Biosynthetic sericin 1-like protein skews dendritic cells to tolerogenic-like phenotype.

Silkworm sericin has been widely exploited in biomaterials due to its favorable biological activities. However, the extraction processes of sericin from silkworm cocoons can alter the biological and biophysical properties, including a structural diversity of natural sericin. In addition, extracted natural sericin is often contaminated with fibroin that may be harmful to human cells. Induction of tolerogenic dendritic cell (DC) has become a strategy in biomaterial fields because this cell type plays a key role in immune modulation and wound healing. To overcome undesired effects of extracted natural sericin and to improve its biological properties, we biosynthesized sericin 1-like protein that contained only functional motifs and tested its biological activity and immunomodulatory properties in fibroblasts and DCs, respectively. In comparison to natural sericin, biosynthetic sericin 1 promoted collagen production in fibroblasts at a late time point. Furthermore, DCs treated with biosynthetic sericin 1 exhibited a tolerogenic-like phenotype with semimaturation and low production of proinflammatory cytokines, but high production of anti-inflammatory cytokine, IL-10. Biosynthetic sericin 1 might be developed as immunomodulator or immunosuppressant.

Chromatographic profiling of silk sericin for biomedical and cosmetic use by complementary hydrophylic, reversed phase and size exclusion chromatographic methods.

Silk sericin (SS) is, together with silk fibroin (SF), one of the two proteins forming the silkworm cocoon. SS is ideal ingredient for cosmetic applications in the formulation of specific products for skin care and hair due to its peculiar physical-chemical composition. SS also showed a great potential in different pharmacological and biotechnological applications, as anticancer drug, anticoagulant, cell culture additive, wound healing agent and drug delivery carrier. Reasons for SS use in biomedical applications derive from its physical-chemical composition. As a consequence, a detailed characterization of SS in terms of average molecular weight, molecular weight distribution and hydro/lipophilic character is crucial to properly address and assess its quality, cosmetic or pharmacological use. In this study, the application of different and complementary chromatographic modes allows a detailed investigation of SS protein isolated from wastewater using two diverse extraction methods. Hydrophilic interaction liquid chromatography (HILIC using an AdvanceBio Glycan Map column) and reverse phase (RP using Symmetry300 C18 column) were applied to intact protein characterization to derive data on protein hydrophilicity and on hydrophobic components of the two SS preparations (SS#1 and SS#2). A higher hydrophilic character of SS#1 was observed by HILIC trace, coherently with the preparation method used, while no significant differences in hydrophobicity were detectable in the RPLC separations. Size distribution was also defined by using a SEC-UV-MS method (using TSKgel SuperSW2000 column) properly optimized to maximize both the size selectivity and the method sensitivity. Taken together, the chromatographic data allowed to better characterize the SS samples obtained by different extraction methods, and the structural properties were correlated to their biological activities.

Quantification of silk protein using phage nanofibers with high binding specificity.

Silk sericin (SS) has emerged as an important silk protein for use in medicine and textiles. However, no sensitive method is available for detecting it. Here, we employed phage nanofibers (∼7 nm wide) as a probe to quantify SS from a dilute aqueous solution by exploiting two properties of the bacteria-infecting phage nanofibers, its use as a platform for discovering SS-binding peptide and its ultrasensitive quantification by a simple titering assay (where the number of phage nanofibers displaying the SS-binding peptide is equal to the number of countable millimeter-sized plaques derived from the phage nanofibers by infecting bacteria through plating). We first discovered a SS-binding peptide and the phage nanofibers (SS-phage) displaying this peptide at the tip. We found that this peptide can even differentiate SS from another silk protein (silk fibroin), showing its high specificity. We then employed SS-phage nanofibers as a probe to bind the SS casted from the aqueous solution. Because SS-phage nanofibers bound to the SS and the SS in the original SS solution were numerically correlated and the number of SS-phage nanofibers can be determined by counting the plaques in a Petri dish by the titering assay, determining the number of phage-derived plaques with the naked eye led to the rapid quantification of SS concentration with a detection limit of 19.50 ng ml-1. This phage-based counting strategy can be potentially applied to the facile detection of other proteins.

Development of novel sericin and alginate-based biosorbents for precious metal removal from wastewater.

In this study, two novel low water-soluble sericin and alginate-based biosorbents were successfully developed for precious metal removal from wastewater: sericin and alginate particles chemically crosslinked by proantocyanidins (SAPAs) and sericin, alginate and polyvinyl alcohol particles (SAPVA). The proportions of proantocynidins (PAs) or polyvinyl alcohol (PVA) added to sericin (2.5% w/v) and alginate (2.0% w/v) blend were 0.5, 1.5, 2.5 and 3.5% w/v. Among these concentrations, particles produced with 0.5% w/v of PVA or 2.5% w/v of PAs presented the lowest water solubility percentages (3.74 ± 0.05 and 3.56 ± 0.21%, respectively) and the following metallic affinity order: AuCl4- > PdCl42- > PtCl62- > Ag+. Then, gold biosorption kinetics by SAPAs was evaluated at three gold initial concentrations (72.88, 187.12, and 273.79 mg/L), and its performance was compared to activated carbon adsorbent uptake. The data modeling revealed that the process follows pseudo-first-order kinetics and is mainly controlled by external diffusion. SAPAs before and after gold biosorption (SAPAs-gold) were characterized by scanning electron microscopy coupled with energy dispersive X-ray spectroscopy, optical microscopy, helium pycnometry, mercury porosimetry, N2 physisorption, and Fourier-transform infrared spectroscopy.

Analysis of proteome dynamics inside the silk gland lumen of Bombyx mori.

The silk gland is the only organ where silk proteins are synthesized and secreted in the silkworm, Bombyx mori. Silk proteins are stored in the lumen of the silk gland for around eight days during the fifth instar. Determining their dynamic changes is helpful for clarifying the secretion mechanism of silk proteins. Here, we identified the proteome in the silk gland lumen using liquid chromatography-tandem mass spectrometry, and demonstrated its changes during two key stages. From day 5 of the fifth instar to day 1 of wandering, the abundances of fibroins, sericins, seroins, and proteins of unknown functions increased significantly in different compartments of the silk gland lumen. As a result, these accumulated proteins constituted the major cocoon components. In contrast, the abundances of enzymes and extracellular matrix proteins decreased in the silk gland lumen, suggesting that they were not the structural constituents of silk. Twenty-five enzymes may be involved in the regulation of hormone metabolism for proper silk gland function. In addition, the metabolism of other non-proteinous components such as chitin and pigment were also discussed in this study.

The characterization of protein release from sericin film in the presence of an enzyme: towards fibroblast growth factor-2 delivery.

Aqueous preparations of silk protein (sericin) films were prepared to evaluate their biodegradation properties. In the absence of trypsin, sericin film swelled rapidly, kept its shape, and remained unaltered for 28 days or longer due to form ß-sheet structures. In the presence of trypsin, sericin film gradually degraded; since the rate depended on the concentration of trypsin, the films likely underwent enzymatic hydrolysis. Sericin film incorporating the model protein drug fluorescein isothiocyanate-albumin (FA) also gradually degraded in the presence of trypsin and resulted in the sustained release of FA for 2 weeks or longer; in contrast, FA release was quite slow in the absence of trypsin. It is expected that sericin film has potential as a biodegradable and drug-releasing carrier. To evaluate the practical applicability of sericin film for the repair of defective tissues, fibroblast growth factor-2 (FGF-2) was incorporated into sericin films and the films were implanted on skull defects in rats. Whereas FGF-2 release was suppressed in the absence of trypsin in vitro, it appears that FGF-2, immobilized by ionic interactions between sericin and FGF-2, can be sustained-released in vivo from films incorporating 2500 or 250 ng of FGF-2 to support the growth of tissue around wounds.

Voltammetric determination of sericin based on its interaction with carmine.

A simple yet sensitive method is developed for the determination of sericin using voltammetry based on the interaction between sericin and carmine for the first time. In the absence of sericin, carmine has a pair of well-defined redox peaks in a pH 1.81 Britton-Robinson buffer solution. Although no new redox peaks appear upon the addition of sericin into a carmine solution, the peak currents of the old peaks reduce while the peak potentials shift positively. This observation is attributed to the decrease in the diffusion coefficient and electrode reaction rate constant of carmine in the presence of sericin. A binding mechanism is proposed and discussed, and the binding constant and binding ratio are calculated as 2.32 x 10(6) L mol(-1) and 1:1, respectively. Furthermore, the decrease in the peak currents is found proportional to the sericin concentration in the range of 32.0-800.0 microg mL(-1) with a detection limit of 13.52 microg mL(-1). The method is further applied to the determination of sericin in degumming wastewater with satisfied average recoveries from 96.7 to 103.3%. The results are in good agreement with those obtained by the conventional Coomassie brilliant blue G-250 spectrophotometric method.

Protective effect of sericin peptide against alcohol-induced gastric injury in mice.

BACKGROUND: Sericin peptide (SP) has shown a powerful anti-oxidant property in a host of studies. The present study was designed to investigate the possible protective effects of SP against alcohol-induced gastric lesions in mice and to explore the potential mechanisms. METHODS: Animals were randomly divided into 5 groups: control, alcohol (56%, 14.2 ml/kg), SP-treated mice (0.2, 0.4, 0.8 g/kg). Mice were pretreated with SP before administering alcohol, the concentration of ethanol in serum and urine, the contents of malondialdehyde (MDA), glutathione (GSH) and the glutathione peroxidase (GSH-PX), catalase (CAT) and superoxide dismutase (SOD) activities in the gastric mucosa were measured, subsequently, the pathological evaluation of stomach was also observed. RESULTS: Of the animals pre-treated with SP (0.4, 0.8 g/kg), the concentration of ethanol in serum was significantly decreased, while increased in urine as compared to the alcohol-administered alone animals. Alcohol administration caused severe gastric damage as indicated by markedly increased MDA levels and decreased antioxidants, such as reduced GSH, GSH-PX and SOD in the gastric tissue while the CAT activity was not altered. On SP administration there was a reversal in these values towards normal. Histopathological studies confirmed the beneficial role of SP, which was in accordance with the biochemical parameters. CONCLUSIONS: SP could protect gastric mucosa from alcohol-induced mucosal injury. These gastroprotective effects might be due to increasing 'first-pass metabolism' in the stomach and hastening ethanol elimination directly through the urine. SP might also play an important role in the protection of the structure and function of gastric mitochondria, at least partly based on their anti-oxidant effect.

Antioxidant potential of silk protein sericin against hydrogen peroxide-induced oxidative stress in skin fibroblasts.

The antioxidant potential of silk protein sericin from the non-mulberry tropical tasar silkworm Antheraea mylitta cocoon has been assessed and compared with that of the mulberry silkworm, Bombyx mori. Skin fibroblast cell line (AH927) challenged with hydrogen peroxide served as the positive control for the experiment. Our results showed that the sericin obtained from tasar cocoons offers protection against oxidative stress and cell viability is restored to that of control on pre-incubation with the sericin. Fibroblasts pre-incubated with non-mulberry sericin had significantly lower levels of catalase; lactate dehydrogenase and malondialdehyde activity when compared to untreated ones. This report indicates that the silk protein sericin from the non-mulberry tropical tasar silkworm, A. mylitta can serve as a valuable antioxidant.

Modification of sericin-free silk fibers for ligament tissue engineering application.

Biomedical application of silk requires the removal of sericin that is the gumming material of native silk fibers. This is because sericin can elicit an adverse immune response after implantation in the human body. However, the removal of sericin causes the silk fiber to fray and weakens its structural property, making it very difficult to knit or braid them into a scaffold for ligament tissue engineering applications. The aim of this study was to replace sericin with gelatin using NDGA as a cross-linking agent to biomimic the natural structure of native silk fibers. The physical properties and biocompatibility of the modified and native silk fibers were compared by in vitro and in vivo models. The mechanical and swelling properties of sericin-free silk fibers were greatly increased after modification with gelatin. Both modified and native silk fibers were shown to be nontoxic by in vitro cytotoxicity tests. The in vivo study demonstrated that the modified silk fibers, after 4 weeks' subcutaneous implantation in rats, caused little or no inflammatory reaction as compared with native silk fibers. The superior mechanical properties and lower inflammatory potential of modified silk fibers make them a promising candidate for ligament tissue engineering applications.

Characterization of amino acids in silk sericin protein from Gonometa rufobrunnae by MEKC with phenyl isothiocyanate derivatization.

An MEKC method was developed for the separation and characterization of phenyl-isothiocyanate (PITC)-labeled amino acids derived from Gonometa rufobrunnae silkworm after microdialysis sample cleanup. The influence of the buffer and SDS concentration on the resolution of the amino acids was investigated. A buffer system consisting of 25 mM phosphate, 10 mM borate buffer at pH 9.00, and 70 mM SDS showed the best results, with 13 PITC-amino acid derivatives being resolved out of 15 possible amino acids that were under study. Microdialysis sampling demonstrated its efficiency as a sample cleanup technique. Sericin protein from G. rufobrunnae was found to be characterized by at least 11 positively identified amino acids. These included His, Tyr, Ser, Ala, Phe, Lys, Gly, Arg, Cys, Glu, and Asp. Leu/Met and Val/Thr were coeluting pairs and hence could not be positively confirmed.

Antioxidative activity of animal and vegetable dietary fibers.

Some dietary fibers originated from insects such as silkworm (Sericin) and others along with constituents of several representative seaweeds such as wakame Undaria pinnatifida; hijiki Hizikia fusifome; and kombu Laminaria japonica, were found to have fairly large reaction rates determined by quenching experiments of emission spectra in the near-infrared region lambdamax 1270 nm for singlet oxygen 1O2, Cypridina luminescence method for superoxide, and peroxide value (POV) for autoxidation. The determined reaction rates are between 10(3)-10(5) (g/L)(-1) s(-1) for the insect and the plant dietary fibers; the larger ones are as large as that of ascorbic acid, 1.93 x 10(4) (g/L)(-1) s(-1) for singlet oxygen. Most of these seaweed constituents also showed antioxidative activity against autoxidation and superoxide as well as their immunological enhancing activity. These results suggest a possibility that dietary fibers that are supposed to prevent the large-intestine cancer by their physical properties may prevent the cancer, at least in parts, by their chemical, antioxidative activity.