Search

PDF file - 10K

PDF file - 293K

PDF file - 83K

PDF file - 406K

PDF file - 19K

PDF file - 96K

PDF file - 227K

Sericin, which is derived from the outer layer of silk thread proteins of the common silkworm (Bombyx mori), functions as a glue to fix fibers of developing cocoons. Although sericin has potential ...

A variety of substituted azoxybenzenes can be obtained by simple photoreduction of the corresponding nitrobenzene using a flow microreactor. The yield and chemoselectivity of each azoxybenzene depends on the type and position of the substituents. In addition, the use of liquid-liquid slug flow conditions, consisting of an organic solution and a fluorous solvent like tetradecafluorohexane (TDFH) or octadecafluorooctane (ODFO), improves both the conversion and yield of this photoreduction compared to those in simple one-phase flow conditions; these results are due to synergy between the very smooth photoreduction process and the highly efficient mixing obtained under continuous-flow conditions.

Growth factors, including fibroblast growth factor-7 (FGF-7), are a group of proteins that stimulate various cellular processes and are often used with carriers to prevent the rapid loss of their activities. Sericin with great biocompatibility has been investigated as a proteinaceous carrier to enhance the stability of incorporated proteins. The difficulties in obtaining intact sericin from silkworm cocoons and the handling of growth factors with poor stability necessitate an efficient technique to incorporate the protein into a sericin-based biomaterial. Here, we report the generation of a transgenic silkworm line simultaneously expressing and incorporating FGF-7 into cocoon shells containing almost exclusively sericin. Growth-factor-functionalized sericin cocoon shells requiring simple lyophilization and pulverization processes were successfully used to induce the proliferation and migration of keratinocytes. Moreover, FGF-7 incorporated into sericin-cocoon powder exhibited remarkable stability, with more than 70% of bioactivity being retained after being stored as a suspension at 25 °C for 3 months. Transgenic sericin-cocoon powder was used to continuously supply biologically active FGF-7 to generate a three-dimensionally cultured keratinocyte model in vitro. The outcomes of this study propound a feasible approach to producing cytokine-functionalized sericin materials that are ready to use for cell cultivation.

The biphasic nucleophilic aromatic substitution of 4-fluoronitrobenzene was carried out using a microreactor. To realize an efficient reaction process, we performed microflow reactions under droplet formation conditions to give the substituted products in excellent yields. The solvent effect was also investigated, and it was found that the use of diisobutyl ketone as an organic phase greatly accelerated the reaction.

The silk glands of silkworms produce large quantities of fibroin, which is a protein that can be physically processed and used as a biodegradable carrier for cell growth factors in tissue engineering applications. Meanwhile, protein microcrystals known as polyhedra, which are derived from cypovirus 1, have been used as a vehicle to protect and release encapsulated cell growth factors. We report the generation of transgenic silkworms that express recombinant fibroblast growth factor‐7 (FGF‐7) fused with the polyhedron‐encapsulating signal in polyhedra produced in the middle (MSG) and posterior (PSG) silk glands. Immunofluorescence showed that polyhedra from silk glands are associated with FGF‐7. The MSG and PSG from transgenic silkworms were processed into fine powdery materials, from which FGF‐7 activity was released to stimulate the proliferation of human keratinocyte epidermal cells. Powders from PSGs exhibited higher FGF‐7 activity than those from MSGs. Moreover, PSG powder showed a gradual release of FGF‐7 activity over a long period and induced keratinocyte proliferation and differentiation in 3D culture to promote the formation of stratified epidermis expressing positive differentiation marker proteins. Our results indicate that powdery materials incorporating the FGF‐7‐polyhedra microcrystals from silk glands are valuable for developing cell/tissue engineering applications in vivo and in vitro.

The iron-catalyzed cross-coupling reaction between an aromatic Grignard reagent and a haloalkane was investigated under micro-flow conditions. The reaction proceeded smoothly by the separate feeding of the substrate solutions to give the desired cross-coupling product. Compared to the previously reported batch reaction, the effect of additives was more predominant in the micro-flow reaction. The flow reactor system appears to contribute to the stabilization and rapid trapping of the unstable catalytic species.

Sericins are derived from the outer layer of silk thread proteins of the common silkworm (Bombyx mori) and act as a glue to fix fibers of the developing cocoon. Although sericin has potential uses ...

Recombinant hemagglutinin-neuraminidase (HN) based subunit vaccine, which is non-infectious and can be produced using insect cell-culture systems, is a potential alternative to conventional live and inactivated Newcastle disease virus (NDV) vaccines. However, process development for manufacture and efficacy assessment of HN subunit vaccines has been hampered by the absence of reference standards, a cornerstone for robust and sensitive quantitative analytical methods. In this work, a downstream purification strategy was developed to obtain NDV HN which was expressed with a hexa-histidine fusion tag (rHN) to facilitate detection using generic antibodies. Highly purified rHN (∼95%) attained after detergent extraction and two-stage ion-exchange-hydroxyapatite column chromatography was subsequently utilized as reference standards for quantitative ELISA development. Recovery of rHN at different stages of purification was monitored. Quantitation of rHN from crude cell lysates was performed for dose-ranging antibody response and protective efficacy studies. A higher dose (1500 ng) of rHN was correlated to a significant reduction in virus shedding and attainment of herd immunity, as indicated by a higher proportion of chickens (92%) with hemagglutination inhibition (HI) antibody titers ≥ log23. The outcome of this study, shows the importance of downstream process development in enabling robust quantitation and efficacy assessment of a recombinant subunit vaccine.

Silk fibroin exhibits high biocompatibility and biodegradability, making it a versatile biomaterial for medical applications. However, contaminated silkworm-derived substances in remnant sericin from the filature and degumming process can result in undesired immune reactions and silk allergy, limiting the widespread use of fibroin. Here, we established transgenic silkworms with modified middle silk glands, in which sericin expression was repressed by the ectopic expression of cabbage butterfly-derived cytotoxin pierisin-1A, to produce cocoons composed solely of fibroin. Intact, nondegraded fibroin can be prepared from the transgenic cocoons without the need for sericin removal by the filature and degumming steps that cause fibroin degradation. A wide-angle X-ray diffraction analysis revealed low crystallinity in the transgenic cocoons. However, nondegraded fibroin obtained from transgenic cocoons enabled the formation of fibroin sponges with varying densities by using 1–5% (v/v) alcohol. The effective chondrogenic differentiation of ATDC5 cells was induced following their cultivation on substrates coated with intact fibroin. Our results showed that intact, allergen-free fibroin can be obtained from transgenic cocoons without the need for sericin removal, providing a method to produce fibroin-based materials with high biocompatibility for biomedical uses.

In this work, the [2+2] photocycloaddition of carbonyl compounds with olefins, the Paterno-Buchi-type photoreaction, was performed in a flow microreactor under slug flow (two-phase flow) conditions...

Nucleophilic aromatic substitution, one of the most common transformation methods for aromatic organic compounds, is often performed under biphasic conditions using a phase-transfer catalyst. Herein, we investigated the synthesis of 4-nitrophenetole from 4-fluoronitrobenzene in a continuous-flow microreactor, which accelerates mass transfer between the two phases. The reaction proceeded smoothly under slug flow conditions and a slight acceleration effect was observed under ambient pressure. By contrast, high temperature and pressure conditions gave the target compound in good yield within a shorter residence time.

Herein, we report a highly selective preparation method of azoxybenzenes from nitrobenzenes by visible-light irradiation. In a batch, this reaction method affords the corresponding aniline derivatives or intermediates (nitrosobenzene or phenyl hydroxylamine); however, in flow microreactors, azoxybenzenes are successfully obtained with very high selectivity.

Homodimerization and crossed-dimerization reactions for ferulic acid and related compounds in an acidic ethanol solution were investigated via a formal [3 + 2] cycloaddition reaction. Arylindanes, as the major products, were produced from some of the acids with hydroxy group and/or methoxy groups at the para and meta positions of the phenyl ring. The formation of arylindane required the following consecutive reactions: (I) protonation of the first produced ester to afford to a benzylic cation, (II) which reacts with another ester to afford a new benzylic cation, and (III) followed by cyclization to produce the dimer. It is suggested that the para-substituent promoted both step I and step II and the meta-substituent accelerated step III. To rationalize the selectivity of homodimerization, the stability of the benzyl cations and the specific electron distribution on the molecular surface of each ester is discussed based on the results of the molecular orbital calculations.

Candida antarctica lipase B (CALB) was cross-linked efficiently, using a reagent containing glutaraldehyde (GA), paraformaldehyde (PA), and a polylysine reagent. For the reaction under micro-flow conditions, CALB was immobilized on the inner surface of a polytetrafluoroethylene (PTFE) tube, by flowing the cross-linking reagents in an appropriate flow rate. The immobilized CALB formed a membrane-like structure on the inner surface of the tube and showed catalytic activity for transesterification. Although the enzyme activity was not high at the immobilized CALB, the productivity of micro-flow reaction using immobilized CALB was higher than that of batch-wise reaction using free CALB.

The molecular structures of in cell protein crystals containing organometallic Pd(allyl) complexes were determined by performing microfocus X-ray diffraction experiments. The coordination sites in a polyhedrin mutant with deletion of selected amino acid residues located at the interface of the polyhedrin trimer are dramatically altered compared to those of the wild-type composite.

Genetically manipulated organisms with dysfunction of specific tissues are crucial for the study of various biological applications and mechanisms. However, the bioengineering of model organisms with tissue-specific dysfunction has not progressed because the challenges of expression of proteins, such as cytotoxins, in living cells of individual organisms need to be overcome first. Here, we report the establishment of a transgenic silkworm (Bombyx mori) with posterior silk glands (PSGs) that was designed to express the cabbage butterfly (Pieris rapae) cytotoxin pierisin-1A (P1A). P1A, a homolog of the apoptosis inducer pierisin-1, had relatively lower DNA ADP ribosyltransferase activity than pierisin-1; it also induced the repression of certain protein synthesis when expressed in B. mori-derived cultured cells. The transgene-derived P1A domain harboring enzymatic activity was successfully expressed in the transgenic silkworm PSGs. The glands showed no apoptosis-related morphological changes; however, an abnormal appearance was evident. The introduced truncated P1A resulted in the dysfunction of PSGs in that they failed to produce the silk protein fibroin. Cocoons generated by the silkworms solely consisted of the glue-like glycoprotein sericin, from which soluble sericin could be prepared to form hydrogels. Embryonic stem cells could be maintained on the hydrogels in an undifferentiated state and proliferated through stimulation by the cytokines introduced into the hydrogels. Thus, bioengineering with targeted P1A expression successfully produced silkworms with a biologically useful trait that has significant application potential.

Cypovirus is an insect virus that is encapsulated in stable cubic protein crystals composed of polyhedrin protein produced in virus-infected cells. Molecular technology developed over the last decade is now able to immobilise proteins of interest on polyhedrin crystals. Modified polyhedrin crystals can be used in cell cultures for implantation in animals and vaccines, among other applications. However, this technique does not work for some proteins. Here, we developed and tested an alternative approach for immobilising foreign proteins in polyhedrin crystals using a linker method; diverse proteins, such as fluorescent proteins, enzymes, antibodies, and streptavidin were successfully contained. The immobilised antibodies retained their binding activity on filter paper, implying their potential for new immunochromatography applications. Moreover, this immobilisation method allows enzymes to be collected from one reaction reagent and transferred to another reagent. These results demonstrate the potential of this immobilisation method and the likelihood of expanding the applications of polyhedrin crystals using this approach.

The spatial and temporal availability of cytokines, and the microenvironments this creates, is critical to tissue development and homeostasis. Creating concentration gradients in vitro using soluble proteins is challenging as they do not provide a self-sustainable source. To mimic the sustained cytokine secretion seen in vivo from the extracellular matrix (ECM), we encapsulated a cargo protein into insect virus-derived proteins to form nanoparticle co-crystals and studied the release of this cargo protein mediated by matrix metalloproteinase-2 (MMP-2) and MMP-8. Specifically, when nerve growth factor (NGF), a neurotrophin, was encapsulated into nanoparticles, its release was promoted by MMPs secreted by a PC12 neuronal cell line. When these NGF nanoparticles were spotted onto a cover slip to create a uniform circular field, movement and alignment of PC12 cells via their extended axons along the periphery of the NGF nanoparticle field was observed. Neural cell differentiation was confirmed by the expression of specific markers of tau, neurofilament, and GAP-43. Connections between the extended axons and the growth cones were also observed, and expression of connexin 43 was consistent with the formation of gap junctions. Extensions and connection of very fine filopodia occurred between growth cones. Our studies indicate that crystalline protein nanoparticles can be utilized to generate a highly stable cytokine gradient microenvironment that regulates the alignment and differentiation of nerve cells. This technique greatly simplifies the creation of protein concentration gradients and may lead to therapies for neuronal injuries and disease.

Specific gene functions have been successfully suppressed by gene silencing or editing in many organisms. However, genetic manipulation to suppress the function of a target tissue has not been achieved using genes such as cytotoxin family members. We established transgenic silkworms with posterior silk glands (PSGs) that express the enzymatic domain of the cytotoxin pierisin-1A (P1A), a recently identified cytotoxic protein from the cabbage butterfly Pieris rapae , which has a relatively lower DNA ADP-ribosylating activity among the pierisin family. Larvae with the modified PSGs produced sericin cocoons with potential utility in tissue engineering. Our observations of an embryonic stem (ES) cell proliferation on fabricated sericin hydrogels demonstrated that such gels can act as a scaffolds to support cell growth and also mimic extracellular matrices to provide cell growth factors. In this highlight paper, we describe a new approach through targeted P1A expression, which could be applicable to the development of biologically-useful model organisms with tissue-specific dysfunction.

Protein and amino acid structures of Norovirus-like particles (NoVLP) have been investigated by Raman spectroscopy before and after encapsulation into Bombyx mori cypovirus (BmCPV) cubic microcrystals, which are usually referred to as cubes or polyhedra. Two different types of tag were used in co-expression, namely VP3 and H1 tags. VP3 tag is derived from a capsid protein VP4 from BmCPV and H1 tag is N-terminal α-helix of BmCPV polyhedrin, respectively. A major capsid protein VP1 of NoVLP G11.4 was fused with H1 or VP3 tags, and then encapsulated into BmCPV polyhedra. Analyses of the spectroscopic data permitted the assignment of conformation-sensitive Raman bands to viral amino acid constituents and the observation of structural similarities or differences between differently tagged samples. Three separate Raman zones were attentioned, namely, the ring-mode structure region (1000–1500 cm−1), the C O and C C double-bond region and its surroundings (1500–1750 cm−1), and the high-frequency C H stretching region (2800–3100 cm−1). Structural fingerprints could be found in specific spectral zones for differently co-expressed samples. One clear characteristic of the H1-tagged VP1 polyhedra was the increase in tyrosine fraction, which played a critical role in binding neighboring strands through its unpaired negatively charged COO– sites. This feature could consistently be detected in different regions, but it was best represented by Raman signals associated with negatively charged COO– sites and H1 helices in the double-bond region. Such peculiar chemical features were revealed by two relatively broad bands at 1570 and 1630 cm−1, which were assigned to COO– anti-symmetric stretching and amide I in 310-helix extensions to α-helices at N-termini, respectively. These specific features did not display in the spectrum of the VP3-tagged VP1 polyhedra. Concurrently, a strong reduction of C H bond Raman signal was noticed in the high frequency stretching region of the Raman spectrum upon H1-tagged VP1 polyhedra. The Raman activity most strikingly also represented fingerprints of tagged NoVLP VP1 after its encapsulation into BmCPV polyhedra, opening thus the possibility to in situ advanced experiments in the fields of drug delivery and regenerative medicine.

Crystalline protein assemblies of polyhedra crystal (PhC) can be utilized as solid enzyme containers for long-term storage of enzymes with retention of their enzymatic activity. The enzymes can be released from the crystals at the optimum pH for the enzymatic activity by dissolution of the crystals using in vivo crystal engineering.

Ferulic acid dimers with phenylindane skeletons were obtained from ferulic acid 1 in an acidic ethanol solution. The reaction conditions used to prepare the dimers or the ester were examined in detail, and the stereochemistries of the dimers were determined. The structural features of the dimers and the diastereoselective formation mechanisms involved in forming the two types of dimers (the racemic diastereomer 8α, which was a major product, and the racemic diastereomer 8γ, which was a minor product) were characterized.

A tunable light source integrated with a DR laser array and an AWG coupler was proposed for digital coherent communication. Fiber output power as high as 19 dBm and spectral linewidth as narrow as 70 kHz were obtained as a tunable light source module.

Crystalline porous materials have been investigated for development of important applications in molecular storage, separations, and catalysis. The potential of protein crystals is increasing as they become better understood. Protein crystals have been regarded as porous materials because they present highly ordered 3D arrangements of protein molecules with high porosity and wide range of pore sizes. However, it remains difficult to functionalize protein crystals in living cells. Here, we report that polyhedra, a natural crystalline protein assembly of polyhedrin monomer (PhM) produced in insect cells infected by cypovirus, can be engineered to extend porous networks by deleting selected amino acid residues located on the intermolecular contact region of PhM. The adsorption rates and quantities of fluorescent dyes stored within the mutant crystals are increased relative to those of the wild-type polyhedra crystal (WTPhC) under both in vitro and in vivo conditions. These results provide a strategy for designing self-assembled protein materials with applications in molecular recognition and storage of exogenous substances in living cell as well as an entry point for development of bioorthogonal chemistry and in vivo crystal structure analysis.

Stable protein microcrystals called polyhedra are produced by certain insect viruses. Cytokines, such as fibroblast growth factors (FGFs), can be immobilized within polyhedra. Here, we investigated three-dimensional (3D) co-cultures of keratinocytes and melanocytes on collagen gel containing FGF-2 and FGF-7 polyhedra. Melanocytes were observed to reside at the base of the 3D cell culture and melanin was also typically observed in the lower layer. The 3D cell culture model with FGF-2 and FGF-7 polyhedra was a useful in vitro model of the epidermis due to effective melanogenesis, proliferation and differentiation of keratinocytes. FGF-7 polyhedra showed a potent cytoprotective effect when keratinocytes were treated with menadione, which is a generator of reactive oxygen species. The cytoprotective effect was activated by the inositol triphosphate kinase-Akt pathway leading to upregulation of the antioxidant enzymes superoxide dismutase and peroxiredoxin 6.

Oleic acid esters of phytosterols (PSs) and triterpene alcohols (TAs), derived from rice bran, were synthesized using lipases under mild conditions. Some lipases, especially from Candida rugosa, type VII, showed very high substrate specificity towards both PSs and TAs, when a mixture of PS and TA (PS/TA mixture) was used as the substrate source. The maximum yield of PS esters was ca. 80 % in each case; however, the maximum yield of TA esters was much lower when the reaction was continued for 7 days. Due to the difficulty in purifying the esters obtained when the PS/TA mixture was used as source of substrate, free PSs and TAs were separated from the PS/TA mixture by silica-gel and reverse-phase chromatography prior to esterification. The pure PSs or TAs were esterified with oleic acid to obtain the corresponding esters with high purity. Differential scanning calorimetric analysis of the resulting esters revealed that their melting points ranged from 7.0 to 42 °C. These values were at least 100 °C lower than those of the free PSs and TAs.

We fabricated TLS module with AWG-DR-LD array for digital coherent application. Separating SOA from LD chip enables us to maximize the performance. We achieved 80mW fiber output power and 80–120 kHz linewidth.

Photoactivatable CO releasing protein crystals were developed by immobilization of Mn carbonyl complexes in polyhedral crystals, which are spontaneously formed in insect cells. The photoactivatable CO release from the engineered protein crystals activates nuclear factor kappa B (NF-κB) upon stimulation by visible light irradiation with suppression of cytotoxicity of the Mn complex.