In the three SFC samples, produced by dissolving SF in CaCl(2)/H(2)O/EtOH solution for dissolution times ranging from 3 to 180 min, the MW of the SFs decreased with increasing dissolution time and a new band appeared at low MW. Interestingly, SFL presented as a relatively transparent aqueous solution with 10-30 nm particle size, whereas the three SFC samples exhibited a turbid solution with 100-300 nm particle size. SF formic acid solutions showed a higher viscosity than SF aqueous solutions and exhibited almost Newtonian fluid behavior, whereas SF aqueous solutions displayed abrupt shear thinning in the low shear rate region (0.1-3 s(-1)).     

Effect of formic acid exposure on keratin fiber derived from poultry feather biomass

Converting poultry feather biomass into useful products presents a new avenue of utilization of agricultural waste material. However, not much is understood about the poultry feather structure or methods to process it. In this study, formic acid vapor is systematically allowed to penetrate the feather fiber structure, which is composed of keratin. The diffusion kinetics show Fickian behavior during absorption. After very long times, i.e., greater than 10(3)h, the absorption experiments are stopped and the formic acid is allowed to desorb from the keratin material. The desorption kinetics of formic acid out of the keratin fiber do not mirror the absorption kinetics, indicating a change in the keratin microstructure. DSC and NMR spectroscopy analyses on the keratin fiber show a reduction in the area of the crystalline melting peak and solubilization of amino acids upon formic acid exposure. This indicates that the crystallinity is disrupted resulting in more amorphous fraction in the keratin polymer.     

Occurrence of beta-chain conformation in poly-gamma-methyl glutamate membranes

Molecular chain conformations of poly-γ-methyl-L -glutamate, poly-γ-methyl-D -glutamate, and poly-γ-methyl-D ,L -glutamate in membranes prepared by using mainly trifluoroacetic acid and formic acid as solvents were investigated by infrared, X-ray diffraction, and optical rotatory dispersion measurements. It was pointed that these polymers exist in the α-helix form in membranes cast from trifluoroacetic acid solutions, but in the β-chain form in membrances swollen in formic acid. The β-chain structure was also observed in crystals precipitated from dilute solutions including formic acid. The formation of the β-chain structure was discussed.     

Studies on chitin. 2. Preparation and properties of chitin membranes

A chitin membrane was prepared by a new procedure involving coagulation of the chitin solution in N,N-dimethyl acetamide, N-methyl 2-pyrrolidone and lithium chloride (DMA-NMP-LiCl) with 2-propanol. The solute permeability, water sorption and mechanical properties were compared with membranes prepared by two previously reported methods (coagulation of a formic acid and dichloroacetic acid (FA-DCA) solution of chitin with 2-propanol; and coagulation of a trichloroacetic acid and dichloroethane (TCA-DCE) solution of chitin with acetone). The permeability coefficients of the three chitin membranes were higher than a regenerated cellulose membrane (Cuprophane®). The membrane prepared from DMA-NMP-LiCl solution had a higher tensile strength (3·3 Mpa) in the wet state than the others. The membrane obtained from TCA-DCE solution absorbed more water (360%) and the membrane prepared from FA-DCA solution was relatively weak (1·8 MPa) in the wet state. It was suggested that 2-propanol was a favourable coagulant for membrane production. In addition, the effect of the origin of chitin on molecular weight and tensile properties of the membranes was studied.     

The role of formic acid in solution stability and crystallization of silk protein polymer

In this paper, the regenerated silk fibroin (SF) solution dissolved in formic acid was used as a model protein to understand the role of formic acid in solution stability and crystallization of protein-based materials. The molecular decomposition of SF did not occur for the dissolution process in formic acid within 1–2 days of storage times. The β-sheet crystallization of SF molecules was occurred by the elimination of formic acid upon drying. The SF molecules in formic acid solution are stable and have low hydrodynamic radius values. This may be closely related to the fact that formic acid has two opposite functions of dissolution and crystallization simultaneously. The turbidity, dynamic light scattering and FTIR measurements elucidate that the solution stability and crystallization of SF are attributed to compact molecular shape of SF in formic acid, resulted from the molecular interactions between formic acid and polar groups in SF molecules.     

Dynamic light scattering and circular dichroism studies on heat-induced gelation of hard-keratin protein aqueous solutions

Animal hairs consist of aggregates of dead cells filled with keratin protein gel. We succeeded in preparing water-soluble hard-keratin proteins and reconstructing the keratin gels by heat-induced disulfide linkages in vitro. Here, the roles of intermolecular hydrophobic interaction and disulfide bonding between the proteins in the gel were discussed. Water-soluble keratin proteins consisting of mixtures of type I ( approximately 48 kDa) and type II ( approximately 61 kDa) were prepared from wool fibers as S-carboxymethyl alanyl disulfide keratin (CMADK). The gelation was achieved by heating an aqueous solution containing at least 0.8 wt % CMADK at 100 degrees C. CMADK solutions with different urea or N-ethylmaleimide concentrations or pH were exposed to dynamic light scattering (DLS) and circular dichroism (CD). DLS clarified the gelation point of CMADK solutions and provided information on the changes in keratin cluster size. DLS suggested two types of gelation mechanism. One was the regenerated chemical disulfide bonding between keratins from CMAD parts of chains. After the gel formed, this bond became important to maintain the gel structure. The other was the physical assembly due to hydrophobic interaction between alpha-helix parts of keratin chains. This hydrophobic assembly also played an important role during gelation. CD confirmed a conformational change in the keratin protein, resulting heat-induced gelation. CD clarified the relationship between keratin protein conformation and gelation, i.e., a rodlike conformation with many alpha-helix structures was necessary to associate keratin chains and form a gel network.     

Focal adhesions are hotspots for keratin filament precursor formation

Recent studies showed that keratin filament (KF) formation originates primarily from sites close to the actin-rich cell cortex. To further characterize these sites, we performed multicolor fluorescence imaging of living cells and found drastically increased KF assembly in regions of elevated actin turnover, i.e., in lamellipodia. Abundant KF precursors (KFPs) appeared within these areas at the distal tips of actin stress fibers, moving alongside the stress fibers until their integration into the peripheral KF network. The earliest KFPs were detected next to actin-anchoring focal adhesions (FAs) and were only seen after the establishment of FAs in emerging lamellipodia. Tight spatiotemporal coupling of FAs and KFP formation were not restricted to epithelial cells, but also occurred in nonepithelial cells and cells producing mutant keratins. Finally, interference with FA formation by talin short hairpin RNA led to KFP depletion. Collectively, our results support a major regulatory function of FAs for KF assembly, thereby providing the basis for coordinated shaping of the entire cytoskeleton during cell relocation and rearrangement.     

Fluctuations of pass band coefficients of water and water salt solutions in the spectral infrared region

The effect of superlow concentrations of KCl and CaCl2 solutions, obtained by diluting starting 1 M solutions of these salts 10(9)-10(15) times, on the fluctuations of pass band coefficients of water was studied. It was found that these solutions are substantially distinguished from bidistilled water by a high intensity of fluctuations of spectra in the infrared region. It was also shown that higher concentrations of solutions of these salts (at dilutions less than 10(8) times) have no strong and specific effect on fluctuations of the infrared spectrum: the intensity of fluctuations of the infrared spectra of these solutions practically coincides with that of control water. Possible reasons for this effect are discussed.     

Formation of silk fibroin matrices with different texture and its cellular response to normal human keratinocytes

Three forms of silk fibroin (SF) matrices, woven (microfiber), non-woven (nanofiber), and film form, were used to perform a conformational analysis and cell culture using normal human oral keratinocytes (NHOK). To obtain the SF microfiber (SF-M) matrix, natural grey silk was degummed, while the SF film (SF-F) and nanofiber (SF-N) matrices were prepared by casting and electrospinning the formic acid solutions of the regenerated SF, respectively. For insolubilization, as-prepared SF-F and SF-N matrices were chemically treated with an aqueous methanol solution of 50%. The conformational structures of as-prepared and chemically treated SF matrices were investigated using attenuated total reflectance infrared spectroscopy (ATR-IR) and solid-state ¹³C CP/MAS nuclear magnetic resonance (NMR) spectroscopy. The as-cast SF-F matrix formed a mainly β-sheet structure that was similar to the SF-M matrix, whereas the as-spun SF-N matrix had a random coil conformation as the predominant secondary structure. Conformational transitions from random coil to β-sheet of the as-spun SF-N occurred rapidly within 10 min following aqueous methanol treatment, and were confirmed by solid-state ¹³C NMR analysis. To assess the cytocompatibility and cells behavior on the different textures of SF, we examined the cell attachment and spreading of NHOK that was seeded onto the SF matrices, as well as the interaction between the cells and SF matrices. Our results indicate that the SF nanofiber matrix may be more preferable than SF film and SF microfiber matrices for biomedical applications, such as wound dressings and scaffolds for tissue engineering.     

Structural studies of Bombyx mori silk fibroin during regeneration from solutions and wet fiber spinning

Regenerated silk fibroin materials show properties dependent on the methods used to process them. The molecular structures of B. mori silk fibroin both in solution and in solid states were studied and compared using X-ray diffraction, FTIR, and (13)C NMR spectroscopy. Some portion of fibroin protein molecules dissolved in formic acid already have a beta-sheet structure, whereas those dissolved in TFA have some helical conformation. Moreover, fibroin molecules were spontaneously assembled into an ordered structure as the acidic solvents were removed from the fibroin-acidic solvent systems. This may be responsible for the improved physical properties of regenerated fibroin materials from acidic solvents. Regenerated fibroin materials have shown poor mechanical properties and brittleness compared to their original form. These problems were technically solved by improving the fiber forming process according to a method reported here. The regenerated fibroin fibers showed much better mechanical properties compared to the native silk fiber and their physical and chemical properties were characterized by X-ray diffraction, solid state (13)C NMR spectroscopy, SinTech tensile testing, and SEM.     

Extraction and electrospinning of gelatin from fish skin

Ultra-fine gelatin fibers were successfully fabricated by electrospinning from the solutions of Nile tilapia (Oreochromis niloticus) skin-extracted gelatin in either acetic acid or formic acid aqueous solutions. The extracted gelatin contained 7.3% moisture, 89.4% protein, 0.3% lipid, and 0.4% ash contents (on the basis of wet weight), while the bloom gel strength, the shear viscosity, and the pH values were 328 g, 17.8 mPa s, and 5.0, respectively. Both the acid concentration and the concentration of the gelatin solutions strongly influenced the properties of the as-prepared solutions and the obtained gelatin fibers. At low acid concentrations (i.e., 15% (w/v) extracted gelatin solutions in 10 and 20% (v/v) acetic acid solvents or 10-60% (v/v) formic acid solvents), a combination between smooth and beaded fibers was observed. At low concentrations of the gelatin solutions in either 40% (v/v) acetic acid solvent or 80% (v/v) formic acid solvent (i.e., 5-11%, w/v), either discrete beads or beaded fibers were obtained, while, at higher concentrations (i.e., 14-29%, w/v), only smooth or a combination of smooth and beaded fibers were obtained. The average diameters of the obtained fibers, regardless of the types of the acid solvents used, ranged between 109 and 761 nm. Lastly, cross-linking of the obtained gelatin fiber mats with glutaraldehyde vapor caused slight shrinkage from their original dimension, and the cross-linked gelatin fiber mats became stiffer.     

Hydrothermal upgrading of biomass to biofuel; studies on some monosaccharide model compounds

During the hydrothermal upgrading of biomass, hydrolysis to glucose is an important step. To elucidate some of the reaction pathways that follow this initial hydrolysis, the hydrothermal treatment (340 degrees C, 27.5 MPa, 25-204 s) of dilute (50 mM) solutions of D-glucose and some other monosaccharides were studied. As a result of the increase of Kw under subcritical conditions, both acid and base catalysed reactions occur. The acid catalysed reactions are mainly dehydrations leading initially to 5-hydroxymethylfurfural. Important base catalysed reactions result in glycolaldehyde and glyceraldehyde. Further fragmentations and dehydrations lead to a variety of low molecular weight compounds such as formic acid, acetic acid, lactic acid, acrylic acid, 2-furaldehyde and 1,2,4-benzenetriol. Important pathways leading to a decrease of the O-content of the liquid reaction products start from the intermediate glyceraldehyde, which forms pyruvaldehyde, which in its turn is converted into formic acid and acetaldehyde. The latter compound can also be formed via isomerisation of glyceraldehyde into lactic acid followed by decarbonylation.     

Biosynthesis of an amphiphilic silk-like polymer

An amphiphilic silk-like protein polymer was efficiently produced in the yeast Pichia pastoris. The secreted product was fully intact and was purified by solubilization in formic acid and subsequent precipitation of denatured host proteins upon dilution with water. In aqueous alkaline solution, the negatively charged acidic polymer assumed extended helical (silk III-like) and unordered conformations. Upon subsequent drying, it assumed a conformation rich in beta-turns. In water at low pH, the uncharged polymer aggregated and the solution became turbid. Concentrated solutions in 70% (v/v) formic acid slowly formed gels. Replacement of the formic acid-water mixture with methanol and subsequent drying resulted in beta-sheets, which stacked into fibril-like structures. The novel polymer instantaneously lowered the air-water interfacial tension under neutral to alkaline conditions and reversed the polarity of hydrophobic and hydrophilic solid surfaces upon adsorption.     

Mutational replacements of conserved amino acid residues in the beta subunit resulted in defective assembly of H+-translocating ATPase (F0F1) in Escherichia coli

Mutant genes for the beta subunit of H+-translocating ATPase (F0F1) were cloned from Escherichia coli strains isolated in this laboratory. Determination of their nucleotide sequence revealed four missense mutations (strain KF39, Glu-41----Lys; strain KF16 and KF42, Glu-185----Lys; strain KF48, Gly-223----Asp; KF26 and 4 other strains, Ser-292----Phe). Two nonsense mutants (strain KF40, Gln-361----end; strain KF20, Gln-397----end) were also identified. Glu-41, Glu-185, and Ser-292 are conserved in the amino acid sequences of the beta subunits so far studied, and Gly-223, Gln-361, and Gln-397 are conserved in beta subunits from bacteria and mitochondria, but not in those from chloroplasts. The amounts of F1 subunits in the membranes of these strains were studied by immunochemical assay and two-dimensional gel electrophoresis. In the mutants studied, the amounts of alpha and beta subunits in the membranes were 69-21 and 46-2%, respectively, of the amounts in wild-type membranes, the amount depending on the strain. No delta and epsilon subunits were detected in membranes of a missense mutant KF16, although reduced amounts of alpha and beta subunits could be detected, suggesting that the F1 portion may not be connected to F0 through the delta and epsilon subunits. The altered residues in missense mutants or missing domains in nonsense mutants may be important for the subunit-subunit interactions or assembly of the entire complex. Genetic experiments on introduction of suppressor tRNA into strains KF40 and KF20 suggested that F1 could be active even when residue 361 or 397 was replaced by a Ser, Leu, or Tyr residue.     

Characterization of a novel cell line from the caudal fin of koi carp Cyprinus carpio

A continuous cell line (KF‐101) derived from the caudal fin of the koi carp Cyprinus carpio was established and characterized. The KF‐101 cell line multiplied abundantly in Leibovitz's L‐15 medium containing 10% foetal bovine serum at 25° C, and was subcultured for >90 passages over a period of 3 years. Immunocytochemistry revealed that the KF‐101 cells contain keratin, junction proteins connexin‐43 and occludin, and ectodermal stem‐cell marker Pax‐6, but not vimentin. Furthermore, the KF‐101 cells reacted with anti‐human DARPP‐32 and anti‐human GATA‐4 antibodies, and the labelling was regulated according to the cell cycle. The labels of the DARPP‐32 and GATA‐4 antibodies in the KF‐101 cells were the suggested phosphatase‐1 inhibitor‐1 and GATA‐3, respectively. In addition, the KF‐101 cells were susceptible to koi herpesvirus but were resistant to eel herpesvirus, iridovirus, grouper nodavirus and chum salmon (Oncorhynchus keta) virus. The results indicate that the KF‐101 cells are suitable materials for investigating biological and virological development.     

Control of kernel weight and kernel water relations by post-flowering source-sink ratio in maize

The maize (Zea mays L.) kernel undergoes large changes in water content during its development. Whether such changes regulate the pattern of kernel development or are simply a consequence of it has not yet been established because other factors, such as assimilate supply, can also affect the rate and duration of kernel growth. This study was conducted to determine whether variation in kernel weight (KW) in response to source-sink treatments is mediated by a change in kernel water relations. Two hybrids were sown at three stand densities (one, eight and 18 plants m-2), and kernel numbers were restricted to control the post-flowering source-sink ratio within each stand density. Kernel development and water relations [water content, water potential (psiw), osmotic potential (psis) and turgor] were monitored throughout grain filling. Final KW varied from 253 to 372 mg per kernel in response to source-sink treatments. For both genotypes, variation in KW was a result of a change in kernel growth rate (r2 = 0.91; P < 0.001) and not in the duration of kernel filling. Final KW was closely correlated with maximum kernel water content (r2 = 0.94; P < 0.001) achieved during rapid dry matter accumulation. However, variation in KW was not reflected in kernel water status parameters (psiw, psis or turgor), which remained fairly stable across treatments. These results indicate that maximum water content provides an easily quantifiable measure of kernel sink capacity in maize. Kernel water status parameters may affect the duration of grain filling, but have no discernible impact on kernel growth rate.