Modulation of aggregation of silk fibroin by synergistic effect of the complex of curcumin and β-cyclodextrin

Amyloid aggregation has been associated with numerous human pathological diseases. A recent study has demonstrated that silk fibroin intermittently endorses amyloidogenesis in vivo. In the current study, we explored the propensity of silk fibroin to undergo amyloid-like aggregation and its prevention using an optimized concoction of curcumin with β-cyclodextrin. Aggregation of silk fibroin resulted in the formation of fibrils with a diameter of ~3.2 nm. However, addition of the optimized concentration of curcumin and β-cyclodextrin to silk fibroin inhibited aggregation and preserved the random coil conformation even under aggregation inducing conditions, as demonstrated by CD and FTIR spectroscopy. Benzene rings of curcumin interact with the aromatic residues of fibroin via hydrophobic interactions. However, β-cyclodextrin preferentially interacts with the non-polar residues, which are the core components for nucleation dependent protein aggregation. The present study demonstrates the ability of the concoction of curcumin and β-cyclodextrin in tuning the self assembly process of fibroin. It also provides a platform to explore the assembly process of nano-fibril and hierarchical structures in vitro along with a novel insight for designing clinically relevant silk-based functional biomaterials.     

Evidence of α-helical coiled coils and β-sheets in hornet silk

α-Helical coiled coil and β-sheet complexes are essential structural building elements of silk proteins produced by different species of the Hymenoptera. Beside X-ray scattering at wide and small angles we applied cryo-electron diffraction and microscopy to demonstrate the presence and the details of such structures in silk of the giant hornet Vespa mandarinia japonica. Our studies on the assembly of the fibrous silk proteins and their internal organization in relation to the primary chain structure suggest a 172 Å pitch supercoil consisting of four intertwined alanine-rich α-helical strands. The axial periodicity may adopt even multiples of the pitch value. Coiled coil motifs form the largest portion of the hornet silk structure and are aligned nearly parallel to the cocoon fiber axis in the same way as the membrane-like parts of the cocoon are molecularly orientated in the spinning direction. Supercoils were found to be associated with β-crystals, predominantly localized in the l-serine-rich chain sequences terminating each of the four predominant silk proteins. Such β-sheet blocks are considered resulting from transformation of random coil molecular sequences due to the action of elongational forces during the spinning process.     

Chemical modification,antioxidant and α-amylase inhibitory activities of corn silk polysaccharides

Water-soluble corn silk polysaccharides (CSPS) were chemically modified to obtain their sulfated, acetylated and carboxymethylated derivatives. Chemical characterization and bioactivities of CSPS and its derivatives were comparatively investigated by chemical methods, gas chromatography, gel filtration chromatography, scanning electron microscope, infrared spectroscopy and circular dichroism spectroscopy, scavenging DPPH free radical assay, scavenging hydroxyl radical assay, ferric reducing power assay, lipid peroxidation inhibition assay and α-amylase activity inhibitory assay, respectively. Among the three derivatives, carboxylmethylated polysaccharide (C-CSPS) demonstrated higher solubility, narrower molecular weight distribution, lower intrinsic viscosity, a hyperbranched conformation, significantly higher antioxidant and α-amylase inhibitory abilities compared with the native polysaccharide and other derivatives. C-CSPS might be used as a novel nutraceutical agent for human consumption.     

The blocked and methylated 5′ terminal sequence of a specific cellular messenger: the mRNA for silk fibroin of bombyx mori

The messenger RNA for silk fibroin, labeled with ³²PO₄ and methyl-³H L-methionine, was purified to near homogeneity from the posterior silk gland of the silkworm Bombyx mori, and the sequence of a methylated, RNAase T2-resistant structure was determined. This sequence is similar structurally to 5′ terminal blocked and methylated sequences found on the total populations of polyadenylated eucaryotic cellular and certain viral mRNAs. The RNAase T2-resistant oligomer from fibroin mRNA was cleaved by nuclease P1 into three components: a blocked and methylated sequence containing three phosphates; a 2′-0-methyl UMP residue (pU^m), and an unmethylated CMP (pC). The blocked and methylated sequence comigrated in three chromatographic systems with the blocked and methylated terminus of silkworm cytoplasmic polyhedrosis virus mRNA, which has the structure m⁷GpppA^m. The fibroin mRNA cap was cleaved by nucleotide pyrophosphatase to yield 7-methyl GMP (pm⁷G) and 2′-0-methyl AMP (pA^m). This sequence also appeared to be terminally located, with the m⁷G joined by a 5′-5′ pyrophosphate linkage to the A^m. It was concluded that the 5′ terminal sequence of fibroin mRNA molecules is m⁷G(5′)ppp(5′)A^mpU^mpCp. The regulation of expression of the highly specialized gene for fibroin is discussed in light of this finding.     

Structure and ultrastructure of the silk glands and spinneret of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

This study provides comprehensive documentation of silk production in the pest moth Helicoverpa armigera from gland secretion to extrusion of silk thread. The structure of the silk glands, accessory structures and extrusion apparatus are reported. The general schema of the paired silk glands follows that found for Lepidoptera. Morphology of the duct, silk press, muscle attachments and spigot are presented as a three-dimensional reconstruction and the cuticular crescent-shaped profile of the silk press is demonstrated in both open and closed forms with attendant muscle blocks, allowing advances in our knowledge of how the silk press functions to regulate the extrusion of silk. Growth of the spigot across instars is documented showing a distinctive developmental pattern for this extrusion device. Its shape and structure are related to use and load-bearing activity.     

A hydrogel–fiber–hydrogel composite scaffold based on silk fibroin with the dual-delivery of oxygen and quercetin

Supplying sufficient oxygen within the scaffolds is one of the essential hindrances in tissue engineering that can be resolved by oxygen-generating biomaterials (OGBs). Two main issues related to OGBs are controlling oxygenation and reactive oxygen species (ROS). To address these concerns, we developed a composite scaffold entailing three layers (hydrogel-electrospun fibers-hydrogel) with antioxidant and antibacterial properties. The fibers, the middle layer, reinforced the composite structure, enhancing the mechanical strength from 4.27 ± 0.15 to 8.27 ± 0.25 kPa; also, this layer is made of calcium peroxide and silk fibroin (SF) through electrospinning, which enables oxygen delivery. The first and third layers are physical SF hydrogels to control oxygen release, containing quercetin (Q), a nonenzymatic antioxidant. This composite scaffold resulted in almost more than 40 mmHg of oxygen release for at least 13 days, and compared with similar studies is in a high range. Here, Q was used for the first time for an OGB to scavenge the possible ROS. Q delivery not only led to antioxidant activity but also stabilized oxygen release and enhanced cell viability. Based on the given results, this composite scaffold can be introduced as a safe and controllable oxygen supplier, which is promising for tissue engineering applications, particularly for bone.     

Regional differences in the apical F-actin cytoskeleton of silk gland cells of Calpodes ethlius (Stöll) (Lepidoptera : Hesperiidae)

Silk glands of the larvae of Calpodes ethlius, Stöll (Lepidoptera : Hesperiidae) were labelled with rhodaminyl-phalloin, which is specific for f-actin. Most f-actin lies below the apical cell surface in bundles oriented in rings around the lumen of the gland. By the fifth larval stadium, the density of bundles differs in the 5 regions of the gland, the greatest density occurring in the narrower anterior regions. The greater bundle density may be correlated with the increased hoop stress where the gland has the smallest diameter. F-actin from the bundles redistributes during larval moults to coat vacuoles in the cytoplasm, except in the 2 most anterior regions where it keeps its arrangement from stage to stage, even through the moult.     

Inducing β-sheets formation in synthetic spider silk fibers by aqueous post-spin stretching

As a promising biomaterial with numerous potential applications, various types of synthetic spider silk fibers have been produced and studied in an effort to produce man-made fibers with mechanical and physical properties comparable to those of native spider silk. In this study, two recombinant proteins based on Nephila clavipes Major ampullate Spidroin 1 (MaSp1) consensus repeat sequence were expressed and spun into fibers. Mechanical test results showed that fiber spun from the higher molecular weight protein had better overall mechanical properties (70 KD versus 46 KD), whereas postspin stretch treatment in water helped increase fiber tensile strength significantly. Carbon-13 solid-state NMR studies of those fibers further revealed that the postspin stretch in water promoted protein molecule rearrangement and the formation of β-sheets in the polyalanine region of the silk. The rearrangement correlated with improved fiber mechanical properties and indicated that postspin stretch is key to helping the spider silk proteins in the fiber form correct secondary structures, leading to better quality fibers.     

The silk road of <Emphasis Type="Italic">Tetranychus urticae</Emphasis>: is it a single or a double lane?

Tetranychus urticae (Acari: Tetranychidae) is a phytophagous mite that forms huge colonies. All active members of a colony (immatures and matures, females and males) spin silken threads. These mites construct a common web that protects the colony from external aggression. The silk coverage is well-known to provide advantages to the colony but very little is known about the characteristics of the threads themselves. Here is the first quantification of the diameter of silken threads spun by two different stages (adult females and larvae) and its relationship with body size of the spinning individuals. Moreover, we observed how silk was deposited on the substrate through their two pedipalps. Threads were observed by means of transmission electron and fluorescence microscopy. Silken threads spun by larvae (0.055 ± 0.018 μm) were significantly thinner than threads spun by adult females (0.111 ± 0.038 μm). In the first step of the silk depositing behaviour, the mite attached the thread to the substrate by putting its pedipalps in contact with the surface (adhesion, double silken threads). When walking, silken threads became detached from the substrate and spitted up (silken threads were free). Finally, silken threads adhered to the surface. The presence of single and double threads makes thread diameter highly variable.     

Electrospun poly (ɛ-caprolactone)/silk fibroin core-sheath nanofibers and their potential applications in tissue engineering and drug release

One of the key tenets of tissue engineering is to develop scaffold materials with favorable biodegradability, surface properties, outstanding mechanical strength and controlled drug release property. In this study, we generated core-sheath nanofibers composed of poly (?-caprolactone) (PCL) and silk fibroin (SF) blends via emulsion electrospinning. Nanofibrous scaffolds were characterized by combined techniques of scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), differential scanning calorimetry (DSC), contact angle and tensile measurements. An in vitro FITC release study was conducted to evaluate sustained release potential of the core-sheath structured nanofibers. We found that the conformation of SF contained in PCL/SF composite nanofibers was transformed from random coil to β-sheet when treated with methanol, leading to improved crystallinity and tensile strength of nanofibrous scaffolds. The hydrophobicity and diameter of nanofibers decreased when we increased the content of SF in PCL/SF composite nanofibers. Furthermore, we evaluated the potential of fabricated PCL/SF composite nanofibers as scaffold in vitro. The results confirmed that fabricated PCL/SF scaffolds improved cell attachment and proliferation. Our results demonstrated the feasibility to generate core-sheath nanofibers composed of PCL and SF using a single-nozzle technique. The produced nanofibrous scaffolds with sustained drug release have potential application in tissue engineering.     

To feed or to wrap? Female silk cues elicit male nuptial gift construction in a semiaquatic trechaleid spider

By wrapping prey and offering it as a nuptial gift, males can obtain mating and/or parental benefits despite some costs. Males of the Neotropical semiaquatic spider Paratrechalea ornata (Trechaleidae) offer females a nuptial gift consisting of a prey item wrapped in silk. What stimulus inhibits males from feeding and elicits gift construction? We hypothesized that signals associated with female silk threads could affect decision-making by males. We investigated three groups of males carrying a captured prey under different experimental treatments. In the treatment S, males were exposed to an arena with female silk; in SF, males were exposed to both silk and a female confined in a cell, and in the control group, males were exposed to a clean arena. Gift construction was observed only in the S and SF groups, with a similar occurrence rate. After touching females (SF group), males did not change their pattern of gift construction. Gift construction occurrence increased with male and female age. The results lead us to assume that the existence of chemical cues associated with female silk elicits male searching behaviour and gift construction, allowing males to decide between eating or wrapping prey according to the possibility of a sexual encounter. Anticipating gift construction, males are ready to mate, diminishing the risks of predation, female desertion or male–male scramble competition. The effect of age on behavioural variation and the rate of construction is discussed.     

The morphology and ultrastructure of “amphipod silk” glands in <Emphasis Type="Italic">Ampithoe rubricata</Emphasis> (Crustacea,Amphipoda, Ampithoidae)

The anatomy and ultrastructure of “amphipod silk” glands in Ampithoe rubricata Montagu 1818 (Ampithoidae) have been studied. The morphology and ultrastructure of the glands in pereopods 3 and 4 have been examined in semithin and ultrathin sections using light and transmission electron microscopy. The glands of two types producing secretions different in their chemical compositions are observed in these pereopods. The ducts of the glands of both types lead to a common reservoir in the dactylus. Each gland comprises several secretory cells and one duct cell. The structure earlier regarded as the chitin wall of the duct is the cytoplasm of the duct cell; the presence of this cell in the studied glands is demonstrated for the first time. The secretory cells contain one or two nuclei and form rows along each duct cell. A new, previously unknown type of crustacean glands is described.     

The antennal benzoic acid receptor cell of the female silk moth <Emphasis Type="Italic">Bombyx mori</Emphasis> L.: structure–activity relationship studies with halogen substitutes

Studies on structure–activity relationships were carried out to characterize the response specificity of the benzoic acid cell of the female of the moth Bombyx mori by means of single sensillum electrophysiological recordings. We demonstrated that this cell type responds best to a natural key substance (benzoic acid) and has similar response profiles for less effective compounds, including various halogen substitutes of benzoic acid, benzaldehyde and other derivates of the key compound. Using different halogen substitutes (F, Cl, Br, I), we showed that the cellular response decreases with increasing atomic size of the substitute and that halogen substitutes were most effective in the meta-position. Thus, m-fluor benzoic acid was even more effective than benzoic acid. These results indicate that a critical feature of the stimulus molecule is the inductive effect generated by the halogen substitutes. Increasing the atomic size of the halogen substitute impairs the recognition of the molecule by the receptor cell, possibly due to steric effects. Decreasing the electron density in the aromatic ring improves the receptor response. The benzoic acid receptor cell can be considered as specialist despite not being involved in pheromone detection as it responds maximally to a key substance and has similar response profiles for less effective compounds.     

A comparative analysis of karyotypes of three species of Macleaya—producers of a complex of isoquinoline alkaloids

Using a set of methods (C-banding, DAPI-staining, fluorescence hybridization in situ (FISH) with probes of 26S and 5S rDNA, and analysis of meiosis), the first comparative cytogenetic study of three species of Macleaya, producers of complex isoquinoline alkaloids, cordate Macleaya cordata (Willd.) R. Br. (2n = 20), small-fruited Macleaya microcarpa (Maxim.) Fedde (2n = 20) and Macleaya kewensis Turrill (2n = 20), was first carried out. On the basis of morphometric analysis, formulas of karyotypes were made for each species. Species ideograms for M. cordata, M. microcarpa, and M. kewensis were constructed taking into account the polymorphic variants of the C-banding patterns and indicating the location of 26S and 5S rDNA sites. A comparative study revealed that the karyotypes of M. microcarpa and M. kewensis have more in common with each other than with M. cordata. Analysis of meiotic chromosomes suggests of genetic stability of Macleaya genomes. The results of chromosome analysis were used to confirm the close relationship of Macleaya and to clarify their phylogenetic relationships.     

Effect of γ-Irradiation on Development of Lachrymator of Onion

A thermosensitive uracil requiring mutant of Bacillus subtilis Marburg 168 thy trp₂ ts42 was examined as to the colony forming ability at the permissive and nonpermissive temperatures. The viability of the mutant cells decreased rapidly at the restrictive temperature in the modified Woese’s (MW) medium. However, the cells retained viability when sodium succinate or potassium chloride was added to the medium at that temperature although uracil deficiency was unchanged. A little but significant incorporation of adenine-8-¹⁴C into RNA still continued even after the incorporation of N-acetyl-³H-d-glucosamine into acid insoluble fraction of the cells terminated in the MW medium at 48°C. Both incorporations as well as increase of absorbance were slowed down in the presence of sodium succinate at 48°C. This mutant, ts42, was more sensitive to deoxycholate (DOC) than the parent strain. The restoration of colony forming ability after the temperature shift back from 48 to 37°C was suppressed by the addition of DOC to the medium. However, the cell became resistant to DOC when uracil was added to the medium prior to the temperature shift.     

Mechanisms of action of the α-amylase of Micromonospora melanosporea

The -amylase of Micromonospora melanosporea was produced extracellularly during batch fermentation in a 5.0-1 fermentor. The absence of an organic nitrogen source in its growth medium facilitated subsequent purification of the enzyme by ammonium sulphate fractionation and two consecutive Superose-12 gel-filtration steps. The enzyme exhibited maxima for activity at pH 7.0 and 55° C and was 72% stable at pH 6.0–12.0 for 30 min at 40° C. It had a relative molecular mass of 45 000 and an isoelectric point at pH 7.6. The enzyme catalyses the conversion of starch to maltose (53%, w/w) as the predominant final end-product. Initial hydrolysis of this substrate, however, gave rise to the formation of maltooligosaccharides in the range maltotriose to maltohexaose. Maximum yields of these intermediate sugars accumulated to between 31 and 42% (w/w) as the reaction proceeded. The action of the M. melanosporea amylase on high concentrations of saccharides larger than maltotriose resulted in the formation of mainly maltose and maltotriose without concomitant glucose production. A combination of hydrolytic and transfer events is postulated to be responsible for this phenomenon and for the high maltose levels achieved. Correspondence to: C. T. Kelly