Assembly of the TgrB1-TgrC1 cell adhesion complex during Dictyostelium discoideum development.

In Dictyostelium discoideum, TgrB1 and TgrC1 are partners of a heterophilic cell-adhesion system. To investigate its assembly process, the split GFP complementation assay was used to track the oligomeric status of both proteins. The ability of TgrC1 to form cis-homodimers spontaneously was demonstrated by fluorescence complementation studies and confirmed by chemical cross-linking.

The Expression, Purification, and Characterization of a Ras Oncogene (Bras2) in Silkworm (Bombyx mori).

The Ras oncogene of silkworm pupae (Bras2) may belong to the Ras superfamily. It shares 77% of its amino acid identity with teratocarcinoma oncogene 21 (TC21) related ras viral oncogene homolog-2 (R-Ras2) and possesses an identical core effector region. The mRNA of Bombyx mori Bras2 has 1412 bp.

TgrC1 mediates cell-cell adhesion by interacting with TgrB1 via mutual IPT/TIG domains during development of Dictyostelium discoideum.

Cell-cell adhesion plays crucial roles in cell differentiation and morphogenesis during development of Dictyostelium discoideum. The heterophilic adhesion protein TgrC1 (Tgr is transmembrane, IPT, IG, E-set, repeat protein) is expressed during cell aggregation, and disruption of the tgrC1 gene results in the arrest of development at the loose aggregate stage. We have used far-Western blotting coupled with MS to identify TgrB1 as the heterophilic binding partner of TgrC1.

Construction of the ie1-Bacmid expression system and its use to express EGFP and BmAGO2 in BmN cells.

The presently available expression tools and vectors (e.g., eukaryotic expression vectors and the adenovirus expression system) for studying the functional genes in Bombyx mori are insufficient.

Characterization of a gene encoding prohibitin in silkworm, Bombyx mori.

Background: Prohibitin (PHB) is an evolutionarily conserved multifunctional protein with ubiquitous expression. However, its molecular roles are largely unknown.

Methods: To better understand the function of prohibitin protein in silkworm (BmPHB), its coding sequence was isolated from a cDNA library of silkworm pupae.

All-trans retinoic acid affects subcellular localization of a novel BmNIF3l protein: functional deduce and tissue distribution of NIF3l gene from silkworm (Bombyx mori).

A novel cDNA sequence encoding a predicted protein of 271 amino acids containing a conserved NIF3 domain was found from a pupal cDNA library of silkworm. The corresponding gene was named BmNIF3l (Bombyx mori NGG1p interacting factor 3-like). It was found by bioinformatics that BmNIF3l gene consisted of five exons and four introns and BmNIF3l had a high degree of homology to other NIF3-like proteins, especially in the N-terminal and C-terminal regions.

In vivo bioassay of recombinant human growth hormone synthesized in B. mori pupae.

The human growth hormone (hGH) has been expressed in prokaryotic expression system with low bioactivity previously. Then the effective B. mori baculovirus system was employed to express hGH identical to mature hGH successfully in larvae, but the expression level was still limited.

Characterization of the gene BmEm4, a homologue of Drosophila E(spl)m4, from the silkworm, Bombyx mori.

The Drosophila E(spl)m4 gene contains some highly conserved motifs (such as the Brd box, GY box, K box, and CAAC motif) in its 3' untranslated region (3' UTR). It was shown to be a microRNA target gene in Drosophila and to play an important role in the regulation of neurogenesis. We identified a homologue of the E(spl)m4 gene from Bombyx mori called BmEm4 and examined the expression patterns of BmEm4 mRNA and protein.

Molecular cloning and expression characterization of translationally controlled tumor protein in silkworm pupae.

A Bombyx mori (B. mori) cDNA was isolated from silkworm pupae cDNA library encoding a homologue of translationally controlled tumor protein (BmTCTPk). BmTCTPk was expressed in E.

Molecular characterization and tissue localization of an F-box only protein from silkworm, Bombyx mori.

The eukaryotic F-box protein family is characterized by an F-box motif that has been shown to be critical for the controlled degradation of regulatory proteins. We identified a gene encoding an F-box protein from a cDNA library of silkworm pupae, which has an ORF of 1821 bp, encoding a predicted 606 amino acids. Bioinformatic analysis on the amino acid sequence shows that BmFBXO21 has a low degree of similarity to proteins from other species, and may be related to the regulation of cell-cycle progression.

Aligning the proteome and genome of the silkworm, Bombyx mori.

A technology of mass spectrometry (MS) was used in this study for the large-scale proteomic identification and verification of protein-encoding genes present in the silkworm (Bombyx mori) genome. Peptide sequences identified by MS were compared with those from an open reading frame (ORF) library of the B. mori genome and a cDNA library, to validate the coding attributes of ORFs.

Bioavailability of orally administered rhGM-CSF: a single-dose, randomized, open-label, two-period crossover trial.

Background: Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is usually administered by injection, and its oral administration in a clinical setting has been not yet reported. Here we demonstrate the bioavailability of orally administered rhGM-CSF in healthy volunteers. The rhGM-CSF was expressed in Bombyx mori expression system (BmrhGM-CSF).

Safety and immunogenicity of H5N1 influenza vaccine based on baculovirus surface display system of Bombyx mori.

Avian influenza virus (H5N1) has caused serious infections in human beings. This virus has the potential to emerge as a pandemic threat in humans. Effective vaccines against H5N1 virus are needed.

A novel method for isolation of membrane proteins: a baculovirus surface display system.

We have developed a novel baculovirus surface display (BVSD) system for the isolation of membrane proteins. We expressed a reporter gene that encoded hemagglutinin gene fused in frame with the signal peptide and transmembrane domain of the baculovirus gp64 protein, which is displayed on the surface of BmNPV virions. The expression of this fusion protein on the virion envelope allowed us to develop two methods for isolating membrane proteins.

Expression analysis and characteristics of profilin gene from silkworm, Bombyx mori.

A recombinant Bombyx mori profilin protein (rBmPFN) was overexpressed in Escherichia coli BL21. Purified rBmPFN was used to generate anti-BmPFN polyclonal antibody, which were used to determine the subcellular localization of BmPFN. Immunostaining indicated that profilin can be found in both the nucleus and cytoplasm but is primarily located in the cytoplasm.

Subcellular localization and RNA interference of an RNA methyltransferase gene from silkworm, Bombyx mori.

RNA methylation, which is a form of posttranscriptional modification, is catalyzed by S-adenosyl-L-methionone-dependent RNA methyltransterases (RNA MTases). We have identified a novel silkworm gene, BmRNAMTase, containing a 369-bp open reading frame that encodes a putative protein containing 122 amino acid residues and having a molecular weight of 13.88 kd.

Identification and characteristics of microRNAs from Bombyx mori.

Background: MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression by targeting messenger RNAs (mRNAs) and causing mRNA cleavage or translation blockage. Of the 355 Arthropod miRNAs that have been identified, only 21 are B. mori miRNAs that were predicted computationally; of these, only let-7 has been confirmed by Northern blotting.

[Construction and expression of recombinant baculovirus with Schistosoma japonicum SjPP gene].

Objective: To express the soluble recombinant Schistosoma japonicum SjPP proteins in TN5B1-4 cells.

Methods: The total RNA was extracted from adult worms of Schistosoma japonicum. The whole coding sequence of SjPP gene was synthesized by RT-PCR and cloned into donor plasmid.

Molecular characterization and immunohistochemical localization of a novel troponin C during silkworm development.

We have cloned and sequenced a novel Bombyx mori gene that encodes a protein having a high degree of homology with other known troponin C (TnC) proteins. The amino acid sequence, DX[DN]X[DSG]X(6)E, a highly conserved putative Ca(2+) -binding motif found in loops within the globular domains of previously identified TnC proteins, is also present in BmTnC. We have expressed and purified to homogeneity a His-tagged BmTnC fusion protein having a molecular weight of approximately 21.

Expression analysis and tissue distribution of two 14-3-3 proteins in silkworm (Bombyx mori).

14-3-3 proteins, which have been identified in a wide variety of eukaryotes, are highly conserved acidic proteins. In this study, we identified two genes in silkworm that encode 14-3-3 proteins (Bm14-3-3zeta and Bm14-3-3epsilon). Category of two 14-3-3 proteins was identified according to phylogenetic analysis.

Complete sequence and organization of Antheraea pernyi nucleopolyhedrovirus, a dr-rich baculovirus.

Background: The completion and reporting of baculovirus genomes is extremely important as it advances our understanding of gene function and evolution. Due to the large number of viral genomes now sequenced it is very important that authors present significantly detailed analyses to advance the understanding of the viral genomes. However, there is no report of the Antheraea pernyi nucleopolyhedrovirus (AnpeNPV) genome.

Large-scale purification of human granulocyte-macrophage colony-stimulating factor expressed in Bombyx mori pupae.

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) acts on many different kinds of cells, including monocytes, macrophages, granulocytes, eosinophils, and multipotential stem cells. To explore further explore pharmaceutical action, we expressed hGM-CSF by the Bombyx mori nucleopolyhedrovirus expression system in silkworm pupae. However, purifying recombinant proteins from silkworm pupae on a large scale has been a big challenge.

Expression of open reading frames in silkworm pupal cDNA library.

A cDNA library containing 2409 singletons was constructed from whole silkworm pupae (Bombyx mori) In addition, the types of genes overexpressed in pupa were analyzed. These genes contained 79 types of proteins with the exception of enzyme, mitochondrial DNA, andribosomal protein. Also analyzed were the expression and nonexpression of open reading frame (ORF) sequences in Escherichia coli.

Expression and functional analysis of the cellular retinoic acid binding protein from silkworm pupae (Bombyx mori).

Cellular retinoic acid binding protein (CRABP) is a member of intracellular lipid-binding protein (iLBP), and closely associated with retinoic acid (RA) activity. We have cloned the CRABP gene from silkworm pupae and studied the interaction between Bombyx mori CRABP (BmCRABP) and all-trans retinoic acid (atRA). The MTT assay data indicated that when BmCRABP is overexpressed in Bm5 cells, the cells dramatically resisted to atRA-induced growth inhibition.

Oral administration activity determination of recombinant osteoprotegerin from silkworm larvae.

Osteoprotegerin (OPG) regulates the formation of osteoclasts and is involved in the regulation of bone resorption and remodeling. To investigate the feasibility of using silkworm (Bombyx mori) larvae to produce recombinant osteoprotegerin as a oral administration drug, the rh-OPG was expressed in the larvae of silkworm through the silkworm baculovirus expression system, and was orally administered to mice. Compared with the control, oral administration of rh-OPG was effective to decrease serum calcium concentration in normal mice, and block the bone loss induced by the loss of estrogen in ovariectomized mice.