The potential of a universal influenza virus-like particle vaccine expressing a chimeric cytokine.

The efficacy of the current influenza vaccines is frequently reduced because of antigenic drift, a trade-off of developing improved vaccines with broad cross-protective activity against influenza A viruses. In this study, we have successfully constructed a chimeric cytokine (CC) comprising the M2 protein, influenza A neuraminidase stalk, and interleukin-12. We produced virus-like particles (VLPs) containing CC and influenza hemagglutinin (HA) proteins using a baculovirus system in Eri silkworm pupae. The protective efficacy of the CCHA-VLP vaccine was evaluated in mice. The CCFkH5HA-VLP vaccine increased the survival rates of BALB/c mice, infected with a lethal dose of PRH1 and HKH5 viruses, to 80% and 100%, respectively. The results suggested that CCHA-VLP successfully induced potent cross-reactive protective immunity against infection with homologous and heterologous subtypes of the influenza A virus. This is the first study to design a CC-containing HA-VLP vaccine and validate its protective efficacy.

Eri silkworm spins mechanically robust silk fibers regardless of reeling speed.

Wild silkworms survive in environmental habitats in which temperature and humidity vary based on the weather. In contrast, domesticated silkworms live in mild environments where temperature and humidity are generally maintained at constant levels. Previous studies showed that the mechanical strengths and molecular orientation of the silk fibers reeled from domesticated silkworms are significantly influenced by the reeling speed. Here, we investigated the effects of reeling speed on the mechanical properties of eri silk fibers produced by wild silkworms, Samia cynthia ricini, which belong to the family of Saturniidae. We found that the structural, morphological and mechanical features of eri silk fibers are maintained irrespective of the reeling speed, in contrast to those of domesticated silkworm silk fibers. The obtained results are useful not only for understanding the biological basis underlying the natural formation of silk fibers but also for contributing to the design of artificial spinning systems for producing synthetic silk fibers.

Long-term preservation of eri and ailanthus silkworms using frozen gonads.

Cryopreservation of eri and ailanthus silkworms using frozen gonads was investigated. First, we evaluated the freeze tolerance of ovary and testis in the eri silkworm, which showed high tolerance. Mating between frozen ovary-transplanted females and frozen testis-transplanted males produced 163.0 eggs, yielding 105.7 larvae per moth. In a second experiment, we tested the use of the eri silkworm as a host insect for gonad transplantation from ailanthus silkworm donors. A high success ratio for laid and hatched eggs was demonstrated for ovary transplantation (97.8 and 51.3 eggs per moth, respectively). For testis transplantation, however, the average number of hatched larvae was low (12.0). Mating between host eri females and males in which both frozen ovary and testis of the ailanthus silkworm had been transplanted produced 6.4 fertilized eggs per host moth. Our success in using cross subspecies cryopreservation between these wild silkworms could lead to the alternative use of hosts between species in other insects.

The silkworm-an attractive BioResource supplied by Japan.

Silkworms have played an important agricultural role in supporting Japan's modernization, and traditionally, Japan has led the world as a repository of silkworm bioresources. The silkworm is a small and highly domesticated insect, which is ideal as a laboratory tool, although it is a bioresource that is relatively infrequently used in experiments at present. In this review, we describe the potential for silkworm resources to contribute to life sciences.

Transgenic silkworms (Bombyx mori) produce recombinant spider dragline silk in cocoons.

Spider dragline silk is a unique fibrous protein with a combination of tensile strength and elasticity, but the isolation of large amounts of silk from spiders is not feasible. In this study, we generated germline-transgenic silkworms (Bombyx mori) that spun cocoons containing recombinant spider silk. A piggyBac-based transformation vector was constructed that carried spider dragline silk (MaSp1) cDNA driven by the sericin 1 promoter. Silkworm eggs were injected with the vector, producing transgenic silkworms displaying DsRed fluorescence in their eyes. Genotyping analysis confirmed the integration of the MaSp1 gene into the genome of the transgenic silkworms, and silk protein analysis revealed its expression and secretion in the cocoon. Compared with wild-type silk, the recombinant silk displayed a higher tensile strength and elasticity. The results indicate the potential for producing recombinant spider silk in transgenic B. mori.

Sequence structure and expression pattern of a novel anionic defensin-like gene from silkworm (Bombyx mori).

A defensin-like gene, BmdefA, was rediscovered in the silkworm genome and expressed sequence tags databases. The open reading frame of BmdefA encodes a prepropeptide consisting of a 22-residue signal peptide, a 34-residue propeptide, and a 36-residue mature peptide with a molecular mass of 4.0 kDa. The mature peptide possesses the characteristic six-cysteine motif of insect defensins, and its predicted isoelectric point is 4.12, indicating it is a novel anionic defensin. An intron is present in BmdefA and several cis-regulatory elements are in the regulating region. It is transcribed constitutively at a high level in the hemocyte, silk gland, head, and ovary of the silkworm larvae, and in the fat body of early-stage pupae and moth. BmdefA is also strongly induced by immune challenge. These results suggest that BmdefA plays an important role in both immunity and metamorphosis.

Expression of EGFP-spider dragline silk fusion protein in BmN cells and larvae of silkworm showed the solubility is primary limit for dragline proteins yield.

Spider dragline silk is a unique fibrous protein with combination of tensile strength and elasticity, but the isolation of large amount of silk from spiders is not feasible. In this paper, we used a newly established Bac-to-Bac/BmNPV Baculovirus expression system to express the recombinant spider (Nephila clavata) dragline silk protein (MaSp1) fused EGFP in BmN cells and larvae of silkworm. A 70 kDa fusion protein was visualized after rBacmid/BmNPV/drag infection by SDS-PAGE and immunoblotting analysis. Fusion protein expressed in the BmN cells probably occupied five percent of the cell total protein; In a silkworm larva, approximately 6 mg fusion proteins were expressed. Solubility analysis of the expressed spider dragline silk protein indicated that 60% fusion protein is insoluble. EGFP fluorescence showed that fusion protein is tend to form aggregate by self assemblage. The results indicated the solubility is the primary limit for spider dragline proteins yield. It also suggested that directly produce fibrous spider silk in the secreting-silk organs of the transgenic silkworm larvae might be a better method.

The Pitx homeobox gene in Bombyx mori: regulation of DH-PBAN neuropeptide hormone gene expression.

The diapause hormone-pheromone biosynthesis activating neuropeptide gene, DH-PBAN, is expressed exclusively in seven pairs of DH-PBAN-producing neurosecretory cells (DHPCs) on the terminally differentiated processes of the subesophageal ganglion (SG). To help reveal the regulatory mechanisms of cell-specific DH-PBAN expression, we identified a cis-regulatory element that regulates expression in DHPCs using the recombinant AcNPV-mediated gene transfer system and a gel-mobility shift assay. Bombyx mori Pitx (BmPitx), a bicoid-like homeobox transcription factor, binds this element and activates DH-PBAN expression. The BmPitx was expressed in various tissues, including DHPCs in the SG. Suppression of DH-PBAN expression by silencing of the BmPitx successfully induced non-diapaused eggs from a diapause egg producer. To the best of our knowledge, this report is the first to identify a neuropeptide-encoding gene as a target of the Pitx transcriptional regulator in invertebrates. Thus, it is tempting to speculate that functional conservation of Pitx family members on neuropeptide gene expression occurs through a "combinational code mechanism" in both vertebrate and invertebrate in neuroendocrine systems.

Novel molecular and mechanical properties of egg case silk from wasp spider, Argiope bruennichi.

Araneoid spiders use specialized abdominal glands to produce up to seven different protein-based silks/glues that have various mechanical properties. To date, the fibroin sequences encoding egg case fibers have not been fully determined. To gain further understanding of a recently reported spider silk protein gene family, several novel strategies were utilized in this study to isolate two full-length cDNAs of egg case silk proteins, cylindrical silk protein 1 (CySp1, 9.1 kb) and cylindrical silk protein 2 (CySp2, 9.8 kb), from the wasp spider, Argiope bruennichi. Northern blotting analysis demonstrated that CySp1 and CySp2 are selectively expressed in the cylindrical glands. The amino acid composition of raw egg case silk was closely consistent with the deduced amino acid composition based on the sequences of CySp1 and CySp2, which supports the assertion that CySp1 and CySp2 represent two major components of egg case silk. CySp1 and CySp2 are primarily composed of remarkable homogeneous assemble repeats that are 180 residues in length and consist of several complex subrepeats, and they contain highly homologous C-termini and markedly different N-termini. Our results suggest a possible link between CySp1 and CySp2. In addition, comparisons of stress/strain curves for dragline and egg case silk from Argiope bruennichi showed obvious differences in ultimate strength and extensibility, and similarities in toughness.

Unique molecular architecture of egg case silk protein in a spider, Nephila clavata.

We describe a unique silk protein secreted from the cylindrical silk glands of the spider Nephila clavata. This silk is primarily composed of three proteins, whose transcripts of approximately 16.0, 14.5 and 13.0 kb are homologous to one another in two termini and repetitive units, as determined on Northern blotting. Its overall organization shows that it is similar to other characterized silk proteins, including in the mainly central repetitive region as well as the non-repetitive N-terminal (166 residues) and C-terminal (176 residues) parts. However, up to 90% of the protein consists of highly ordered repetitive structures that are not found in other silks. The repetitive region mainly consists of several types of complexes and remarkably conserved polypeptide repeats. The assembled repeat units (A1B1) contain a high proportion of Ala (30.41%), Ser (25.15%), and residues with hydrophobic side chains (22.22% for Gly, Leu, Ile, Val and Phe combined). The presence of Ser-rich and GVGAGASA motifs suggests the formation of a beta-sheet. The repetitive region is characterized by alternating arrays of hydrophobic and hydrophilic blocks. The results suggested that this egg case silk is an exceptional protein when compared with previously investigated spider silks.

Myocyte enhancer factor 2 (MEF2) is a key modulator of the expression of the prothoracicotropic hormone gene in the silkworm, Bombyx mori.

Prothoracicotropic hormone (PTTH) plays a central role in controlling molting, metamorphosis, and diapause termination in insects by stimulating the prothoracic glands to synthesize and release the molting hormone, ecdysone. Using Autographa californica nucleopolyhedrovirus (AcNPV)-mediated transient gene transfer into the central nervous sytem (CNS) of the silkworm, Bombyx mori, we identified two cis-regulatory elements that participate in the decision and the enhancement of PTTH gene expression in PTTH-producing neurosecretory cells (PTPCs). The cis-element mediating the enhancement of PTTH gene expression binds the transcription factor Bombyx myocyte enhancer factor 2 (BmMEF2). The BmMEF2 gene was expressed in various tissues including the CNS. In brain, the BmMEF2 gene was expressed at elevated levels in two types of lateral neurosecretory cells, namely PTPCs and corazonin-like immunoreactive lateral neurosecretory cells. Overexpression of BmMEF2 cDNA caused an increase in the transcription of PTTH. Therefore, BmMEF2 appears to be particularly important in the brain where it is responsible for the differentiation of lateral neurosecretory cells, including the enhancement of PTTH gene expression. This is the first report to identify a target gene of MEF2 in the invertebrate nervous system.

Baculovirus-mediated expression and isolation of human ribosomal phosphoprotein P0 carrying a GST-tag in a functional state.

We constructed an overexpression system for human ribosomal phosphoprotein P0, together with P1 and P2, which is crucially important for translation. Genes for these proteins, fused with the glutathione S-transferase (GST)-tag at the N-terminus, were inserted into baculovirus and introduced to insect cells. The fusion proteins, but not the proteins without the tag, were efficiently expressed into cells as soluble forms. The fusion protein GST.P0 as well as GST.P1/GST.P2 was phosphorylated in cells as detected by incorporation of (32)P and reactivity with monoclonal anti-phosphoserine antibody. GST.P0 expressed in insect cells, but not the protein obtained in Escherichia coli, had the ability to form a complex with P1 and P2 proteins and to bind to 28S rRNA. Moreover, the GST.P0-P1-P2 complex participated in high eEF-2-dependent GTPase activity. Baculovirus expression systems appear to provide recombinant human P0 samples that can be used for studies on the structure and function.