Microarray analysis of gene expression profile in resistant and susceptible Bombyx mori strains reveals resistance-related genes to nucleopolyhedrovirus

To investigate the molecular mechanism of silkworm resistance to BmNPV infection, we constructed a near-isogenic line (BC8) with BmNPV resistance using highly resistant (NB) and highly susceptible parental strains (306). We investigated variations in the gene expression in the midguts of BmNPV-infected BC8 and 306 at 12 h pi using the microarray. 92 differentially expressed genes were identified. Real-time qPCR analysis confirmed that 10 genes were significantly up-regulated or down-regulated in the midguts of BC8 and NB compared to 306. To our knowledge, we first defined the role of the amino acid transporter and 26S proteasome in insect antiviral. However, serine protease was not completely consistent with data of reported previously in insect antiviral. The role of the 5 genes (Bm123, Bm122, COP β′, aquaporin, glycoside hydrolases) was also demonstrated in insect antiviral. Our results provided new insights into the molecular mechanism of the Bombyx mori immune response against BmNPV infection.     

Functional study of active residues scorpion insect toxin BmK IT from Buthus martensii Karsch

Chinese scorpion Buthus martensii Karsch (BmK) venom is a rich source of neurotoxins which bind to various ion channels with high affinity and specificity and thus widely used as compounds to modulate channel gating. An excitatory insect toxin, BmK IT, is not conserved with a glutamate residue at the preceding position of the third Cys residue, and is a toxin with a non-glutamate residue at the relevant position in the excitatory scorpion β-toxin subfamily. In this study, the mutants of recombinant BmK IT (BmK IT (I25E), BmK IT (E15G), BmK IT C-terminal (TKSYCDVQIN) truncated) were achieved by site-directed mutagenesis. Biological activity of BmK IT and its mutants confirmed these residues or peptides played key roles in BmK IT. BmK IT (I25E) could increase the sensitivity of BmK IT, but BmK IT(E15G) could decrease the sensitivity of BmK IT on Sf9 cells. BmK IT truncated C-terminal hydrophobic amino acids could cross the species boundaries and was effective on mammalian C6 cells. To date, several excitatory insect toxins have been isolated and identified from the venom of Buthus martensii Karsch. However, no functional data are available and therefore its classification in the family of excitatory insect toxins remains putative and is just based on its high similarity with the other toxins of this family. These results verified I25, E15 and C-terminal (TKSYCDVQIN) in BmK IT played key roles in the interaction of the BmK IT and its receptor- sodium channels on the surface of insect cells and laid a foundation for further structural and functional analysis of BmK IT.     

pEGFP-N1-mediated BmK CT expression suppresses the migration of glioma

Gliomas can diffuse into the normal brain and this invasion of glioma cells involves modification of receptor-mediated adhesive properties of tumor cells, degradation and remodeling of extracellular matrix by tumor-secreted metalloproteinase (MMPs) such as MMP-2, consequently creating an intercellular space for invasion of glioma cells. BmK CT, one of the key toxins in scorpion Buthus martensii Karsch venom, is a novel blocker of the chloride ion channel and MMP-2. In this report, a recombinant plasmid pEGFP-N1-BmK CT was constructed and characterized by in vitro studies. The results showed that pEGFP-N1 mediated BmK CT expression displayed a high activity in suppressing cell migration via MMP-2. The potential therapeutic effect of pEGFP-N1 mediated BmK CT against rat glioma C6 cells was assessed and its potential mechanism was elucidated. It represented an approach for developing a novel therapeutic agent—recombinant plasmid pEGFP-N1-BmK CT as an efficient and powerful adjuvant.     

Surface Display and Bioactivity of Bombyx mori Acetylcholinesterase on Pichia pastoris

A Pichia pastoris (P. pastoris) cell surface display system of Bombyx mori acetylcholinesterase (BmAChE) was constructed and its bioactivity was studied. The modified Bombyx mori acetylcholinesterase gene (bmace) was fused with the anchor protein (AGα1) from Saccharomyces cerevisiae and transformed into P. pastoris strain GS115. The recombinant strain harboring the fusion gene bmace-AGα1 was induced to display BmAChE on the P. pastoris cell surface. Fluorescence microscopy and flow cytometry assays revealed that the BmAChE was successfully displayed on the cell surface of P. pastoris GS115. The enzyme activity of the displayed BmAChE was detected by the Ellman method at 787.7 U/g (wet cell weight). In addition, bioactivity of the displayed BmAChE was verified by inhibition tests conducted with eserine, and with carbamate and organophosphorus pesticides. The displayed BmAChE had an IC₅₀ of 4.17×10⁻⁸ M and was highly sensitive to eserine and five carbamate pesticides, as well as seven organophosphorus pesticides. Results suggest that the displayed BmAChE had good bioactivity.     

Mutation of a novel ABC transporter gene is responsible for the failure to incorporate uric acid in the epidermis of ok mutants of the silkworm,Bombyx mori

ok mutants of the silkworm, Bombyx mori, exhibit highly translucent larval skin resulting from the inability to incorporate uric acid into the epidermal cells. Here we report the identification of a gene responsible for the ok mutation using positional cloning and RNAi experiments. In two independent ok mutant strains, we found a 49-bp deletion and a 233-bp duplication, respectively, in mRNAs of a novel gene, Bm-ok, which encodes a half-type ABC transporter, each of which results in translation of a truncated protein in each mutant. Although the Bm-ok sequence was homologous to well-known transporter genes, white, scarlet, and brown in Drosophila, the discovery of novel orthologs in the genomes of lepidopteran, hymenopteran, and hemipteran insects identifies it as a member of a new distinct subfamily of transporters. Embryonic RNAi of Bm-ok demonstrated that repression of Bm-ok causes a translucent phenotype in the first-instar silkworm larva. We discuss the possibility that Bm-ok forms a heterodimer with another half-type ABC transporter, Bmwh3, and acts as a uric acid transporter in the silkworm epidermis.     

Codon Optimization Enhances Protein Expression of Bombyx mori Nucleopolyhedrovirus DNA Polymerase in E. coli

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major viral agent that causes deadly grasserie disease in silkworms, while BmNPV DNA polymerase (BmNPV-pol), encoded by ORF53 gene, plays a central role in viral DNA replication. Efficacy studies of BmNPV-POL are limited because of poor heterologous protein expression in E. coli. Here, we redesigned the BmNPV-pol to preferentially match codon frequencies of E. coli without altering the amino acid sequence. Following de novo synthesis, codon-optimized BmNPV-pol (co-BmNPV-pol) gene was cloned into pET32a and pGEX-4T-2 vector. The expression of co-BmNPV-POL in E. coli was significantly increased when BmNPV-POL was fused with GST protein rather than a His-tag. The co-BmNPV-POL fusion proteins were isolated using GST affinity chromatography and Mono Q iron exchange chromatography. Protein purity and identity were confirmed by western blot and MALDI-TOF analyses. The biological activity of purified proteins was measured on a poly(dA)/oligo(dT) primer/template. The specific polymerasing activity of the recombinant BmNPV-POL was 6,329 units/mg at optimal conditions. Thus, a large amount of purified protein as a soluble form with high activity would provide many benefits for the functional research and application of BmNPV-POL.     

Silencing of FABP3 Inhibits Proliferation and Promotes Apoptosis in Embryonic Carcinoma Cells

Fatty acid–binding protein 3 (FABP3) facilitates the movement of fatty acids in cardiac muscle. Previously, we reported that FABP3 is highly upregulated in the myocardium of ventricular septal defect patients and overexpression of FABP3 inhibited proliferation and promoted apoptosis in embryonic carcinoma cells (P19 cells). In this study, we aimed to investigate the effect of FABP3 gene silencing on P19 cell differentiation, proliferation and apoptosis. We used RNA interference and a lentiviral-based vector system to create a stable FABP3-silenced P19 cell line; knockdown of FABP3 was confirmed by quantitative real-time PCR. Expression analysis of specific differentiation marker genes using quantitative real-time PCR and observation of morphological changes using an inverted microscope revealed that knockdown of FABP3 did not significantly affect the differentiation of P19 cells into cardiomyocytes. CCK-8 proliferation assays and cell cycle analysis demonstrated that FABP3 gene silencing significantly inhibited P19 cell proliferation. Furthermore, Annexin V-FITC/propidium iodide staining and the caspase-3 activity assay revealed that FABP3 gene silencing significantly promoted serum starvation–induced apoptosis in P19 cells. In agreement with our previous research, these results demonstrate that FABP3 may play an important role during embryonic heart development, and that either overexpression or silencing of FABP3 will lead to an imbalance between proliferation and apoptosis, which may result in embryonic cardiac malformations.     

Effects of the m6Am methyltransferase PCIF1 on cell proliferation and survival in gliomas

BackgroundPrevious studies have suggested an important role for N6-methyladenosine (m6A) modification in the proliferation of glioma cells. N6, 2′-O-dimethyladenosine (m6Am) is another methylated form affecting the fate and function of most RNA. PCIF1 has recently been identified as the sole m6Am methyltransferase in mammalian mRNA. However, it remains unknown about the role of PCIF1 in the growth and survival of glioma cells.MethodsWe constructed glioma cell lines that stably downregulated/upregulated PCIF1, established intracranial xenograft models using these cell lines, and employed the following methods for investigations: CCK-8, EdU, colony formation, flow cytometry, qRT-PCR, Western blot, and immunohistochemistry.FindingsDownregulating PCIF1 promoted glioma cell proliferation, while overexpressing PCIF1 showed the opposite effects. Overexpression of PCIF1 blocked cell cycle progression and induced apoptosis in glioma cells, which was further confirmed by alterations in the expression of cell checkpoint proteins and apoptotic markers. Interestingly, disruption of PCIF1 methyltransferase activity slightly reversed the effect of PCIF1 overexpression on cell proliferation, but had no significant reversal effects on cell cycle progression or apoptosis. Knockdown of PCIF1 promoted the growth of gliomas, while overexpressing PCIF1 inhibited tumor growth and prolonged the survival time of tumor-bearing mice. In addition, the mRNA and protein levels of PCIF1 were gradually decreased with the increase of WHO grade in glioma tissues, but there was no significant correlation with patient survival.InterpretationThese results indicated that PCIF1 played a suppressing role in glioma growth and survival, which may not entirely depend on its methyltransferase activity.     

Effects of recombinant baculovirus AcMNPV-BmK IT on the formation of early cables and nuclear polymerization of actin in Sf9 cells

Autographa californica nuclearpoly hedrosis virus (AcMNPV) is one of the most important baculoviridae. However, the application of AcMNPV as a biocontrol agent has been limited. Previously, we engineered Buthus martensii Karsch insect toxin (BmK IT) gene into the genome of AcMNPV. The bioassay data indicated that the recombinant baculovirus AcMNPV-BmK IT significantly enhanced the anti-insect efficacy of the virus. The actin cytoskeleton is the major component beneath the surface of eukaryotic cells. In this report, the effects of AcMNPV-BmK IT on the formation of early cables of actin and nuclear filamentous-actin (F-actin) were studied. The results indicated that these baculovirus induced rearrangement of the actin cytoskeleton of host cells during infection and actin might participate in the transportation of baculovirus from cytoplasm to the nuclei. AcMNPV-BmK IT delayed the formation of early cables of actin and nuclear F-actin and accelerated the clearance of actin in the nuclei.     

Human Immunodeficiency Virus Type 1 Tat Induces Apoptosis and Increases Sensitivity to Apoptotic Signals by Up-Regulating FLICE/Caspase-8

Apoptosis contributes to the loss of CD4 cells during human immunodeficiency virus type 1 (HIV-1) infection. Although the product of the env gene, gp160/gp120, is known to play a role in cell death mediated by HIV-1, the role of other HIV-1 genes in the process is unclear. We found that HIV-1 lacking the env gene (HIVΔenv) still induced apoptosis in T-cell lines and primary CD4 T cells. The ability to induce apoptosis was attributable to Tat, a viral regulatory protein. Tat induction of apoptosis was separate from the transactivation function of Tat, required expression of the second exon of Tat, and was associated with the increased expression and activity of caspase-8 (casp-8), a signaling molecule in apoptotic pathways. Moreover, induction of apoptosis could be prevented by treating cells with an inhibitor of casp-8. In addition, we show that HIV-1Δenv infection and Tat expression increased the sensitivity of cells to Fas-mediated apoptosis, an apoptotic pathway that signals via casp-8. The up-regulation of casp-8 by HIV-1 Tat expression may contribute to the increased apoptosis and sensitivity to apoptotic signals observed in the cells of HIV-1-infected persons.     

Combination of lithium chloride and pEGFP-N1-BmK CT effectively decreases proliferation and migration of C6 glioma cells

Deleterious invasiveness of glioma cells into the normal brain tissue is endorsed by its inherent ability to regulate the receptor-mediated adhesive properties, extracellular matrix degradation and remodeling and elevated secretory ability of metalloproteinase (MMPs) such as MMP-2. By doing so, it will create an intercellular space for the invasion of glioma cells. Here, we reported that combination of gene therapy Buthus martensii Karsch (BmK) CT, a type of scorpion toxin peptide, with lithium chloride (LiCl), clinically used as mood stabilizer, could inhibit the migration and invasion of C6 glioma cells. The results showed that concomitant administration of LiCl and pEGFP-N1-BmK CT on glioma cells would hamper pro-MMP2 secretion and in the meantime, inhibited its proliferation in a synergistic manner. These results try to extrapolate the potential interplay between the combined treatment of LiCl and BmK CT with signaling pathways β-catenin, MMP, GSK-3 in C6 glioma cells. This strategy can stand for a novel approach designated for the development of a new method for glioma therapy.     

The Mitochondria-Mediate Apoptosis of Lepidopteran Cells Induced by Azadirachtin

Mitochondria have been shown to play an important role in apoptosis using mammalian cell lines. However, this seems not to be the case in Drosophila, an insect model organism; thus more in-depth studies of insect cell apoptosis are necessary. In the present study, mitochondrial involvement during azadirachtin- and camptothecin-induced apoptosis in Spodoptera frugiperda Sf9 cells (isolated from Spodoptera frugiperda pupal ovarian tissue) was investigated. The results showed that both azadirachtin and camptothecin could induce apoptosis in Sf9 cells. Reactive oxygen species (ROS) generation, activation of mitochondrial permeability transition pores (MPTPs) and loss of mitochondrial membrane potential (MMP) were observed very early during apoptosis and were followed subsequently by the release of cytochrome-c from the mitochondria. Furthermore, the results also revealed that the opening of MPTPs and the loss of MMP induced by azadirachtin could be significantly inhibited by the permeability transition pore (PTP) inhibitor cyclosporin A (CsA), which was used to identify the key role of mitochondria in the apoptosis of Sf9 cells. However, in camptothecin-treated Sf9 cells, CsA could not suppress the opening of MPTPs and the loss of MMP when apoptosis was induced. The data from caspase-3 and caspase-9 activity assays and detection of apoptosis by morphological observation and flow cytometry also uncovered the different effect of CsA on the two botanical apoptosis inducers. Although different mechanisms of apoptosis induction exist, our study revealed that mitochondria play a crucial role in insect cell line apoptosis.     

Effects of WT1 gene downregulation on apoptosis in porcine fetal fibroblasts

Wilms’ tumor gene 1 (WT1) is located on chromosome 11p13. Besides a role in the development of Wilms’ tumor, specific mutations in the Zn finger region are found in Denys-Drash syndrome and Frasier syndrome, both characterized by urogenital abnormalities, sometimes in combination with Wilms’ tumor. Our past study shows that WT1 is expressed in porcine kidney fibroblasts (PKFs) and swine testis cells (ST cells) and is essential for the maintenance of the development and survival of PKFs and ST cells. But we do not know whether WT1 gene was expressed in porcine fetal fibroblasts or not. To further explore whether WT1 was expressed in porcine fetal fibroblasts (PFFs) and its contribution to cell apoptosis, RT-PCR, immunocytochemical staining, and Western blot were used to detect the expression of WT1, the recombinant plasmids of pLV3-WT1 short hairpin ribonucleic acid (shRNA) were used to downregulate the WT1 gene in porcine fetal fibroblasts, and the role of WT1 in cell proliferation was examined by apoptosis analysis also. Our results indicated that WT1 was expressed in PFFs, the pLV3-WT1 shRNA dramatically reduced the expression of WT1, and downregulation of WT1 directly led to early cell apoptosis by downregulating the expression of antiapoptotic gene Bcl-2 and upregulating the expression of proapoptotic gene Bax in PFFs. Our results demonstrate that WT1 is also essential for the maintenance of the survival of PFFs.     

Expression profiles of glutathione <Emphasis Type="Italic">S</Emphasis>-transferase genes in larval midgut of <Emphasis Type="Italic">Bombyx mori</Emphasis> exposed to insect hormones

Glutathione S-transferases (GSTs) are believed to play a role in the detoxification of xenobiotics, resistance to insect viruses and pesticides, intracellular transport, biosynthesis of hormones and protection against oxidative stress. In this study, we used quantitative real time RT-PCR to examine expression profiles of the silkworm Bombyx mori GST-Sigma (BmGSTS2) and GST-Delta (BmGSTD2) genes in the larval midgut of the silkworm after exposure to 2-hydroxyecdysone (20E) and juvenile hormone analog (JHA). In concentration-course study, 20E at higher concentrations (1.0 and 2.0 μg/μl) caused significant upregulation of BmGSTD2, and all concentrations (0.5–2.0 μg/μl) of 20E caused significant upregulation of BmGSTS2. However, JHA in all concentrations downregulated the expression of BmGSTD2 and BmGSTS2. When exposed to either 20E (2.0 μg/μl) or JHA (2.0 μg/μl) on the third day of the fifth instar, the silkworm had higher BmGSTD2 at later time points: 15, 18, and 24 h for 20E and 24 h for JHA. BmGSTS2 expression was downregulated within 24 h after exposure to JHA and showed a time-dependent response after exposure to 20E. We also did a stage-dependent study, in which JHA downregulated BmGSTD2 expression and upregulated BmGSTS2 expression significantly at both day 1 and day 3 of the fifth instar. 20E upregulated the expression of BmGSTD2 and BmGSTS2 at the two stages. These findings imply that hormones have an important role in the regulation of basal GST expression. However, further validation and field trials should be carried out on the regulatory elements relevant to BmGSTD2 and BmGSTS2 gene expression.     

Biochemical characterization of maintenance DNA methyltransferase DNMT-1 from silkworm,Bombyx mori

DNA methylation is an important epigenetic mechanism involved in gene expression of vertebrates and invertebrates. In general, DNA methylation profile is established by de novo DNA methyltransferases (DNMT-3A, -3B) and maintainance DNA methyltransferase (DNMT-1). DNMT-1 has a strong substrate preference for hemimethylated DNA over the unmethylated one. Because the silkworm genome lacks an apparent homologue of de novo DNMT, it is still unclear that how silkworm chromosome establishes and maintains its DNA methylation profile. As the first step to unravel this enigma, we purified recombinant BmDNMT-1 using baculovirus expression system and characterized its DNA-binding and DNA methylation activity. We found that the BmDNMT-1 preferentially methylates hemimethylated DNA despite binding to both unmethylated and hemimethylated DNA. Interestingly, BmDNMT-1 formed a complex with DNA in the presence or absence of methyl group donor, S-Adenosylmethionine (AdoMet) and the AdoMet-dependent complex formation was facilitated by Zn²⁺ and Mn²⁺. Our results provide clear evidence that BmDNMT-1 retained the function as maintenance DNMT but its sensitivity to metal ions is different from mammalian DNMT-1.     

Synthesis and anticancer activity of a novel series of 9-O-substituted berberine derivatives: A lipophilic substitute role

To alter its hydrophobicity, a series of compounds bearing 9-O-alkyl- or 9-O-terpenyl- substituted berberine were synthesized and evaluated for anticancer activity against human cancer HepG2 and HT29 cell lines. We found that the lipophilic substitute of 9-O-alkyl- and 9-O-terpenyl berberine derivatives plays a role in inhibiting the human cancer cell growth and its activity could be maximized with the optimized substitute type and chain length. Most strikingly, nonetheless, of the six compounds prepared, sample 8, a farnesyl 9-O-substituted berberine, showed either comparable or better cytotoxic activity against human cancer HepG2 cell line than that of berberine. Compound 8 had also shown a 104-fold antiproliferation activity in compare with berberine against human hepatoma HepG2 cell lines after 48 incubation hours. Further, in Hoechst 33258 and annexin V-FITC/PI staining analyses it induced apoptosis in HepG2 cells at lower concentration than that of berberine for 24 h. Take all; farnesyl 9-O-substituted berberine could be a potential candidate for new anticancer drug development.